Skip to main content

Advertisement

SpringerLink
Log in

Tocilizumab Reduces Vasospasms, Neuronal Cell Death, and Microclot Formation in a Rabbit Model of Subarachnoid Hemorrhage

  • Original Article
  • Published:
Translational Stroke Research Aims and scope Submit manuscript

Abstract

Early brain injury (EBI), delayed cerebral vasospasm (DCVS), and delayed cerebral ischemia (DCI) are common complications of subarachnoid hemorrhage (SAH). Inflammatory processes in the cerebrospinal fluid (CSF) are one of the causes for such complications. Our aim to study the effects of an IL-6 receptor antagonist (Tocilizumab) examines the occurrence of DCVS, neuronal cell death, and microclot formation in an acute SAH rabbit model. Twenty-nine New Zealand white rabbits were randomized into one of three groups as the SAH, SAH + Tocilizumab, and sham groups. In SAH groups, hemorrhage was induced by extracranial-intracranial arterial blood shunting from the subclavian artery into the cisterna magna under intracranial pressure (ICP) monitoring. In the second group, Tocilizumab was given once intravenously 1 h after SAH induction. Digital subtraction angiography was performed, and CSF and blood were sampled before and after (day 3) SAH induction. IL-6 plasma and CSF levels were measured. TUNEL, FJB, NeuN, and caspase-3 immunostaining were used to assess cell apoptosis, neurodegeneration, and neuronal cell death, respectively. Microclot formation was detected by fibrinogen immunostaining. Between baseline and follow-up, there was a significant reduction of angiographic DCVS (p < 0.0001) in the Tocilizumab compared with the SAH group. Tocilizumab treatment resulted in decreased neuronal cell death in the hippocampus (p = 0.006), basal cortex (p = 0.001), and decreased microclot formation (p = 0.02). Tocilizumab reduced DCVS, neuronal cell death, and microclot formation in a rabbit SAH model, and could be a potential treatment to prevent DCVS and DCI in SAH patients.

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

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

Similar content being viewed by others

Data Availability

The authors declare that all supporting data are available within the article and its online supplementary files, including the detailed protocols for anesthesia, surgery, and angiography previously described [5, 12–16].

Abbreviations

ABGA:

Arterial blood gas analysis

BA:

Basilar artery

BBB:

Blood brain barrier

CPP:

Cerebral perfusion pressure

CSF:

Cerebrospinal fluid

DCI:

Delayed cerebral ischemia

DCVS:

Delayed cerebral vasospasm

DSA:

Digital subtraction angiography

EBI:

Early brain injury

ELISA:

Enzyme-linked immunosorbent assay

ET-1:

Endothelin-1

FJB:

Fluoro Jade B

HE:

Hematoxylin and eosin

ICP:

Intracranial pressure

IL:

Interleukin

MAP:

Mean arterial pressure

ROI:

Regions of interest

RR:

Respiratory rate

SAH:

Subarachnoid hemorrhage

SD:

Standard deviation

vWF:

von Willebrand factor

References

  1. Niwa A, Osuka K, Nakura T, Matsuo N, Watabe T, Takayasu M. Interleukin-6, MCP-1, IP-10, and MIG are sequentially expressed in cerebrospinal fluid after subarachnoid hemorrhage. Journal of Neuroinflammation. Journal of Neuroinflammation; 2017;:1–6.

  2. Kassell NF, Sasaki T, Colohan AR, Nazar G. Cerebral vasospasm following aneurysmal subarachnoid hemorrhage. Stroke. 1985;16:562–72.

    Article  CAS  PubMed  Google Scholar 

  3. Konczalla J, Kashefiolasl S, Brawanski N, Bruder M, Gessler F, Senft C, et al. Cerebral vasospasm-dependent and cerebral vasospasm-independent cerebral infarctions predict outcome after nonaneurysmal subarachnoid hemorrhage: a single-center series with 250 patients. World Neurosurg. 2017;106:861–4.

    Article  PubMed  Google Scholar 

  4. Al-Tamimi YZ, Bhargava D, Orsi NM, Teraifi A, Cummings M, Ekbote UV, et al. Compartmentalisation of the inflammatory response following aneurysmal subarachnoid haemorrhage. Cytokine. 2019;123:154778.

    Article  CAS  PubMed  Google Scholar 

  5. Croci D, Nevzati E, Danura H, Schöpf S, Fandino J, Marbacher S, et al. The relationship between IL-6, ET-1 and cerebral vasospasm, in experimental rabbit subarachnoid hemorrhage. J Neurosurg Sci. 2019;63(3):245–50.

    Article  PubMed  Google Scholar 

  6. Fassbender K, Hodapp B, Rossol S, Bertsch T, Schmeck J, Schütt S, et al. Inflammatory cytokines in subarachnoid haemorrhage: association with abnormal blood flow velocities in basal cerebral arteries. J Neurol Neurosurg Psychiatry BMJ Publishing Group. 2001;70:534–7.

    Article  CAS  Google Scholar 

  7. Muroi C, Seule M, Sikorski C, Dent W, Keller E. Systemic interleukin-6 levels reflect illness course and prognosis of patients with spontaneous nonaneurysmal subarachnoid hemorrhage. Acta Neurochir Suppl Vienna: Springer Vienna. 2013;115:77–80.

    Google Scholar 

  8. Grignani G, Maiolo A. Cytokines and hemostasis. Haematologica. 2000;85:967–72.

    CAS  PubMed  Google Scholar 

  9. Sehba FA, Hou J, Pluta RM, Zhang JH. The importance of early brain injury after subarachnoid hemorrhage. Prog Neurobiol. 2012;97:14–37.

    Article  PubMed  PubMed Central  Google Scholar 

  10. Andereggen L, Neuschmelting V, Gunten von M, Widmer HR, Fandino J, Marbacher S. The role of microclot formation in an acute subarachnoid hemorrhage model in the rabbit. Biomed Res Int Hindawi. 2014;2014:161702–10.

    Google Scholar 

  11. Stein SC, Browne KD, Chen X-H, Smith DH, Graham DI. Thromboembolism and delayed cerebral ischemia after subarachnoid hemorrhage: an autopsy study. Neurosurgery. 2006;59:781–7 –discussion787–8.

    Article  PubMed  Google Scholar 

  12. Andereggen L, Neuschmelting V, Gunten von M, Widmer HR, Takala J, Jakob SM, et al. The rabbit blood-shunt model for the study of acute and late sequelae of subarachnoid hemorrhage: technical aspects. J Vis Exp. 2014;:e52132.

  13. Marbacher S, Nevzati E, Croci D, Erhardt S, Muroi C, Jakob SM, et al. The rabbit shunt model of subarachnoid haemorrhage. Transl Stroke Res. Springer US. 2014;5:669–80.

    Article  CAS  Google Scholar 

  14. Marbacher S, Neuschmelting V, Andereggen L, Widmer HR, Gunten von M, Takala J, et al. Early brain injury linearly correlates with reduction in cerebral perfusion pressure during the hyperacute phase of subarachnoid hemorrhage. Intensive Care Med Exp. Springer International Publishing; 2014;2:30.

  15. Marbacher S, Fathi A-R, Muroi C, Coluccia D, Andereggen L, Neuschmelting V, et al. The rabbit blood shunt subarachnoid haemorrhage model. Acta Neurochir Suppl Cham: Springer International Publishing. 2015;120:337–42.

    Google Scholar 

  16. Marbacher S, Sherif C, Neuschmelting V, Schläppi J-A, Takala J, Jakob SM, et al. Extra-intracranial blood shunt mimicking aneurysm rupture: intracranial-pressure-controlled rabbit subarachnoid hemorrhage model. J Neurosci Methods. 2010;191:227–33.

    Article  PubMed  Google Scholar 

  17. Endo S, Branson PJ, Alksne JF. Experimental model of symptomatic vasospasm in rabbits. Stroke. 1988;19:1420–5.

    Article  CAS  PubMed  Google Scholar 

  18. Marbacher S, Andereggen L, Neuschmelting V, Widmer HR, Gunten von M, Takala J, et al. A new rabbit model for the study of early brain injury after subarachnoid hemorrhage. J Neurosci Methods. 2012;208:138–45.

    Article  PubMed  Google Scholar 

  19. Croci DM, Wanderer S, Strange F, Grüter BE, Casoni D, Sivanrupan S, et al. Systemic and CSF interleukin-1α expression in a rabbit closed cranium subarachnoid hemorrhage model: an exploratory study. Brain Sci Multidisciplinary Digital Publishing Institute. 2019;9:249.

    CAS  Google Scholar 

  20. Zhang Z-W, Yanamoto H, Nagata I, Miyamoto S, Nakajo Y, Xue J-H, et al. Platelet-derived growth factor-induced severe and chronic vasoconstriction of cerebral arteries: proposed growth factor explanation of cerebral vasospasm. Neurosurgery. 2010;66:728–35 –discussion735.

    Article  PubMed  Google Scholar 

  21. Marbacher S, Milavec H, Neuschmelting V, Andereggen L, Erhardt S, Fandino J. Outer skull landmark-based coordinates for measurement of cerebral blood flow and intracranial pressure in rabbits. J Neurosci Methods. 2011;201:322–6.

    Article  PubMed  Google Scholar 

  22. NC3Rs Reporting Guidelines Working Group. Animal research: reporting in vivo experiments: the ARRIVE guidelines. J. Physiol. (Lond.). Blackwell Publishing Ltd; 2010. pp. 2519–21.

  23. Osuka K, Suzuki Y, Tanazawa T, Hattori K, Yamamoto N, Takayasu M, et al. Interleukin-6 and development of vasospasm after subarachnoid haemorrhage. Acta Neurochir. 1998;140:943–51.

    Article  CAS  PubMed  Google Scholar 

  24. Bowman G, Dixit S, Bonneau RH, Chinchilli VM, Cockroft KM, et al. Neurosurgery. 2004;54:719–25 –discussion725–6.

    Article  PubMed  Google Scholar 

  25. Fassbender K, Hodapp B, Rossol S, Bertsch T, Schmeck J, Schütt S, et al. Endothelin-1 in subarachnoid hemorrhage: an acute-phase reactant produced by cerebrospinal fluid leukocytes. Stroke. Lippincott Williams & Wilkins. 2000;31:2971–5.

    CAS  Google Scholar 

  26. Zhou C, Yamaguchi M, Kusaka G, Schonholz C, Nanda A, Zhang JH. Caspase inhibitors prevent endothelial apoptosis and cerebral vasospasm in dog model of experimental subarachnoid hemorrhage. J. Cereb. Blood Flow Metab. SAGE Publications Sage UK: London, England. 2004;24:419–31.

    CAS  Google Scholar 

  27. Zhou C, Yamaguchi M, Colohan ART, Zhang JH. Role of p53 and apoptosis in cerebral vasospasm after experimental subarachnoid hemorrhage. J. Cereb. Blood Flow Metab. SAGE Publications Sage UK: London, England. 2005;25:572–82.

    Article  CAS  PubMed  Google Scholar 

  28. Li S-J, Liu W, Wang J-L, Zhang Y, Zhao D-J, Wang T-J, et al. The role of TNF-α, IL-6, IL-10, and GDNF in neuronal apoptosis in neonatal rat with hypoxic-ischemic encephalopathy. Eur Rev Med Pharmacol Sci. 2014;18:905–9.

    PubMed  Google Scholar 

  29. Ribeiro MC, Bezerra TDS, Soares AC, Boechat-Ramos R, Carneiro FP, Vianna LM d S, et al. Hippocampal and cerebellar histological changes and their behavioural repercussions caused by brain ischaemic hypoxia experimentally induced by sodium nitrite. Behav Brain Res. 2017;332:223–32.

    Article  CAS  PubMed  Google Scholar 

  30. Powell J, Kitchen N, Heslin J, Greenwood R. Psychosocial outcomes at 18 months after good neurological recovery from aneurysmal subarachnoid haemorrhage. J Neurol Neurosurg Psychiatry. 2004;75:1119–24.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Treggiari MM, Walder B, Suter PM, Romand J-A. Systematic review of the prevention of delayed ischemic neurological deficits with hypertension, hypervolemia, and hemodilution therapy following subarachnoid hemorrhage. J Neurosurg Journal of Neurosurgery Publishing Group. 2003;98:978–84.

    Google Scholar 

  32. Macdonald RL, Pluta RM, Zhang JH. Cerebral vasospasm after subarachnoid hemorrhage: the emerging revolution. Nat Clin Pract Neurol Nature Publishing Group. 2007;3:256–63.

    Article  CAS  Google Scholar 

  33. Pisapia JM, Xu X, Kelly J, Yeung J, Carrion G, Tong H, et al. Microthrombosis after experimental subarachnoid hemorrhage: time course and effect of red blood cell-bound thrombin-activated pro-urokinase and clazosentan. Exp Neurol. 2012;233:357–63.

    Article  CAS  PubMed  Google Scholar 

  34. Muroi C, Fujioka M, Mishima K, Irie K, Fujimura Y, Nakano T, et al. Effect of ADAMTS-13 on cerebrovascular microthrombosis and neuronal injury after experimental subarachnoid hemorrhage. J Thromb Haemost. John Wiley & Sons, Ltd. 2014;12:505–14.

    Article  CAS  PubMed  Google Scholar 

  35. Senchenkova EY, Komoto S, Russell J, Almeida-Paula LD, Yan L-S, Zhang S, et al. Interleukin-6 mediates the platelet abnormalities and thrombogenesis associated with experimental colitis. Am J Pathol. 2013;183:173–81.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Cahill J, Cahill WJ, Calvert JW, Calvert JH, Zhang JH. Mechanisms of early brain injury after subarachnoid hemorrhage. J. Cereb. Blood Flow Metab. SAGE Publications Sage UK: London, England. 2006;26:1341–53.

    Article  CAS  PubMed  Google Scholar 

  37. Interleukin-6 Receptor Mendelian Randomisation Analysis (IL6R MR) Consortium, Swerdlow DI, Holmes MV, Kuchenbaecker KB, Engmann JEL, Shah T, et al. The interleukin-6 receptor as a target for prevention of coronary heart disease: a Mendelian randomisation analysis. Lancet. 2012;379:1214–24.

  38. Bernardo A, Ball C, Nolasco L, Moake JF, Dong J-F. Effects of inflammatory cytokines on the release and cleavage of the endothelial cell-derived ultralarge von Willebrand factor multimers under flow. Blood. 2004;104:100–6.

    Article  CAS  PubMed  Google Scholar 

  39. Vergouwen MDI, Bakhtiari K, van Geloven N, Vermeulen M, Roos YBWEM, Meijers JCM. Reduced ADAMTS13 activity in delayed cerebral ischemia after aneurysmal subarachnoid hemorrhage. J Cereb Blood Flow Metab. 2009;29:1734–41.

    Article  CAS  PubMed  Google Scholar 

  40. Yamashita A, Asada Y. A rabbit model of thrombosis on atherosclerotic lesions. J Biomed Biotechnol Hindawi. 2011;2011:424929–15.

    Google Scholar 

  41. Claassen J, Carhuapoma JR, Kreiter KT, Du EY, Connolly ES, Mayer SA. Global cerebral edema after subarachnoid hemorrhage: frequency, predictors, and impact on outcome. Stroke Lippincott Williams & Wilkins. 2002;33:1225–32.

    Google Scholar 

  42. Suwatcharangkoon S, Meyers E, Falo C, Schmidt JM, Agarwal S, Claassen J, et al. Loss of consciousness at onset of subarachnoid hemorrhage as an important marker of early brain injury. JAMA Neurol. 2016;73:28–35.

    Article  PubMed  Google Scholar 

  43. Marbacher S, Grüter B, Schöpf S, Croci D, Nevzati E, D'Alonzo D, et al. Systematic review of in vivo animal models of subarachnoid hemorrhage: species, standard parameters, and outcomes. Transl Stroke Res Springer US. 2018;10:250–8.

    Article  Google Scholar 

  44. Breu M, Glatter S, Höftberger R, Freilinger M, Kircher K, Kasprian G, et al. Two cases of pediatric AQP4-antibody positive neuromyelitis optica spectrum disorder successfully treated with tocilizumab. Neuropediatrics Georg Thieme Verlag KG. 2019;50:193–6.

    Google Scholar 

  45. Ringelstein M, Ayzenberg I, Harmel J, Lauenstein A-S, Lensch E, Stögbauer F, et al. Long-term therapy with interleukin 6 receptor blockade in highly active neuromyelitis optica spectrum disorder. JAMA Neurol. 2015;72:756–8.

    Article  PubMed  Google Scholar 

  46. Toyota Y, Wei J, Xi G, Keep RF, Hua Y. White matter T2 hyperintensities and blood-brain barrier disruption in the hyperacute stage of subarachnoid hemorrhage in male mice: the role of lipocalin-2. CNS Neurosci Ther. 2019;25:1207–14.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Kanamaru H, Suzuki H. Potential therapeutic molecular targets for blood-brain barrier disruption after subarachnoid hemorrhage. Neural Regen Res Medknow Publications. 2019;14:1138–43.

    Article  Google Scholar 

Download references

Acknowledgments

We are deeply grateful to: the team of Prof. Hans-Ruedi Widmer, PhD, at the Neurosurgical Research Institute, Department of Neurosurgery, University and University Hospital of Bern, Switzerland, for their assistance in histological staining; Mary Kemper for editing and proofreading and the team of the Experimental Surgical Facility and Central Animal Facility, Department of Biomedical Research, University of Bern, for animal care, anesthesia, and perioperative assistance.

Funding

The current project has been financially supported by the European Association of Neurological Surgeons (EANS) research grant; the research fund of the department of Neurosurgery Kantonsspital Aarau, Switzerland; the HANELA Foundation, Switzerland; and the research fund of the department of Neurosurgery University Hospital Basel.

Author information

Authors and Affiliations

  1. Department of Neurosurgery, University Hospital Basel, Basel, Switzerland

    Davide M. Croci & Luigi Mariani

  2. Department of Neurosurgery, Kantonsspital Aarau, Tellstrasse, 5001, Aarau, Switzerland

    Davide M. Croci, Stefan Wanderer, Fabio Strange, Basil E. Grüter, Lukas Andereggen, Javier Fandino & Serge Marbacher

  3. Cerebrovascular Research Group, Department of Biomedical Research, University of Bern, Bern, Switzerland

    Davide M. Croci, Stefan Wanderer, Fabio Strange, Basil E. Grüter, Sivani Sivanrupan, Lukas Andereggen, Javier Fandino & Serge Marbacher

  4. Department of Biomedical Research, University of Bern, Bern, Switzerland

    Daniela Casoni

  5. Institute of Pathology Länggasse, Bern, Switzerland

    Michael von Gunten

  6. Department of Neurosurgery, Inselspital, Bern University Hospital, University of Bern, Bern, Switzerland

    Hans Rudolf Widmer & Stefano Di Santo

Authors
  1. Davide M. Croci
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Stefan Wanderer
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Fabio Strange
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Basil E. Grüter
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Sivani Sivanrupan
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Lukas Andereggen
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Daniela Casoni
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Michael von Gunten
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Hans Rudolf Widmer
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Stefano Di Santo
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Javier Fandino
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Luigi Mariani
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Serge Marbacher
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Conception and design: Croci, Marbacher. Experimental procedures: Croci, Marbacher, Grueter, Strange; Histological sample preparation and analysis: Croci, Widmer, Di Santo, von Gunten, Wanderer, Andereggen, Sivanrupan. Drafting the article: Croci, Wanderer, Marbacher; Statistical analysis and interpretation of data: Croci, Wanderer, Andereggen, Marbacher; Critically revising the article: Fandino, Widmer, Marbacher, Mariani; Administrative support: Fandino, Mariani.

Corresponding author

Correspondence to Davide M. Croci.

Ethics declarations

Conflict of Interest

The authors declare that they have no conflict of interest.

Ethics Approval

The project has been performed according to the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines [22] and was performed in accordance with the National Institutes of Health Guidelines for the care and use of experimental animals and with the approval of the Animal Care Committee of the Canton Bern, Switzerland (Approval Nr. BE58/17).

Consent for Publication

All the authors agree for the publication of the manuscript.

Additional information

Publisher’s Note

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

Supplementary Information

ESM 1

(PDF 16290 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Croci, D.M., Wanderer, S., Strange, F. et al. Tocilizumab Reduces Vasospasms, Neuronal Cell Death, and Microclot Formation in a Rabbit Model of Subarachnoid Hemorrhage. Transl. Stroke Res. 12, 894–904 (2021). https://doi.org/10.1007/s12975-020-00880-3

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12975-020-00880-3

Keywords

Search

Navigation