Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Neonatal hypoxia ischemia redistributes L1 cell adhesion molecule into rat cerebellar lipid rafts

Abstract

Background

Hypoxic–ischemic encephalopathy (HIE) is a devastating disease with lifelong disabilities. Hypothermia is currently the only treatment. At term, the neonatal cerebellum may be particularly vulnerable to the effects of HIE. At this time, many developmental processes depend on lipid raft function. These microdomains of the plasma membrane are critical for cellular signaling and axon extension. We hypothesized that HIE alters the protein content of lipid rafts in the cerebellum.

Methods

Postnatal day (PN) 10 animals, considered human term equivalent, underwent hypoxic–ischemic (HI) injury by a right carotid artery ligation followed by hypoxia. For some animals, LPS was administered on PN7, and hypothermia (HT) was conducted for 4 h post-hypoxia. Lipid rafts were isolated from the right and left cerebella. The percent of total L1 cell adhesion molecule in lipid rafts was determined 4 and 72 h after hypoxia.

Results

No sex differences were found. HI alone caused significant increases in the percent of L1 in lipid rafts which persisted until 72 h in the right but not the left cerebellum. A small but significant effect of LPS was detected in the left cerebellum 72 h after HI. Hypothermia had no effect.

Conclusions

Lipid rafts may be a new target for interventions of HIE.

Impact

  • This article investigates the effect of neonatal exposure to hypoxic–ischemic encephalopathy (HIE) on the distribution of membrane proteins in the cerebellum.

  • This article explores the effectiveness of hypothermia as a prevention for the harmful effects of HIE on membrane protein distribution.

  • This article shows an area of potential detriment secondary to HIE that persists with current treatments, and explores ideas for new treatments.

This is a preview of subscription content

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Fig. 1: Schematic of experimental timeline.
Fig. 2: Representative immunoblot showing the altered distribution of L1 in lipid raft portions of CGN in the left and right halves of the cerebellum 4 h after hypoxia (immediately after hypothermia).
Fig. 3: Hypoxia–ischemia (HI) leads to a redistribution of L1 cell adhesion molecule in lipid rafts in the cerebellum 4 h after hypoxia.
Fig. 4: Hypoxia–ischemia (HI) leads to a redistribution of L1 cell adhesion molecule (L1CAM) in lipid rafts in the cerebellum 72 h after hypoxia.

References

  1. Fatemi, A., Wilson, M. A. & Johnston, M. V. Hypoxic-ischemic encephalopathy in the term infant. Clin. Perinatol. 36, 835–858 (2009).

    PubMed  PubMed Central  Google Scholar 

  2. Graham, E. M., Ruis, K. A., Hartman, A. L., Northington, F. J. & Fox, H. E. A systematic review of the role of intrapartum hypoxia-ischemia in the causation of neonatal encephalopathy. Am. J. Obstet. Gynecol. 199, 587–595 (2008).

    CAS  PubMed  Google Scholar 

  3. Bonifacio, S. L. & Hutson, S. The term newborn: evaluation for hypoxic-ischemic encephalopathy. Clin. Perinatol. 48, 681–695 (2021).

    PubMed  Google Scholar 

  4. Shankaran, S. et al. Whole-body hypothermia for neonates with hypoxic-ischemic encephalopathy. N. Engl. J. Med. 353, 1574–1584 (2005).

    CAS  PubMed  Google Scholar 

  5. Davidson, J. O., Wassink, G., Van Den Heuij, L. G., Bennet, L. & Gunn, A. J. Therapeutic hypothermia for neonatal hypoxic-ischemic encephalopathy - where to from here? Front. Neurol. 6, 198 (2015).

    PubMed  PubMed Central  Google Scholar 

  6. Schreglmann, M., Ground, A., Vollmer, B. & Johnson, M. J. Systematic review: long-term cognitive and behavioural outcomes of neonatal hypoxic–ischaemic encephalopathy in children without cerebral palsy. Acta Paediatr. Int. J. Paediatr. 109, 20–30 (2020).

    Google Scholar 

  7. Askalan, R., Wang, C., Shi, H., Armstrong, E. & Yager, J. Y. The effect of postischemic hypothermia on apoptotic cell death in the neonatal rat brain. Dev. Neurosci. 33, 320–329 (2011).

    CAS  PubMed  Google Scholar 

  8. Wassink, G., Gunn, E. R., Drury, P. P., Bennet, L. & Gunn, A. J. The mechanisms and treatment of asphyxial encephalopathy. Front. Neurosci. 8, 40 (2014).

    PubMed  PubMed Central  Google Scholar 

  9. Wassink, G. et al. A working model for hypothermic neuroprotection. J. Physiol. 596, 5641–5654 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Chevin, M. et al. Neuroprotective effects of hypothermia in inflammatory-sensitized hypoxic-ischemic encephalopathy. Int. J. Dev. Neurosci. 55, 1–8 (2016).

    PubMed  Google Scholar 

  11. Colbourne, F., Sutherland, G. & Corbett, D. Postischemic hypothermia. A critical appraisal with implications for clinical treatment. Mol. Neurobiol. 14, 171–201 (1997).

    CAS  PubMed  Google Scholar 

  12. Benitez, S. G., Castro, A. E., Patterson, S. I., Muñoz, E. M. & Seltzer, A. M. Hypoxic preconditioning differentially affects GABAergic and glutamatergic neuronal cells in the injured cerebellum of the neonatal rat. PLoS ONE 9, e102056 (2014).

    PubMed  PubMed Central  Google Scholar 

  13. Biran, V. et al. Cerebellar abnormalities following hypoxia alone compared to hypoxic-ischemic forebrain injury in the developing rat brain. Neurobiol. Dis. 41, 138–146 (2011).

    PubMed  Google Scholar 

  14. Sanches, E. F., Van De Looij, Y., Toulotte, A., Sizonenko, S. V. & Lei, H. Mild neonatal brain hypoxia-ischemia in very immature rats causes long-term behavioral and cerebellar abnormalities at adulthood. Front. Physiol. 10, 1–12. (2019).

    Google Scholar 

  15. Altman, J. Morphological development of the rat cerebellum and some of its mechanisms. Exp. Brain Res. 47, 8–49 (1982).

    Google Scholar 

  16. Altman, J. & Das, G. D. Autoradiographic and histological studies of postnatal neurogenesis migration and transformation of cells incorporating tritiated thymidine in neonate rats, with special reference to postnatal neurogenesis in some brain regions. J. Comp. Neurol. 126, 337–390. (1966).

    CAS  PubMed  Google Scholar 

  17. Peng, J. H. F., Feng, Y., LeBlanc, M. H., Rhodes, P. G. & Parker, J. C. Apoptosis and necrosis in developing cerebellum and brainstem induced after focal cerebral hypoxic-ischemic injury. Dev. Brain Res. 156, 87–92 (2005).

    CAS  Google Scholar 

  18. Volpe, J. J. Cerebellum of the premature infant: rapidly developing, vulnerable, clinically important. J. Child Neurol. 24, 1085–1104 (2009).

    PubMed  PubMed Central  Google Scholar 

  19. Sathyanesan, A. et al. Emerging connections between cerebellar development, behaviour and complex brain disorders. Nat. Rev. Neurosci. 20, 298–313 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Wang, V. Y. & Zoghbi, H. Y. Genetic regulation of cerebellar development. Nat. Rev. Neurosci. 2, 484–491 (2001).

    CAS  PubMed  Google Scholar 

  21. Levine, S. Anoxic-ischemic encephalopathy in rats. Am. J. Pathol. 36, 1–17 (1960).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Rice, J. E., Vannucci, R. C. & Brierley, J. B. The influence of immaturity on hypoxic-ischemic brain damage in the rat. Ann. Neurol. 9, 131–141 (1981).

    PubMed  Google Scholar 

  23. Geddes, R., Vannucci, R. C. & Vannucci, S. J. Delayed cerebral atrophy following moderate hypoxia-ischemia in the immature rat. Dev. Neurosci. 23, 180–185 (2001).

    CAS  PubMed  Google Scholar 

  24. Towfighi, J., Zec, N., Yager, J., Housman, C. & Vannucci, R. C. Temporal evolution of neuropathologic changes in an immature rat model of cerebral hypoxia: a light microscopic study. Acta Neuropathol. 90, 375–386 (1995).

    CAS  PubMed  Google Scholar 

  25. Burden-Gulley, S. M., Pendergast, M. & Lemmon, V. The role of cell adhesion molecule L1 in axonal extension, growth cone motility, and signal transduction. Cell Tissue Res. 290, 415–422 (1997).

    CAS  PubMed  Google Scholar 

  26. Hortsch, M. Structural and functional evolution of the L1 family: are four adhesion molecules better than one? Mol. Cell. Neurosci. 15, 1–10 (2000).

    CAS  PubMed  Google Scholar 

  27. Katidou, M., Vidaki, M., Strigini, M. & Karagogeos, D. The immunoglobulin superfamily of neuronal cell adhesion molecules: lessons from animal models and correlation with human disease. Biotechnol. J. 3, 1564–1580 (2008).

    CAS  PubMed  Google Scholar 

  28. Tang, N. et al. Ethanol causes the redistribution of L1 cell adhesion molecule in lipid rafts. J. Neurochem. 119, 859–867 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Kitchen, S. T. et al. Bilirubin inhibits lipid raft dependent functions of L1 cell adhesion molecule in rat pup cerebellar granule neurons. Pediatr. Res. 89, 1389–1395 (2021).

    CAS  PubMed  Google Scholar 

  30. Milstone, A. M. et al. Chlorhexidine inhibits L1 cell adhesion molecule-mediated neurite outgrowth in vitro. Pediatr. Res. 75, 8–13 (2014).

    CAS  PubMed  Google Scholar 

  31. Tsui-Pierchala, B. A., Encinas, M., Milbrandt, J. & Johnson, E. M. Lipid rafts in neuronal signaling and function. Trends Neurosci. 25, 412–417 (2002).

    CAS  PubMed  Google Scholar 

  32. Nakai, Y. & Kamiguchi, H. Migration of nerve growth cones requires detergent-resistant membranes in a spatially defined and substrate-dependent manner. J. Cell Biol. 159, 1097–1108 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Semple, B. D., Blomgren, K., Gimlin, K., Ferriero, D. M. & Noble-Haeusslein, L. J. Brain development in rodents and humans: identifying benchmarks of maturation and vulnerability to injury across species. Prog. Neurobiol. 106–107, 1–16 (2013).

    PubMed  Google Scholar 

  34. Chow, J. C., Young, D. W., Golenbock, D. T., Christ, W. J. & Gusovsky, F. Toll-like receptor-4 mediates lipopolysaccharide-induced signal transduction. J. Biol. Chem. 274, 10689–10692 (1999).

    CAS  PubMed  Google Scholar 

  35. Rietschel, E. T. et al. Bacterial endotoxin: molecular relationships of structure to activity and function. FASEB J. 8, 217–225 (1994).

    CAS  PubMed  Google Scholar 

  36. Olsson, S. & Sundler, R. The role of lipid rafts in LPS-induced signaling in a macrophage cell line. Mol. Immunol. 43, 607–612 (2006).

    CAS  PubMed  Google Scholar 

  37. Miller, Y. I., Navia-Pelaez, J. M., Corr, M. & Yaksh, T. L. Lipid rafts in glial cells: role in neuroinflammation and pain processing. J. Lipid Res. 61, 655–666 (2020).

    CAS  PubMed  Google Scholar 

  38. Eklind, S., Mallard, C., Arvidsson, P. & Hagberg, H. Lipopolysaccharide induces both a primary and a secondary phase of sensitization in the developing rat brain. Pediatr. Res. 58, 112–116 (2005).

    CAS  PubMed  Google Scholar 

  39. Eklind, S. et al. Bacterial endotoxin sensitizes the immature brain to hypoxic-ischaemic injury. Eur. J. Neurosci. 13, 1101–1106 (2001).

    CAS  PubMed  Google Scholar 

  40. Wang, X., Rousset, C. I., Hagberg, H. & Mallard, C. Lipopolysaccharide-induced inflammation and perinatal brain injury. Semin. Fetal Neonatal Med. 11, 343–353 (2006).

    PubMed  Google Scholar 

  41. Martinello, K. A. et al. Hypothermia is not therapeutic in a neonatal piglet model of inflammation-sensitized hypoxia–ischemia. Pediatr. Res. https://doi.org/10.1038/s41390-021-01584-6 (2021).

  42. Corry, K. A. et al. Evaluating neuroprotective effects of uridine, erythropoietin, and therapeutic hypothermia in a ferret model of inflammation-sensitized hypoxic-ischemic encephalopathy. Int. J. Mol. Sci. 22, 9841 (2021).

  43. Sobesky, J. et al. Crossed cerebellar diaschisis in acute human stroke: a PET study of serial changes and response to supratentorial reperfusion. J. Cereb. Blood Flow. Metab. 25, 1685–1691 (2005).

    PubMed  Google Scholar 

  44. Patel, S. D. et al. Therapeutic hypothermia and hypoxia-ischemia in the term-equivalent neonatal rat: characterization of a translational preclinical model. Pediatr. Res. 78, 264–271 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Lingwood, D. & Simons, K. Lipid rafts as a membrane-organizing principle. Science 327, 46–50 (2010).

    CAS  Google Scholar 

  46. Maness, P. F. & Schachner, M. Neural recognition molecules of the immunoglobulin superfamily: signaling transducers of axon guidance and neuronal migration. Nat. Neurosci. 10, 19–26 (2006).

    Google Scholar 

  47. Edwards, A. B. et al. Modification to the Rice-Vannucci perinatal hypoxic-ischaemic encephalopathy model in the P7 rat improves the reliability of cerebral infarct development after 48 h. J. Neurosci. Methods 288, 62–71 (2017).

    PubMed  Google Scholar 

  48. Vannucci, R. C., Lyons, D. T. & Vasta, F. Regional cerebral blood flow during hypoxia-ischemia in immature rats. Stroke 19, 245–250 (1988).

    CAS  PubMed  Google Scholar 

  49. Wood, T. et al. Monitoring of cerebral blood flow during hypoxia-ischemia and resuscitation in the neonatal rat using laser speckle imaging. Physiol. Rep. 4, e12749 (2016).

    PubMed  PubMed Central  Google Scholar 

  50. Ohshima, M., Tsuji, M., Taguchi, A., Kasahara, Y. & Ikeda, T. Cerebral blood flow during reperfusion predicts later brain damage in a mouse and a rat model of neonatal hypoxic–ischemic encephalopathy. Exp. Neurol. 233, 481–489 (2012).

    PubMed  Google Scholar 

  51. Charriaut-Marlangue, C. et al. Sildenafil mediates blood-flow redistribution and neuroprotection after neonatal hypoxia-ischemia. Stroke 45, 850–856 (2014).

    CAS  PubMed  Google Scholar 

  52. Stone, B. S. et al. Delayed neural network degeneration after neonatal hypoxia-ischemia. Ann. Neurol. 64, 535–546 (2008).

    PubMed  PubMed Central  Google Scholar 

  53. Tang, S. et al. Neuroprotective effects of acetyl-L-carnitine on neonatal hypoxia ischemia-induced brain injury in rats. Dev. Neurosci. 38, 384–396 (2017).

  54. Taylor, D. L., Joashi, U. C., Sarraf, C., Edwards, A. D. & Mehmet, H. Consequential apoptosis in the cerebellum following injury to the developing rat forebrain. Brain Pathol. 16, 195–201 (2006).

    PubMed  PubMed Central  Google Scholar 

  55. Shamoto, H. & Chugani, H. T. Glucose metabolism in the human cerebellum: an analysis of crossed cerebellar diaschisis in children with unilateral cerebral injury. J. Child Neurol. 12, 407–414 (2016).

    Google Scholar 

  56. Onat, F. & Çavdar, S. Cerebellar connections: hypothalamus. Cerebellum 2, 263–269 (2003).

    PubMed  Google Scholar 

  57. Çavdar, S. et al. Cerebellar connections to the rostral reticular nucleus of the thalamus in the rat. J. Anat. 201, 485–491 (2002).

    PubMed  PubMed Central  Google Scholar 

  58. Watson, T. C. et al. Anatomical and physiological foundations of cerebello-hippocampal interaction. Elife 8, 1–28. (2019).

    Google Scholar 

  59. Xiao, L., Bornmann, C., Hatstatt-Burklé, L. & Scheiffele, P. Regulation of striatal cells and goal-directed behavior by cerebellar outputs. Nat. Commun. 9, 1–14. (2018).

    Google Scholar 

  60. Annink, K. V. et al. Cerebellar injury in term neonates with hypoxic–ischemic encephalopathy is underestimated. Pediatr. Res. 89, 1171–1178 (2021).

    PubMed  Google Scholar 

  61. Kwan, S. et al. Injury to the cerebellum in term asphyxiated newborns treated with hypothermia. Am. J. Neuroradiol. 36, 1542–1549 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  62. Lemmon, M. E. et al. Diffusion tensor imaging detects occult cerebellar injury in severe neonatal hypoxic-ischemic encephalopathy. Dev. Neurosci. 39, 207–214 (2017).

    CAS  PubMed  Google Scholar 

  63. Le Strange, E., Saeed, N., Cowan, F. M., Edwards, A. D. & Rutherford, M. A. MR imaging quantification of cerebellar growth following hypoxic-ischemic injury to the neonatal. Brain Am. J. Neuroradiol. 25, 463–468 (2004).

    PubMed  Google Scholar 

  64. Wang, S. S. H., Kloth, A. D. & Badura, A. The cerebellum, sensitive periods, and autism. Neuron 83, 518–532 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  65. Zhou, K. Q., Davidson, J. O., Bennet, L. & Gunn, A. J. Combination treatments with therapeutic hypothermia for hypoxic-ischemic neuroprotection. Dev. Med. Child Neurol. 62, 1131–1137 (2020).

    PubMed  Google Scholar 

  66. Xue, J. et al. Sphingomyelin synthase 2 inhibition ameliorates cerebral ischemic reperfusion injury through reducing the recruitment of toll-like receptor 4 to lipid rafts. J. Am. Heart Assoc. 8, 1–14. (2019).

    CAS  Google Scholar 

  67. Berman, D. R. et al. Docosahexaenoic acid augments hypothermic neuroprotection in a neonatal rat asphyxia model. Neonatology 104, 71–78 (2013).

    CAS  PubMed  Google Scholar 

  68. Wassall, S. R. et al. Docosahexaenoic acid regulates the formation of lipid rafts: A unified view from experiment and simulation. Biochim. Biophys. Acta Biomembr. 1860, 1985–1993 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  69. Kinnun, J. J., Bittman, R., Shaikh, S. R. & Wassall, S. R. DHA modifies the size and composition of raftlike domains: a solid-state 2H NMR study. Biophys. J. 114, 380–391 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  70. Shaikh, S. R., Kinnun, J. J., Leng, X., Williams, J. A. & Wassall, S. R. How polyunsaturated fatty acids modify molecular organization in membranes: insight from NMR studies of model systems. Biochim. Biophys. Acta Biomembr. 1848, 211–219 (2015).

    CAS  Google Scholar 

  71. Shaikh, S. R., Rockett, B. D., Salameh, M. & Carraway, K. Docosahexaenoic acid modifies the clustering and size of lipid rafts and the lateral organization and surface expression of MHC class I of EL4 cells. J. Nutr. 139, 1632–1639 (2009).

    CAS  PubMed  Google Scholar 

  72. Hou, T. Y. et al. N-3 polyunsaturated fatty acids suppress CD4+ T cell proliferation by altering phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2] organization. Biochim. Biophys. Acta Biomembr. 1858, 85–96 (2016).

    CAS  Google Scholar 

  73. Levental, I. & Veatch, S. L. The continuing mystery of lipid rafts. J. Mol. Biol. 428, 4749–4764 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  74. Schley, P. D., Brindley, D. N. & Field, C. J. (n-3) PUFA alter raft lipid composition and decrease epidermal growth factor receptor levels in lipid rafts of human breast cancer cells. J. Nutr. 137, 548–553 (2007).

  75. Huun, M. U. et al. DHA reduces oxidative stress following hypoxia-ischemia in newborn piglets: a study of lipid peroxidation products in urine and plasma. J. Perinat. Med. 46, 209–217 (2018).

    CAS  PubMed  Google Scholar 

  76. Jalili, M. & Hekmatdoost, A. Dietary ω-3 fatty acids and their influence on inflammation via Toll-like receptor pathways. Nutrition 85, 111070 (2021).

Download references

Funding

This study was funded by the NIH/NICHD P01 HD085928.

Author information

Authors and Affiliations

Authors

Contributions

J.W.: Substantial contributions to gathering and interpretation of data, drafting and revising article critically for important intellectual content, and final approval of the version to be published. N.C.R.: Substantial contribution to drafting the article or revising it critically for important intellectual content. M.H.: Substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data. N.T.: Substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data. C.F.B.: Substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data, drafting the article or revising it critically for important intellectual content; and final approval of the version to be published.

Corresponding author

Correspondence to Cynthia F. Bearer.

Ethics declarations

Competing interests

The authors declare no competing interests.

Disclaimer

This material is original and has not been previously published nor has it been submitted for publication elsewhere while under consideration.

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

Waddell, J., Rickman, N.C., He, M. et al. Neonatal hypoxia ischemia redistributes L1 cell adhesion molecule into rat cerebellar lipid rafts. Pediatr Res (2022). https://doi.org/10.1038/s41390-022-01974-4

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1038/s41390-022-01974-4

Search

Quick links