Basic Neuroscience
A new method of quantitatively assessing the opening of the blood–brain barrier in murine animal models

https://doi.org/10.1016/j.jneumeth.2012.03.012Get rights and content

Abstract

The blood–brain barrier (BBB) restricts the delivery of drugs into the brain. Different strategies have been developed to circumvent this obstacle. One such approach, the osmotic BBB disruption (BBBD), has been under pre-clinical study since the 70's. Typically, qualitative ex vivo assessment of the extent of BBBD has been performed using Evan's blue staining technique. In this study, we describe a simple quantitative technique based on albumin indirect immunohistochemistry to measure the extent of BBB breach. Thirty Fischer rats were assigned to one of 6 groups: a control group, and BBBD groups with escalation in IA mannitol infusion rate: 0.06, 0.08, 0.10, 0.12 and 0.15 cc/s. Fifteen minutes after the BBBD procedure, the animals were sacrificed, brain harvested and sections stained for albumin. Using an image analysis software, isolated albumin staining pixels were expressed as a fraction of the treated hemisphere. This ratio was used as a percentage value in the intensity of the BBB permeabilization. All sections studied harbored staining, averaging 0.37% for the controls (group 1), 5.69% for group 2 (0.06 cc/s), 10.44% for group 3 (0.08 cc/s), 6.99% for group 4 (0.1 cc/s), 18.50% for group 5 (0.12 cc/s) and reaching 61.70% for group 6 (0.15 cc/s). Important variations were observed between animals. A threshold effect was observed, and animals in group 6 presented a significant increase in BBB permeabilization compared to the other groups. We hereby detail a simple technique that can be applied to quantitatively measure the extent of the BBB breach notwithstanding the pathological process.

Highlights

► We describe a new quantitative method to assess the extent of the BBB disruption. ► The method relies on the detection of albumin by indirect-immunohistochemistry. ► The extent of the BBBD was studied using increasing mannitol infusion rates. ► At 0.15 cc/s, we observed a threshold effect on the BBB permeability.

Introduction

The BBB is a complex physiological entity located at the level of the cerebral endothelial cell presenting a surface area of approximatively 20 m2 in the human. The BBB derives its restrictive function in delivery from multiple anatomical and physiological characteristics, such as the presence of tight junctions, the expression of different efflux pumps, a luminal negative charge of the endothelial cells, the presence of basal lamina and of the astrocytic podophilic projections. Altogether, the BBB limits the passage of water soluble molecules presenting a molecular weight greater than 180 Da (Kroll et al., 1996, Pardridge, 2005). In fact, it is estimated that 98% of all the therapeutic molecules cannot reach the brain parenchyma in pharmacologically-relevant concentrations (Neuwelt et al., 2008). This ‘neurovascular unit’ thus impacts the treatment of different pathologies by greatly limiting the entry of therapeutic molecules and restricting the arsenal at our disposal. As a consequence, a significant number of molecules with the activity in vitro, such as antineoplastic agents against glioma cell lines, have limited efficacy because of limited brain bioavailability in vivo. Therefore, the concept of transitorily breaching the permeability or the function of the BBB to increase delivery of therapeutics is highly relevant (Boyle et al., 2004, Bradford et al., 1997). Different approaches have been developed to circumvent the BBB, such as intra-arterial infusion of hyperosmolar solutions, infusion of bradykinin receptor agonists and convection enhanced delivery (Kraemer et al., 2002). The intra-arterial infusion of hyperosmolar solutions, or blood–brain barrier disruption (BBBD), has been studied and characterized, both in clinical and pre-clinical studies (Blanchette et al., 2009, Fortin, 2003, Fortin, 2004, Fortin et al., 2005, Fortin and Neuwelt, 2003, Kraemer et al., 2002, Kroll and Neuwelt, 1998, Neuwelt, 1989, Neuwelt, 1980, Neuwelt et al., 1980, Neuwelt et al., 1986, Pardridge, 2005). Our clinical team commonly uses this approach in the treatment of primary CNS lymphoma, malignant gliomas and brain metastasis (Fortin et al., 2005, Fortin et al., 2007).

The BBBD procedure involves the intra-arterial infusion of a hyperosmolar solution (mannitol 25%) to produce a transient increase in the permeability of the BBB. This is typically followed by the intra-arterial infusion of the therapeutic agents (e.g. chemotherapy). The effectiveness of the procedure can be influenced by different factors, including hemodynamic variables, type of anesthesia and rate of hyperosmolar solution infusion (Fortin et al., 2004, Remsen et al., 1999). It is thus paramount to monitor the degree of the barrier permeabilization obtained after a procedure, as it can be highly variable from patient to patient, and even with repeated procedures in the same subject.

In the clinic, a CT scan with an IV radiographic contrast agent infusion is performed after a BBBD procedure to evaluate the degree of the BBB permeabilization in a semi-quantitative fashion, based on an analog scale. The validity of this monitoring approach has been established (Neuwelt et al., 1980, Roman-Goldstein et al., 1994). Using this technique, some authors have reported an association between the degree of the BBBD as a surrogate of drug delivery, and survival of patients bearing primary central nervous system lymphomas treated with the BBBD procedure (Kraemer et al., 2001).

In pre-clinical studies however, the most widely used approach to study the extent of the BBBD process is an ex vivo technique requiring the intravenous infusion of a 2% solution of Evans blue prior to the BBBD (Fortin, 2003, Fortin, 2004, Kroll and Neuwelt, 1998). This in vivo marker binds tightly but reversibly to albumin, which does not normally cross the BBB (Neuwelt, 1989). Thus, the increase in the BBB permeability allows diffusion of the albumin-Evans blue complex into the brain parenchyma. Once the brain is harversted after the BBBD procedure, the intensity distribution of the blue coloration in the treated hemisphere can than be qualitatively assessed, and a visual score is attributed (Fortin, 2003). The qualitative and subjective aspect of this approach decreases the reliability of this evaluation method (Fortin, 2003, Fortin, 2004).

Recently, we developed a new technique to monitor the dynamic process of the BBBD by DCE-MRI in pre-clinical research (Blanchette et al., 2009) that possesses the invaluable advantage of allowing in vivo assessment. However, not all research groups have access to an animal MRI scanner in their facilities, and thus ex vivo qualitative or semi-quantitative techniques to evaluate the efficacy of the BBBD procedure remain pertinent and useful.

To improve the knowledge of the BBBD process, its evaluation has to be revisited. A quantitative measurement of the degree of BBBD would allow a better analysis in the intensity of delivery against efficacy in any given experiment regarding CNS treatments. Accordingly, in the design of new glioma treatment strategies, it is essential to distinguish between the different factors at play, such as the delivery impediment and the efficacy of the therapeutic molecule. Only by isolating and analyzing theses two variables individually will we be able to optimize the design of new treatment approaches. It is common knowledge that albumin does not cross the normal BBB, and that it is easily labeled by indirect immunohistochemistry. The aim of this study was thus to develop a simple, accurate and quantitative technique to measure the intensity of delivery produced by the BBBD procedure.

Section snippets

Materials and methods

The experimental protocol was approved by the institutional ethical committee and conformed to regulations of the Canadian Council on animal care.

Results

Mean staining values obtained for each animal are presented in Fig. 3. As can be appreciated from this data, all groups depicted significantly different staining values. As expected, the saline infused animals (group 1) presented insignificant albumin staining, with an average of staining ratio of 0.37%. Animals from groups 2–6 presented increased staining when compared to the control group (Fig. 3). However, this difference was statistically significant only when mannitol was administered at a

Discussion

Osmotic BBBD is one of the strategies designed to bypass the blood–brain barrier, and improve delivery of therapeutic molecules to the central nervous system. The aspect of delivery is under-emphasized in the literature, and has not always received the proper attention it deserves (Pardridge, 1997, Pardridge, 2005). Even now, delivery across the BBB is overlooked as an important cause of failure in the treatment of many CNS diseases (Fortin et al., 2005). Admittedly, researchers working in the

Source of funding

This work was supported by grant from the CIHR (Canadian Institute of Health Research) and by the Research Chair on Brain Tumor Therapies National Bank of Canada.

Conflict of interest

The authors declare no conflict of interests.

References (26)

  • D. Fortin et al.

    Enhanced chemotherapy delivery by intraarterial infusion and blood–brain barrier disruption in the treatment of cerebral metastasis

    Cancer

    (2007)
  • D. Fortin et al.

    Unexpected neurotoxicity of etoposide phosphate administered in combination with other chemotherapeutic agents after blood–brain barrier modification to enhance delivery, using propofol for general anesthesia, in a rat model

    Neurosurgery

    (2000)
  • D. Fortin et al.

    Therapeutic manipulation of the blood–brain barrier

    Neurobase-neurosurgery. Medlink CD-ROM

    (2003)
  • Cited by (0)

    View full text