Additional increased effects of mannitol-temozolomide combined treatment on blood-brain barrier permeability

https://doi.org/10.1016/j.bbrc.2018.02.149Get rights and content

Highlights

  • The blood−brain barrier (BBB) poses a major obstacle to therapeutic drug delivery.

  • Mannitol-temozolomide (TMZ) combined treatment induced decreased tight junction protein expression.

  • Mannitol-TMZ combined treatment increased BBB permeability.

  • This combined treatment may improve intracerebral delivery of various drugs.

Abstract

The blood−brain barrier (BBB) is major obstacle in drug or stem cell treatment in chronic stroke. We hypothesized that adding mannitol to temozolomide (TMZ) is a practically applicable method for resolving the low efficacy of intravenous mannitol therapy. In this study, we investigated whether BBB permeability could be increased by this combined treatment. First, we established a chronic ischemic stroke rat model and examined changes in leakage of Evans blue dye within a lesion site, and in expression of tight junction proteins (TJPs), by this combined treatment. Additionally, in an in vitro BBB model using trans-wells, we analyzed changes in diffusion of a fluorescent tracer and in expression of TJPs. Mannitol-TMZ combined treatment not only increased the amount of Evans blue dye within the stroke lesion site, but also reduced occludin expression in rat brain microvessels. The in vitro study also showed that combined treatment increased the permeability for two different-sized fluorescent tracers, especially large size, and decreased expression of TJPs, such as occludin and ZO-1. Increased BBB permeability effects were more prominent with combined than with single treatments. Mannitol-TMZ combined treatment induced a decrease of TJPs with a consequent increase in BBB permeability. This combined treatment is clinically useful and might provide new therapeutic options by enabling efficient intracerebral delivery of various drugs that could not otherwise be used to treat many CNS diseases due to their inability to penetrate the BBB.

Introduction

The blood−brain barrier (BBB) is a specific structure that is closely regulated by complicated interactions among vascular endothelial cells, glial cells, and nerve cells, and it also functions in protecting the brain from harmful substances [1,2]. The importance of BBB function has been highlighted in studies on the patho-mechanism of various central nervous system (CNS) diseases [[3], [4], [5]]. In the case of ischemic stroke, there is a rapid influx of inflammatory cells into the brain, due to the BBB disruption that occurs in the acute phase; subsequently, this is exacerbated in the damaged area and lasts for 4 weeks [6,7]. Consequently, many studies have found that stem cell treatment in the acute phase of stroke improves recovery of neurological deficits, since stem cells not only decrease inflammation and neuronal injury, but also increase regeneration of neurons and vessels. However, stem cell treatment in chronic stroke has no or little effect, due to the recovery of the BBB and its decreased permeability.

Therefore, there have been many attempts to increase BBB permeability, for instance by using hyperosmotic change, transient inflammatory response, and physiological stimulation (focused ultrasound) in vivo studies [[8], [9], [10], [11]]. Among of them, mannitol is still the most popular agent for transient BBB opening. However, the intra-arterial dosage of mannitol used in previous experiments is too high and toxic to apply clinically. On the other hand, intravenous (IV) administration of mannitol is an alternative and safe method; however, many reports have indicated that it has little effect in stem cell treatment for chronic stroke, with the exception of one report [12].

We hypothesized that combination of mannitol with another drug may help to overcome this difficulty. Temozolomide (TMZ) is well-known anti-cancer drug for brain tumor and blocks drug efflux by inhibiting function of the ABCB1 transporter in the cell membrane on the BBB [13]. Furthermore, it has reported that combined treatment of TMZ and mesenchymal stem cell extended the life span of animals with brain tumors [14]. We therefore hypothesized that a transient increase of BBB permeability, through mannitol-TMZ combined treatment, in chronic stroke would enhance the efficacy of delivery of intravenously administered stem cells as well as the factors they secrete.

In this study, we therefore investigated whether mannitol-TMZ combined treatment would have an additional effect in increasing BBB permeability, both in vivo and in vitro.

Section snippets

Ethics

All the experimental animals were handled by appropriately trained researchers and experiments were performed in accordance with the Institutional Animal Care and Use Committee of CHA University (IACUC No.: 160066).

Animal surgery and treatment

Male Sprague−Dawley rats, weighing 280–300 g, were used to produce an animal model of ischemic stroke in the chronic phase. The surgery involved transient insertion of a filament into the middle cerebral artery (middle cerebral artery occlusion [MCAo] method) [15]. To assess the

Change in BBB permeability in chronic ischemic stroke rat models induced by mannitol and TMZ treatment

To investigate the change in BBB permeability depending on different treatments with mannitol and TMZ in vivo, an EBD extravasation experiment was performed in chronic stroke rat models at 4 weeks after MCAo induction. As shown in Fig. 1A and B, there was no difference in EBD leakage in the contra-hemisphere among the different groups, while EBD leakage in the ipsi-hemisphere was more notable than that in the contra-hemisphere, in comparison to the control group. Leakage of EBD in the

Discussion

In this study, we showed that mannitol-TMZ combined treatment induced an increase in BBB permeability, which was more prominent than the effects of either single drug treatments. In addition, a decrease in expression of TJP associated with BBB integrity was confirmed in both in vivo and in vitro experiments.

Mannitol has frequently been used in studies on increasing BBB permeability. Most of these studies showed that intra-arterial injection of mannitol led to an effective BBB permeability

Author contribution

Choi C, and Kim OJ designed the study concept, design, and wrote the draft. Choi C, Kim HM and Shon JH performed the acquisition of data and analysis. Choi C, Kim HM, Shon JH, Park J, Kim HT, Oh SH, Kim NK and Kim OJ, conducted the interpretation. Choi C, Oh SH and Kim OJ made substantial revision of the report. All authors have given final approval of the manuscript for publication.

Conflicts of interest

None.

Acknowledgments

This study was supported by grants of the Korea Healthcare Technology R&D project though the Korea Health Industry Development Institute (KHIDI), funded by the Ministry for Health, Welfare, & Family Affairs, Republic of Korea (HI16C1559).

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