Intravenous mannitol does not increase blood brain barrier permeability to inert dyes in the adult rat forebrain

Copyright: 123 Year: 2013

Dochead: Clinical neuroscience and neuropathology Language: English(en)

Intravenous mannitol does not increase blood–brain barrier permeability to

inert dyes in the adult rat forebrainNeuroReportIntravenous mannitol on

BBB Chen et al.

Kuen-Bao

Chen

_,

_Vivi

_Chiali

_Wei

_,

_Lola

_Fenghuei

_Yen

_,

_Kin-Shing

_Poon

_,

_Yu-Cheng

_Liu

_,

Ka-Shun

Cheng

_,

_Chia-Sheng

_Chang

_,

_Ted

_Weita

_Lai

a

_{Graduate Institute of Clinical Medical Science, China Medical University}

_{Department of Anesthesiology}

c

_{Translational Medicine Research Center, China Medical University Hospital, Taichung, Taiwan}

_{Correspondence to Ted Weita Lai, PhD, Graduate Institute of Clinical Medical Science, China}

Medical University, 40402 Taichung, TaiwanTel: +886 4 22052121 x7638; fax: +886 4 22052121

x7837; e-mail: ted.weita@me.com

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Intravenous mannitol (IV-M) is widely administered in the clinic to lower intracranial pressure in patients with brain trauma and stroke. However, intracarotid arterial mannitol (ICA-M) is known to potently open the blood–brain barrier (BBB) to serum protein tracers such as the Evans blue dye (EBD). In this study, we aimed to determine the potential effect of IV-M on BBB permeability to EBD and a small molecular tracer sodium fluorescein dye (NaF). Rats received intravenous EBD/NaF injections, and after a 30-min equilibration time, they received mannitol (20%, 0.5 g/kg) through either route of administration. At 90 min after the mannitol injection, the rats were perfused to rid their circulations of the tracers, and the tracers extravasated into the brain parenchyma were measured by photospectrometry. As expected, ICA-M considerably increased EBD extravasation into the rat forebrain regions, including the motor cortex (P=0.0069), the striatum (P=0.0097), and the hippocampus (P=0.0281; student’s t-test). In marked contrast, IV-M exerted no effect on EBD extravasation into these forebrain regions. To increase the power of the IV-M study, we repeated the experiments in two independent trials of experiments (n=6–

9/group/trial) and found the same result. Finally, consistent with no effect on EBD extravasation, IV-M had no effect on NaF extravasation into the rat forebrain. In conclusion, we report direct evidence that IV-M, at a dose used clinically, in contrast to the same dose of ICA-M, exerted no effect on BBB permeability to protein and small molecular tracers.

Keywords:

Introduction

Intravenous mannitol (IV-M), which osmotically draws fluid from the edematous brain into the circulation, is the most widely used drug for lowering intracranial pressure (ICP) in the clinic [1–3]. Importantly, the effectiveness of osmotic agents such as IV-M depends on their exclusion by the blood– brain barrier (BBB) [4,5], and inadvertent extravasation of osmotic agents into the brain parenchyma can trigger an adverse increase in ICP [1,5]. Thus, IV-M is most effective in lowering ICP in brain tissues in which the BBB is intact [4,5].

In comparison, intracarotid arterial mannitol (ICA-M), administered at about the same doses as IV-M used to lower ICP, has been investigated extensively as a measure to open the BBB to facilitate drug and protein delivery into the brain [6–8]. In particular, ICA-M opens the BBB in experimental animals to circulating Evans blue dye (EBD), which labels serum albumin through high-affinity binding once injected into the circulation [9,10]. This effect of ICA-M on BBB permeability to EBD is reversible [9], and exerts no long-term damaging effect on the brain [11]. More recently, ICA-M was found to improve the

therapeutic efficacy of chemotherapy against brain tumors in several clinical studies, and this was believed to be because of ICA-M-mediated increase in BBB permeability to chemotherapy [6–8].

Given the similar dose used in lowering ICP and opening BBB, albeit through different routes of administration, we aim to investigate whether IV-M at doses clinically used to lower ICP has an effect on BBB permeability to widely used experimental tracers: (a) EBD for labeling large serum proteins and (b) sodium fluorescein dye (NaF) as a small inert tracer.

Materials and methods

Animals

Male Sprague–Dawley rats (P50–P71; 220–410 g) were used in this study, and all experiments were conducted in accordance to the Institutional Guidelines of the China Medical University for the Care and Use of Experimental Animals, and have been approved by the Institutional Animal Care and Use

Committee of the China Medical University (Taichung, Taiwan) (Protocol No.: 101-270-N).

Intra-arterial and intravenous mannitol

Each rat was anesthetized with urethane (3–4 g/kg; intraperitoneally), and body temperature was maintained at ∼37C by means of a heating pad that received active feedback from a rectal probe. The femoral vein was cannulated to facilitate intravenous injections of EBD, NaF, and/or IV-M. To administer ICA-M, the external carotid artery was cannulated to allow a retrograde infusion of mannitol into the carotid bifurcation, such that mannitol was allowed to flow into the internal carotid artery in an anterograde manner.

Experimental procedure

Each rat received a bolus intravenous injection of 4% dye (either EBD or NaF), which was allowed to circulate in the blood for 30 min before a single bolus intra-arterial or an intravenous injection of (a) 20% mannitol (at 0.5 g/kg) dissolved in 0.9% saline, or (b) equal volume of 0.9% saline. Mannitol and saline were prewarmed to 37C before injection and supplemented with an additional dose of 0.5 ml 0.9% saline immediately after injection. At 120 min after dye injection, a blood sample was collected and stored on ice. Thereafter, each rat was subjected to intracardiac perfusion of saline into the left ventricle at a rate of 30–33 ml/min to rid its circulation of dye, and this was confirmed by an outflow of colorless perfusate from the severed right jugular vein.

Sample collection and photospectrometry

The perfused forebrain was carefully isolated and coronal sectioned to allow isolation of the motor cortex, the striatum, and the hippocampus. The brain specimens were then dry weighted, suspended in a 1 : 3 volume of 50% trichloroacetic acid (TCA; dissolved in saline), and homogenized by metal beads

propelled with a homogenizer. The homogenate was centrifuged at 10 000 rpm for 10 min at 4C, and the supernatant was dissolved in 1 : 3 volume of 95% ethanol to allow photospectrometric analysis of EBD (440 nm excitation and 525 nm emission) or NaF (420 nm excitation and 680 nm emission) fluorescence.

The liver sample was similarly prepared, except that it was first homogenized in a 1 : 1.5 volume of 0.9% saline before adding an equal volume of 100% TCA. The homogenate was similarly centrifuged, and the supernatant was further diluted (25) with 50% TCA before photospectrometric analysis of dye fluorescence (in 1 : 3 volume of 95% ethanol).

The blood sample was centrifuged at 10 000 rpm for 10 min at 4C, and the plasma supernatant was collected and diluted with a 1 : 500 volume of 50% TCA, before photospectrometric analysis of dye fluorescence (in a 1 : 3 volume of 95% ethanol).

Data presentation and analysis

Data are presented as meanSEM. Comparisons were made using the unpaired student’s t-test.

Results

In this study, we measured BBB permeability by injecting rats with the widely used tracer EBD [9– 11], which quickly binds to endogenous serum album once administered into the circulation. As expected, EBD remained mostly in the blood (600–1100 g of EBD/ml of blood), with considerable distribution into peripheral organs such as the liver (20–40 g of EBD/g of liver tissue) (Fig. 1). In comparison, EBD was largely restricted from entering the brain parenchyma of control rats (<1 g of EBD/g of brain tissue) (Figs

1 and 2).

As expected, ICA-M (0.5 g/kg; 20% in 0.9% saline) produced single-sided extravasation of EBD into the ipsilateral cerebral hemisphere (Fig. 1a and b), and this resulted in a three- to nine-fold increase in EBD extravasation into the motor cortex (5.51.2 g/g), the striatum (2.70.6 g/g), and the hippocampus (3.71.1 g/g), compared with that in control rats that received saline (motor cortex: 0.60.1 g/g,

P=0.0069,

Fig. 1c; striatum: 0.50.1 g/g, P=0.0097, Fig. 1d; hippocampus: 0.50.1 g/g, P=0.0281,

Fig. 1e; n=4/group). The effect of ICA-M on EBD extravasation was brain-specific, because there was no

Fig. 1g).

In comparison, IV-M (0.5 g/kg; 20% in 0.9% saline) caused no noticeable EBD extravasation into the rat brain (Fig. 2a and b). This lack of EBD extravasation into the brain parenchyma was confirmed by photospectrometric analysis of EBD concentrations in the motor cortex (0.50.1 g/g), the striatum (0.50.1 g/g), and the hippocampus (0.50.1 g/g) of rats that received IV-M, compared with rats that received saline (motor cortex: 0.40.1 g/g, P=0.2923, Fig. 2c; striatum: 0.40.1 g/g, P=0.3820, Fig. 2d; hippocampus: 0.60.1 g/g, P=0.8656, Fig. 2e; n=9/group). To confirm this finding, we repeated the study in a second trial of experiments, and again, IV-M exerted no effect on EBD extravasation into the motor cortex (0.50.1 g/g), the striatum (0.40.1 g/g), and the hippocampus (0.50.04 g/g), compared with the saline control (motor cortex: 0.50.04 g/g, P=0.2642, Fig. 2f; striatum: 0.40.1 g/g, P=0.8260, Fig.

2g; hippocampus: 0.40.1 g/g, P=0.5622, Fig. 2h; n=6–7/group).

In lieu of large protein tracers such as EBD, we sought to investigate whether IV-M increased extravasation of small inert tracers such as NaF across the BBB and into the brain parenchyma. Consistent with the notion that IV-M has no effect on BBB permeability, IV-M had no effect on NaF extravasation into the motor cortex (0.270.03 g/g), the striatum (0.250.02 g/g), and the hippocampus (0.250.02 g/g), compared with the saline control (motor cortex: 0.230.02 g/g, P=0.3136, Fig. 2i; striatum: 0.240.02 g/g, P=0.6990, Fig. 2j; hippocampus: 0.250.02 g/g, P=0.9489, Fig. 2k; n=10/group).

Discussion

Head trauma and stroke are leading causes of death and disability in developed countries [2], and the abrupt increase in ICP following these head injuries strongly correlates with increased morbidity and mortality [2,4]. To date, IV-M remains the most widely used method to lower ICP in many clinics. Given that the ICP-lowering efficacy of IV-M depends on the integrity of the BBB [4,5], as extravasation of mannitol can cause a secondary increase in ICP [1,5], this treatment is most effective when the BBB is intact [4,5].

Intriguingly, recent studies provide indirect evidence that IV-M, at doses similar to those used clinically to lower ICP, can directly open the BBB [12–16]. Specifically, IV-M improved cerebral gene

expression and therapy by intravenous recombinant adeno-associated viral vectors injected in mice [12,13]. In addition, IV-M increased the cerebral neurotrophic factor concentration and the therapeutic efficacy of intravenous human umbilical cord blood in rats subjected to cerebral ischemia [14,15]. Although IV-M may indeed confer these effects, including improved gene expression, neurotrophic factor concentration, and therapeutic efficacy by disrupting the BBB, there remains no direct evidence that BBB was indeed disrupted or whether these effects could simply be because of the ICP-lowering effect of IV-M.

Given the prominent use of IV-M in many clinics including our own, we carried out this study to investigate whether IV-M indeed disrupts the BBB at doses commonly used in the clinic to lower ICP. Notably, the same dose ICA-M is an experimental treatment extensively demonstrated to osmotically disrupt the BBB to large protein tracers such as EBD [9], and is being developed to facilitate drug and protein delivery across the BBB [6–8]. In line with this notion, ICA-M served as our positive control, and it indeed considerably increased EBD extravasation into the rat brain parenchyma in our study. When

administered as IV-M, prepared in the same way and treated at the same dose as ICA-M, it had no effect on EBD extravasation into the rat forebrain. To double confirm our findings, we repeated the same study in two independent experimental trials, and found the same results on both trials.

Because IV-M-mediated lowering of ICP depends on the exclusion of mannitol by the BBB [4,5], the treatment becomes less effective in damaged brain tissue where extravasation of mannitol could in turn lead to an increase in ICP [1,5]. To investigate whether IV-M may increase extravasation of small

molecules such as mannitol, we tested for a possible increase in BBB permeability to the small molecular tracer NaF following IV-M administration. Consistent with the findings from the EBD study, IV-M exerted no effect on NaF extravasation into the brain. Taken together, here, we report direct evidence that IV-M, at doses used clinically to lower ICP and at equal doses to ICA-M used to disrupt the BBB, has no effect on BBB permeability to both large proteins and small molecules.

The findings here can have several important experimental and clinical implications for future studies. In light of recent studies showing that IV-M improved the therapeutic efficacy of gene therapy against neurological disorders [12,13], more experiments should be conducted to determine whether such an effect indeed resulted from BBB disruption or rather because of lowering of ICP. Similarly, increased neurotrophic factor concentration in the ischemic brain by IV-M coupled with intravenous cord blood injection, reported recently [14,15], may not necessary be because of a global disruption of the BBB. Finally, although we report here that IV-M had no effect on BBB permeability to large protein and small molecules, our study was carried out in control rats rather than rats subjected to brain injuries. In the latter case, BBB can already be compromised. Given the prominent use of mannitol in lowering ICP and the risk of a secondary increase in ICP associated with BBB disruption, future studies should examine the effect of IV-M and perhaps also ICA-M on BBB permeability in experimental models of head trauma and stroke.

Acknowledgements

The authors thank Eva Yuhua Kuo and Ya Lan Yang from Dr Ted Weita Lai laboratory at the China Medical University for preparing, randomizing, and blinding the treatments.

This work was supported by research grants from the China Medical University Hospital (DMR-95-058), the Taiwan Department of Health Clinical Trial and Research Center of Excellence (DOH102-TD-B-111-004), and the National Research Council of Taiwan (NSC100-2632-B-039-001-MY3; NSC101-2321-B-039-008).

Conflicts of interest

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Fig. 1Intracarotid arterial mannitol (ICA-M) increased Evans blue dye (EBD) extravasation into the brain. (a) Represented images of a whole brain from rats subjected to ICA-M, with blue EBD staining. (b) Represented images of 2-mm thick coronal brain sections from rats subjected to ICA-M, with blue EBD staining. (c–e) EBD concentrations in the motor cortex (c), striatum (d), and hippocampus (e) isolated from the perfused brain parenchyma from rats subjected to ICA-administered saline or mannitol. (f) EBD concentrations in the perfused liver tissues. (g) EBD concentrations in the blood samples 90 min after ICA-M. Data are expressed as meanSEICA-M. *_{P<0.05 and} **_{P<0.01, comparison by unpaired student’s t-test.}

Fig. 2Intravenous mannitol (IV-M) had no effect on Evans blue dye (EBD) and sodium fluorescein dye (NaF) extravasation into the brain. (a) Represented images of a whole brain from rats subjected to IV-M, with no noticeable blue EBD staining. (b) Represented images of 2-mm thick coronal brain sections from rats subjected to IV-M, with no noticeable blue EBD staining. EBD concentrations in the motor cortex (c), striatum (d), and hippocampus (e) isolated from the perfused brain parenchyma from rats subjected to intravenous (i.v.) administered saline or mannitol. (f–h) Repeated study trial on the basis of

experiments described in (c–e). NaF concentrations in the motor cortex (i), striatum (j), and hippocampus (k) isolated from the perfused brain parenchyma from rats subjected to i.v. administered saline or mannitol. Data are expressed as meanSEM. No significant difference when compared by unpaired student’s t-test.