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.

Hyperosmolar blood–brain barrier opening using intra-arterial injection of hyperosmotic mannitol in mice under real-time MRI guidance

Abstract

The blood–brain barrier (BBB) is the main obstacle to the effective delivery of therapeutic agents to the brain, compromising treatment efficacy for a variety of neurological disorders. Intra-arterial (IA) injection of hyperosmotic mannitol has been used to permeabilize the BBB and improve parenchymal entry of therapeutic agents following IA delivery in preclinical and clinical studies. However, the reproducibility of IA BBB manipulation is low and therapeutic outcomes are variable. We demonstrated that this variability could be highly reduced or eliminated when the procedure of osmotic BBB opening is performed under the guidance of interventional MRI. Studies have reported the utility and applicability of this technique in several species. Here we describe a protocol to open the BBB by IA injection of hyperosmotic mannitol under the guidance of MRI in mice. The procedures (from preoperative preparation to postoperative care) can be completed within ~1.5 h, and the skill level required is on par with the induction of middle cerebral artery occlusion in small animals. This MRI-guided BBB opening technique in mice can be utilized to study the biology of the BBB and improve the delivery of various therapeutic agents to the brain.

Your institute does not have access to this article

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Fig. 1: BBBO under real-time MRI guidance in mice.
Fig. 2: Schematic illustration of BBBO under real-time MRI guidance in large animals and humans.
Fig. 3: Schematic diagram of the catheter assembly.
Fig. 4: Schematic diagram of the preoperative preparation of the mouse.
Fig. 5: Surgical procedures before catheter insertion.
Fig. 6: Schematic diagram of catheter cannulation.
Fig. 7: Mouse MRI setup.
Fig. 8: Use of real-time MRI to ensure an effective infusion rate via IA injection to predict perfusion territory in a mouse brain.
Fig. 9: Variability of cortical involvement during IA infusion of a contrast agent in the mouse brain.
Fig. 10: Prediction of mannitol-induced BBBO territory.
Fig. 11: MRI and histological assessment post-BBBO.

Data availability

Source data are provided with this paper. The other data that support the findings of this study are available from the corresponding author upon reasonable request.

Code availability

The code used in this study is provided in Supplementary Data 1. We have also deposited the code and a demonstration of image processing at https://github.com/dychuchengyan/ChengyanMRI.

References

  1. Nduom, E. K., Yang, C., Merrill, M. J., Zhuang, Z. & Lonser, R. R. Characterization of the blood–brain barrier of metastatic and primary malignant neoplasms. J. Neurosurg. 119, 427–433 (2013).

    PubMed  PubMed Central  Google Scholar 

  2. Budde, M. D., Janes, L., Gold, E., Turtzo, L. C. & Frank, J. A. The contribution of gliosis to diffusion tensor anisotropy and tractography following traumatic brain injury: validation in the rat using Fourier analysis of stained tissue sections. Brain 134, 2248–2260 (2011).

    PubMed  PubMed Central  Google Scholar 

  3. Pardridge, W. M. The blood–brain barrier: bottleneck in brain drug development. NeuroRx 2, 3–14 (2005).

    PubMed  PubMed Central  Google Scholar 

  4. Goldstein, G. W. & Betz, A. L. The blood–brain barrier. Sci. Am. 255, 74–7 (1986).

    CAS  PubMed  Google Scholar 

  5. Chakraborty, S. et al. Superselective intraarterial cerebral infusion of cetuximab after osmotic blood/brain barrier disruption for recurrent malignant glioma: phase I study. J. Neurooncol. 128, 405–415 (2016).

    CAS  PubMed  Google Scholar 

  6. Gonzales-Portillo, G. S. et al. Mannitol-enhanced delivery of stem cells and their growth factors across the blood–brain barrier. Cell Transplant. 23, 531–539 (2014).

    PubMed  Google Scholar 

  7. Brightman, M. W., Hori, M., Rapoport, S. I., Reese, T. S. & Westergaard, E. Osmotic opening of tight junctions in cerebral endothelium. J. Comp. Neurol. 152, 317–325 (1973).

    CAS  PubMed  Google Scholar 

  8. Foley, C. P. et al. Intra-arterial delivery of AAV vectors to the mouse brain after mannitol mediated blood brain barrier disruption. J. Control. Release 196, 71–78 (2014).

    CAS  PubMed  Google Scholar 

  9. Burkhardt, J. K. et al. Intra-arterial delivery of bevacizumab after blood–brain barrier disruption for the treatment of recurrent glioblastoma: progression-free survival and overall survival. World Neurosurg. 77, 130–134 (2012).

    PubMed  Google Scholar 

  10. Joshi, S. et al. Inconsistent blood brain barrier disruption by intraarterial mannitol in rabbits: implications for chemotherapy. J. Neurooncol. 104, 11–19 (2011).

    CAS  PubMed  Google Scholar 

  11. Janowski, M., Walczak, P. & Pearl, M. S. Predicting and optimizing the territory of blood–brain barrier opening by superselective intra-arterial cerebral infusion under dynamic susceptibility contrast MRI guidance. J. Cereb. Blood Flow. Metab. 36, 569–575 (2016).

    CAS  PubMed  Google Scholar 

  12. Kaya, M. et al. The effects of magnesium sulfate on blood–brain barrier disruption caused by intracarotid injection of hyperosmolar mannitol in rats. Life Sci. 76, 201–212 (2004).

    CAS  PubMed  Google Scholar 

  13. Yang, W. L. et al. Evaluation of systemically administered radiolabeled epidermal growth factor as a brain tumor targeting agent. J. Neuro-Oncol. 55, 19–28 (2001).

    CAS  Google Scholar 

  14. Tajiri, N., Lee, J. Y., Acosta, S., Sanberg, P. R. & Borlongan, C. V. Breaking the blood–brain barrier with mannitol to aid stem cell therapeutics in the chronic stroke brain. Cell Transplant. 25, 1453–1460 (2016).

    PubMed  Google Scholar 

  15. Seyfried, D. M. et al. Mannitol enhances delivery of marrow stromal cells to the brain after experimental intracerebral hemorrhage. Brain Res. 1224, 12–19 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Fu, H. et al. Self-complementary adeno-associated virus serotype 2 vector: global distribution and broad dispersion of AAV-mediated transgene expression in mouse brain. Mol. Ther. 8, 911–917 (2003).

    CAS  PubMed  Google Scholar 

  17. Janowski, M. et al. Cell size and velocity of injection are major determinants of the safety of intracarotid stem cell transplantation. J. Cereb. Blood Flow. Metab. 33, 921–927 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Walczak, P. et al. Real-time MRI for precise and predictable intra-arterial stem cell delivery to the central nervous system. J. Cereb. Blood Flow Metab. 37, 2346–2358 (2016).

    PubMed  PubMed Central  Google Scholar 

  19. Zawadzki, M. et al. Real-time MRI guidance for intra-arterial drug delivery in a patient with a brain tumor: technical note. BMJ Case Rep. 12, e014469 (2019).

    PubMed  PubMed Central  Google Scholar 

  20. Doyle, A., McGarry, M. P., Lee, N. A. & Lee, J. J. The construction of transgenic and gene knockout/knockin mouse models of human disease. Transgenic Res. 21, 327–349 (2012).

    CAS  PubMed  Google Scholar 

  21. Rivera, J., Sobey, C. G., Walduck, A. K. & Drummond, G. R. Nox isoforms in vascular pathophysiology: insights from transgenic and knockout mouse models. Redox Rep. 15, 50–63 (2010).

    CAS  PubMed  Google Scholar 

  22. Bartke, A. New findings in gene knockout, mutant and transgenic mice. Exp. Gerontol. 43, 11–14 (2008).

    CAS  PubMed  Google Scholar 

  23. Chu, C. et al. Real-time MRI guidance for reproducible hyperosmolar opening of the blood–brain barrier in mice. Front. Neurol. 9, 921 (2018).

    PubMed  PubMed Central  Google Scholar 

  24. Chu, C. et al. Optimization of osmotic blood–brain barrier opening to enable intravital microscopy studies on drug delivery in mouse cortex. J. Control. Release 317, 312–321 (2020).

    CAS  PubMed  Google Scholar 

  25. Lesniak, W. G. et al. PET imaging of distinct brain uptake of a nanobody and similarly-sized PAMAM dendrimers after intra-arterial administration. Eur. J. Nucl. Med. Mol. Imaging 46, 1940–1951 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Lesniak, W. G. et al. A distinct advantage to intraarterial delivery of (89)Zr-bevacizumab in PET imaging of mice with and without osmotic opening of the blood–brain barrier. J. Nucl. Med. 60, 617–622 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Liu, R., Martuza, R. L. & Rabkin, S. D. Intracarotid delivery of oncolytic HSV vector G47Delta to metastatic breast cancer in the brain. Gene Ther. 12, 647–654 (2005).

    CAS  PubMed  Google Scholar 

  28. Choonara, Y. E., Kumar, P., Modi, G. & Pillay, V. Improving drug delivery technology for treating neurodegenerative diseases. Expert Opin. Drug Deliv. 13, 1029–1043 (2016).

    CAS  PubMed  Google Scholar 

  29. Niu, X., Chen, J. & Gao, J. Nanocarriers as a powerful vehicle to overcome blood–brain barrier in treating neurodegenerative diseases: focus on recent advances. Asian J. Pharm. Sci. 14, 480–496 (2019).

    PubMed  Google Scholar 

  30. Cerri, S. et al. Intracarotid infusion of mesenchymal stem cells in an animal model of parkinson’s disease, focusing on cell distribution and neuroprotective and behavioral effects. Stem Cells Transl. Med. 4, 1073–1085 (2015).

    PubMed  PubMed Central  Google Scholar 

  31. Kijima, N. & Kanemura, Y. Mouse models of glioblastoma. in Glioblastoma (ed. De Vleeschouwer, S.) Ch. 7 (Codon Publications, 2017).

  32. Lan, X. et al. Modeling human pediatric and adult gliomas in immunocompetent mice through costimulatory blockade. Oncoimmunology 9, 1776577 (2020).

    PubMed  PubMed Central  Google Scholar 

  33. Hall, A. M. & Roberson, E. D. Mouse models of Alzheimer’s disease. Brain Res. Bull. 88, 3–12 (2012).

    CAS  PubMed  Google Scholar 

  34. Schober, A. Classic toxin-induced animal models of Parkinson’s disease: 6-OHDA and MPTP. Cell Tissue Res. 318, 215–224 (2004).

    PubMed  Google Scholar 

  35. Lipsman, N. et al. Blood–brain barrier opening in Alzheimer’s disease using MR-guided focused ultrasound. Nat. Commun. 9, 2336 (2018).

    PubMed  PubMed Central  Google Scholar 

  36. Mainprize, T. et al. Blood–brain barrier opening in primary brain tumors with non-invasive MR-guided focused ultrasound: a clinical safety and feasibility study. Sci. Rep. 9, 321 (2019).

    PubMed  PubMed Central  Google Scholar 

  37. Meng, Y. et al. Safety and efficacy of focused ultrasound induced blood–brain barrier opening, an integrative review of animal and human studies. J. Control. Release 309, 25–36 (2019).

    CAS  PubMed  Google Scholar 

  38. Silburt, J., Lipsman, N. & Aubert, I. Disrupting the blood–brain barrier with focused ultrasound: perspectives on inflammation and regeneration. Proc. Natl Acad. Sci. USA. 114, E6735–E6736 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Polycarpou, A. et al. Adaptation of the cerebrocortical circulation to carotid artery occlusion involves blood flow redistribution between cortical regions and is independent of eNOS. Am. J. Physiol. Heart Circ. Physiol. 311, H972–H980 (2016).

    PubMed  Google Scholar 

  40. Yoshizaki, K. et al. Chronic cerebral hypoperfusion induced by right unilateral common carotid artery occlusion causes delayed white matter lesions and cognitive impairment in adult mice. Exp. Neurol. 210, 585–591 (2008).

    PubMed  Google Scholar 

  41. Lacolley, P. et al. Occipital artery injections of 5-HT may directly activate the cell bodies of vagal and glossopharyngeal afferent cell bodies in the rat. Neuroscience 143, 289–308 (2006).

    CAS  PubMed  Google Scholar 

  42. Gillilan, L. A. Potential collateral circulation to the human cerebral cortex. Neurology 24, 941–948 (1974).

    CAS  PubMed  Google Scholar 

  43. Cuccione, E., Padovano, G., Versace, A., Ferrarese, C. & Beretta, S. Cerebral collateral circulation in experimental ischemic stroke. Exp. Transl. Stroke Med. 8, 2 (2016).

    PubMed  PubMed Central  Google Scholar 

  44. McGraw, C. P. & Howard, G. Effect of mannitol on increased intracranial pressure. Neurosurgery 13, 269–271 (1983).

    CAS  PubMed  Google Scholar 

  45. Schwarz, S., Schwab, S., Bertram, M., Aschoff, A. & Hacke, W. Effects of hypertonic saline hydroxyethyl starch solution and mannitol in patients with increased intracranial pressure after stroke. Stroke 29, 1550–1555 (1998).

    CAS  PubMed  Google Scholar 

  46. Cosolo, W. C., Martinello, P., Louis, W. J. & Christophidis, N. Blood–brain barrier disruption using mannitol: time course and electron microscopy studies. Am. J. Physiol. 256, R443–R447 (1989).

    CAS  PubMed  Google Scholar 

  47. Fredericks, W. R. & Rapoport, S. I. Reversible osmotic opening of the blood–brain barrier in mice. Stroke 19, 266–268 (1988).

    CAS  PubMed  Google Scholar 

  48. Doolittle, N. D., Muldoon, L. L., Culp, A. Y. & Neuwelt, E. A. Delivery of chemotherapeutics across the blood–brain barrier: challenges and advances. Adv. Pharmacol. 71, 203–243 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Guzman, R., Janowski, M. & Walczak, P. Intra-arterial delivery of cell therapies for stroke. Stroke 49, 1075–1082 (2018).

    PubMed  PubMed Central  Google Scholar 

  50. Golubczyk, D. et al. Endovascular model of ischemic stroke in swine guided by real-time MRI. Sci. Rep. 10, 17318 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Yang, W. et al. Enhanced survival of glioma bearing rats following boron neutron capture therapy with blood–brain barrier disruption and intracarotid injection of boronophenylalanine. J. Neurooncol. 33, 59–70 (1997).

    CAS  PubMed  Google Scholar 

  52. Neuwelt, E. A. et al. Delivery of melanoma-associated immunoglobulin monoclonal antibody and Fab fragments to normal brain utilizing osmotic blood–brain barrier disruption. Cancer Res. 48, 4725–4729 (1988).

    CAS  PubMed  Google Scholar 

  53. Kozler, P., Riljak, V., Jandova, K. & Pokorny, J. CT imaging and spontaneous behavior analysis after osmotic blood–brain barrier opening in Wistar rat. Physiol. Res. 63, S529–S534 (2014).

    CAS  PubMed  Google Scholar 

  54. Chi, O. Z., Liu, X. & Weiss, H. R. Effects of mild hypothermia on blood–brain barrier disruption during isoflurane or pentobarbital anesthesia. Anesthesiology 95, 933–938 (2001).

    CAS  PubMed  Google Scholar 

  55. Godinho, B. et al. Transvascular delivery of hydrophobically modified siRNAs: gene silencing in the rat brain upon disruption of the blood–brain barrier. Mol. Ther. 26, 2580–2591 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. Martin, J. A.; Maris, A. S.; Ehtesham, M.; Singer, R. J., Rat model of blood–brain barrier disruption to allow targeted neurovascular therapeutics. J. Vis. Exp. 2012, e50019.

  57. Bhattacharjee, A. K., Nagashima, T., Kondoh, T. & Tamaki, N. Quantification of early blood–brain barrier disruption by in situ brain perfusion technique. Brain Res. Brain Res. Protoc. 8, 126–131 (2001).

    CAS  PubMed  Google Scholar 

  58. Ju, F. et al. Increased BBB permeability enhances activation of microglia and exacerbates loss of dendritic spines after transient global cerebral ischemia. Front. Cell Neurosci. 12, 236 (2018).

    PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This work was financially supported by 2017-MSCRFF-3942, 2019-MSCRFF-5031, NIH R01NS091110, R01NS102675 and R21NS091599. We thank I.-H. Wu for preparing Fig. 1 and B. Pocta for editorial assistance.

Author information

Authors and Affiliations

Authors

Contributions

P.W., M.J., M.P., T.M., S.L. and C.C. contributed to conception and design; C.C., A.J., W.J., Y.G. and X.L. conducted the experiments; C.C., Y.G., G.L. and Y.L. analyzed and interpreted the data; C.C. drafted the manuscript, with revision from A.J., Y.G., X.L., Y.L., W.J., G.L., S.L., T.M., M.P., M.J. and P.W.

Corresponding author

Correspondence to Piotr Walczak.

Ethics declarations

Competing interests

M.P., M.J. and P.W. are founders and equity holders in Intra-ART. M.J. and P.W. are founders and equity holders in Ti-Com.

Additional information

Peer review information Nature Protocols thanks Mark S. Bolding, Laura M. Vecchio and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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

Related links

Key references using this protocol

Chu, C. et al. Front. Neurol. 9, 921 (2018): https://doi.org/10.3389/fneur.2018.00921

Lesniak, W. G. et al. J. Nucl. Med. 60, 617–622 (2019): https://doi.org/10.2967/jnumed.118.218792

Janowski, M. et al. J. Cereb. Blood Flow Metab. 36, 569–575 (2016): https://doi.org/10.1177/0271678X15615875

Supplementary information

Supplementary Information

Supplementary Figs. 1–4 and Supplementary Methods.

Reporting Summary

Supplementary Data 1

Matlab code and a demonstration of image processing

Supplementary Video 1

Temporary ligation of ECA and OA cauterization

Supplementary Video 2

Temporary ligation of PPA

Supplementary Video 3

Catheter cannulation

Supplementary Video 4

Brain perfusion of a contrast agent under real-time MRI

Supplementary Video 5

Postoperative procedures

Source data

Source Data Fig. 8

Statistical source data.

Source Data Fig. 9

Statistical source data.

Source Data Fig. 10

Statistical source data.

Source Data Fig. 11

Statistical source data.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Chu, C., Jablonska, A., Gao, Y. et al. Hyperosmolar blood–brain barrier opening using intra-arterial injection of hyperosmotic mannitol in mice under real-time MRI guidance. Nat Protoc 17, 76–94 (2022). https://doi.org/10.1038/s41596-021-00634-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41596-021-00634-x

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing