Download PDF
CC BY-NC-ND 4.0 · Neuropediatrics 2022; 53(06): 423-431
DOI: 10.1055/a-1889-8544
Original Article

No Differences in Cerebral Immunohistochemical Markers following Remote Ischemic Postconditioning in Newborn Piglets with Hypoxia–Ischemia

Hannah B. Andersen   *
1   Department of Paediatrics and Adolescent Medicine, Aarhus University Hospital, Aarhus, Denmark
,
Mads Andersen    *
1   Department of Paediatrics and Adolescent Medicine, Aarhus University Hospital, Aarhus, Denmark
,
Kristine Bennedsgaard
1   Department of Paediatrics and Adolescent Medicine, Aarhus University Hospital, Aarhus, Denmark
,
Sigrid Kerrn-Jespersen
1   Department of Paediatrics and Adolescent Medicine, Aarhus University Hospital, Aarhus, Denmark
,
Kasper J. Kyng
1   Department of Paediatrics and Adolescent Medicine, Aarhus University Hospital, Aarhus, Denmark
,
Ida E. Holm
2   Department of Pathology, Randers Regional Hospital, Randers, Denmark
,
Tine B. Henriksen
1   Department of Paediatrics and Adolescent Medicine, Aarhus University Hospital, Aarhus, Denmark
› Author Affiliations Funding The study was supported by the Aase and Ejnar Danielsens Foundations, The Health Research Foundation of Central Denmark Region, Aarhus University Research Foundation, Randers Regional Hospital, and The Elsass Foundation.
› Further Information
   Permissions and Reprints

Abstract

Background Despite therapeutic hypothermia, neonates with hypoxic–ischemic encephalopathy still develop neurological disabilities. We have previously investigated neuroprotection by remote ischemic postconditioning (RIPC) in newborn piglets following hypoxia–ischemia (HI). The aim of this study was to further investigate potential effects of RIPC on cerebral immunohistochemical markers related to edema, apoptosis, and angiogenesis.

Methods Brain expression of aquaporin 4, caspase-3, B-cell lymphoma 2, and vascular endothelial growth factor was analyzed by immunohistochemistry in 23 piglets, randomly selected from a larger study of RIPC after HI. Twenty animals were subjected to 45 minutes of HI and randomized to treatment with and without RIPC, while three animals were randomized to sham procedures. RIPC was conducted by four conditioning cycles of 5-minute ischemia and reperfusion. Piglets were euthanized 72 hours after the HI insult.

Results Piglets subjected to HI treated with and without RIPC were similar at baseline and following the HI insult. However, piglets randomized to HI alone had longer duration of low blood pressure during the insult. We found no differences in the brain expression of the immunohistochemical markers in any regions of interest or the whole brain between the two HI groups.

Conclusion RIPC did not influence brain expression of markers related to edema, apoptosis, or angiogenesis in newborn piglets at 72 hours after HI. These results support previous findings of limited neuroprotective effect by this RIPC protocol. Our results may have been affected by the time of assessment, use of fentanyl as anesthetic, or limitations related to our immunohistochemical methods.

Keywords

hypoxia–ischemia - hypoxic–ischemic encephalopathy - remote ischemic postconditioning - immunohistochemistry - piglet model

* These authors have contributed equally to this work and share first authorship.


Supplementary Material



Publication History

Received: 07 December 2021

Accepted: 02 June 2022

Accepted Manuscript online:
01 July 2022

Article published online:
28 August 2022

© 2022. The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution-NonDerivative-NonCommercial License, permitting copying and reproduction so long as the original work is given appropriate credit. Contents may not be used for commercial purposes, or adapted, remixed, transformed or built upon. (https://creativecommons.org/licenses/by-nc-nd/4.0/)

Georg Thieme Verlag KG
Rüdigerstraße 14, 70469 Stuttgart, Germany

 

  • References

  • 1 Lee AC, Kozuki N, Blencowe H. et al. Intrapartum-related neonatal encephalopathy incidence and impairment at regional and global levels for 2010 with trends from 1990. Pediatr Res 2013; 74 (Suppl. 01) 50-72
    CrossrefPubMedGoogle Scholar
  • 2 Kurinczuk JJ, White-Koning M, Badawi N. Epidemiology of neonatal encephalopathy and hypoxic-ischaemic encephalopathy. Early Hum Dev 2010; 86 (06) 329-338
    CrossrefPubMedGoogle Scholar
  • 3 Jacobs SE, Berg M, Hunt R, Tarnow-Mordi WO, Inder TE, Davis PG. Cooling for newborns with hypoxic ischaemic encephalopathy. Cochrane Database Syst Rev 2013; (01) CD003311
    PubMedGoogle Scholar
  • 4 Edwards AD, Brocklehurst P, Gunn AJ. et al. Neurological outcomes at 18 months of age after moderate hypothermia for perinatal hypoxic ischaemic encephalopathy: synthesis and meta-analysis of trial data. BMJ 2010; 340: c363
    CrossrefPubMedGoogle Scholar
  • 5 Pauliah SS, Shankaran S, Wade A, Cady EB, Thayyil S. Therapeutic hypothermia for neonatal encephalopathy in low- and middle-income countries: a systematic review and meta-analysis. PLoS One 2013; 8 (03) e58834
    CrossrefPubMedGoogle Scholar
  • 6 Thayyil S, Pant S, Montaldo P. et al; HELIX consortium. Hypothermia for moderate or severe neonatal encephalopathy in low-income and middle-income countries (HELIX): a randomised controlled trial in India, Sri Lanka, and Bangladesh. Lancet Glob Health 2021; 9 (09) e1273-e1285
    CrossrefPubMedGoogle Scholar
  • 7 Andelius TCK, Henriksen TB, Kousholt BS, Kyng KJ. Remote ischemic postconditioning for neuroprotection after newborn hypoxia-ischemia: systematic review of preclinical studies. Pediatr Res 2022; 91 (07) 1654-1661
    CrossrefPubMedGoogle Scholar
  • 8 Kyng KJ, Kerrn-Jespersen S, Bennedsgaard K. et al. Short-term outcomes of remote ischemic postconditioning 1 h after perinatal hypoxia-ischemia in term piglets. Pediatr Res 2021; 89 (01) 150-156
    CrossrefPubMedGoogle Scholar
  • 9 Thayyil S, Chandrasekaran M, Taylor A. et al. Cerebral magnetic resonance biomarkers in neonatal encephalopathy: a meta-analysis. Pediatrics 2010; 125 (02) e382-e395
    CrossrefPubMedGoogle Scholar
  • 10 Lally PJ, Montaldo P, Oliveira V. et al; MARBLE consortium. Magnetic resonance spectroscopy assessment of brain injury after moderate hypothermia in neonatal encephalopathy: a prospective multicentre cohort study. Lancet Neurol 2019; 18 (01) 35-45
    CrossrefPubMedGoogle Scholar
  • 11 Ezzati M, Bainbridge A, Broad KD. et al. Immediate remote ischemic postconditioning after hypoxia ischemia in piglets protects cerebral white matter but not grey matter. J Cereb Blood Flow Metab 2016; 36 (08) 1396-1411
    CrossrefPubMedGoogle Scholar
  • 12 Ferrari DC, Nesic O, Perez-Polo JR. Perspectives on neonatal hypoxia/ischemia-induced edema formation. Neurochem Res 2010; 35 (12) 1957-1965
    CrossrefPubMedGoogle Scholar
  • 13 Northington FJ, Chavez-Valdez R, Martin LJ. Neuronal cell death in neonatal hypoxia-ischemia. Ann Neurol 2011; 69 (05) 743-758
    CrossrefPubMedGoogle Scholar
  • 14 Yan F, Zhang M, Meng Y. et al. Erythropoietin improves hypoxic-ischemic encephalopathy in neonatal rats after short-term anoxia by enhancing angiogenesis. Brain Res 2016; 1651: 104-113
    CrossrefPubMedGoogle Scholar
  • 15 Yu L, Fan SJ, Liu L. et al. Effect of ischemic postconditioning on cerebral edema and the AQP4 expression following hypoxic-eschemic brain damage in neonatal rats. World J Pediatr 2015; 11 (02) 165-170
    CrossrefPubMedGoogle Scholar
  • 16 Han D, Sun M, He PP, Wen LL, Zhang H, Feng J. Ischemic postconditioning alleviates brain edema after focal cerebral ischemia reperfusion in rats through down-regulation of aquaporin-4. J Mol Neurosci 2015; 56 (03) 722-729
    CrossrefPubMedGoogle Scholar
  • 17 Li S, Hu X, Zhang M. et al. Remote ischemic post-conditioning improves neurological function by AQP4 down-regulation in astrocytes. Behav Brain Res 2015; 289: 1-8
    CrossrefPubMedGoogle Scholar
  • 18 Zhang W, Wang B, Zhou S, Qiu Y. The effect of ischemic post-conditioning on hippocampal cell apoptosis following global brain ischemia in rats. J Clin Neurosci 2012; 19 (04) 570-573
    CrossrefPubMedGoogle Scholar
  • 19 Xing B, Chen H, Zhang M. et al. Ischemic postconditioning inhibits apoptosis after focal cerebral ischemia/reperfusion injury in the rat. Stroke 2008; 39 (08) 2362-2369
    CrossrefPubMedGoogle Scholar
  • 20 Esposito E, Hayakawa K, Ahn BJ. et al. Effects of ischemic post-conditioning on neuronal VEGF regulation and microglial polarization in a rat model of focal cerebral ischemia. J Neurochem 2018; 146 (02) 160-172
    CrossrefPubMedGoogle Scholar
  • 21 Zhu T, Zhan L, Liang D. et al. Hypoxia-inducible factor 1α mediates neuroprotection of hypoxic postconditioning against global cerebral ischemia. J Neuropathol Exp Neurol 2014; 73 (10) 975-986
    CrossrefPubMedGoogle Scholar
  • 22 Adstamongkonkul D, Hess DC. Ischemic Conditioning and neonatal hypoxic ischemic encephalopathy: a literature review. Cond Med 2017; 1 (01) 9-16
    PubMedGoogle Scholar
  • 23 Kilkenny C, Browne WJ, Cuthill IC, Emerson M, Altman DG. Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research. PLoS Biol 2010; 8 (06) e1000412
    CrossrefPubMedGoogle Scholar
  • 24 Kyng KJ, Skajaa T, Kerrn-Jespersen S, Andreassen CS, Bennedsgaard K, Henriksen TB. A piglet model of neonatal hypoxic-ischemic encephalopathy. J Vis Exp 2015; e52454 (99) e52454
    PubMedGoogle Scholar
  • 25 Thoresen M, Haaland K, Løberg EM. et al. A piglet survival model of posthypoxic encephalopathy. Pediatr Res 1996; 40 (05) 738-748
    CrossrefPubMedGoogle Scholar
  • 26 Takeuchi K, Ogata S, Nakanishi K. et al. LAT1 expression in non-small-cell lung carcinomas: analyses by semiquantitative reverse transcription-PCR (237 cases) and immunohistochemistry (295 cases). Lung Cancer 2010; 68 (01) 58-65
    CrossrefPubMedGoogle Scholar
  • 27 Bösmüller H, Fischer A, Pham DL. et al. Detection of the BRAF V600E mutation in serous ovarian tumors: a comparative analysis of immunohistochemistry with a mutation-specific monoclonal antibody and allele-specific PCR. Hum Pathol 2013; 44 (03) 329-335
    CrossrefPubMedGoogle Scholar
  • 28 Rocha-Ferreira E, Rudge B, Hughes MP, Rahim AA, Hristova M, Robertson NJ. Immediate remote ischemic postconditioning reduces brain nitrotyrosine formation in a piglet asphyxia model. Oxid Med Cell Longev 2016; 2016: 5763743
    CrossrefPubMedGoogle Scholar
  • 29 Zhou Y, Fathali N, Lekic T. et al. Remote limb ischemic postconditioning protects against neonatal hypoxic-ischemic brain injury in rat pups by the opioid receptor/Akt pathway. Stroke 2011; 42 (02) 439-444
    CrossrefPubMedGoogle Scholar
  • 30 Drunalini Perera PN, Hu Q, Tang J. et al. Delayed remote ischemic postconditioning improves long term sensory motor deficits in a neonatal hypoxic ischemic rat model. PLoS One 2014; 9 (02) e90258
    CrossrefPubMedGoogle Scholar
  • 31 Andelius TCK, Pedersen MV, Andersen HB. et al. No added neuroprotective effect of remote ischemic postconditioning and therapeutic hypothermia after mild hypoxia-ischemia in a piglet model. Front Pediatr 2020; 8: 299
    CrossrefPubMedGoogle Scholar
  • 32 Banasiak KJ, Cronin T, Haddad GG. bcl-2 prolongs neuronal survival during hypoxia-induced apoptosis. Brain Res Mol Brain Res 1999; 72 (02) 214-225
    CrossrefPubMedGoogle Scholar
  • 33 Wang X, Karlsson JO, Zhu C, Bahr BA, Hagberg H, Blomgren K. Caspase-3 activation after neonatal rat cerebral hypoxia-ischemia. Biol Neonate 2001; 79 (3,4): 172-179
    CrossrefPubMedGoogle Scholar
  • 34 Konno A, Nishimura A, Nakamura S. et al. Continuous monitoring of caspase-3 activation induced by propofol in developing mouse brain. Int J Dev Neurosci 2016; 51: 42-49
    CrossrefPubMedGoogle Scholar
  • 35 Robertson NJ, Martinello K, Lingam I. et al. Melatonin as an adjunct to therapeutic hypothermia in a piglet model of neonatal encephalopathy: a translational study. Neurobiol Dis 2019; 121: 240-251
    CrossrefPubMedGoogle Scholar
  • 36 Pang R, Martinello KA, Meehan C. et al. Proton magnetic resonance spectroscopy lactate/N-acetylaspartate within 48 h predicts cell death following varied neuroprotective interventions in a piglet model of hypoxia-ischemia with and without inflammation-sensitization. Front Neurol 2020; 11 (883) 883
    CrossrefPubMedGoogle Scholar
  • 37 Huang YF, Zhuang SQ, Chen DP, Liang YJ, Li XY. Angiogenesis and its regulatory factors in brain tissue of neonatal rat hypoxic-ischemic encephalopathy [in Chinese]. Zhonghua Er Ke Za Zhi 2004; 42 (03) 210-214
    PubMedGoogle Scholar
  • 38 Beach TG, Tago H, Nagai T, Kimura H, McGeer PL, McGeer EG. Perfusion-fixation of the human brain for immunohistochemistry: comparison with immersion-fixation. J Neurosci Methods 1987; 19 (03) 183-192
    CrossrefPubMedGoogle Scholar
  • 39 Hilbig H, Bidmon HJ, Oppermann OT, Remmerbach T. Influence of post-mortem delay and storage temperature on the immunohistochemical detection of antigens in the CNS of mice. Exp Toxicol Pathol 2004; 56 (03) 159-171
    CrossrefPubMedGoogle Scholar
  • 40 Smith AL, Alexander M, Rosenkrantz TS, Sadek ML, Fitch RH. Sex differences in behavioral outcome following neonatal hypoxia ischemia: insights from a clinical meta-analysis and a rodent model of induced hypoxic ischemic brain injury. Exp Neurol 2014; 254: 54-67
    CrossrefPubMedGoogle Scholar