Elsevier

Brain Research

Volume 1120, Issue 1, 20 November 2006, Pages 172-182
Brain Research

Research Report
Biphasic cytoarchitecture and functional changes in the BBB induced by chronic inflammatory pain

https://doi.org/10.1016/j.brainres.2006.08.085Get rights and content

Abstract

The blood–brain barrier (BBB) is a dynamic system which maintains brain homeostasis and limits CNS penetration via interactions of transmembrane and intracellular proteins. Inflammatory pain (IP) is a condition underlying several diseases with known BBB perturbations, including stroke, Parkinson's, multiple sclerosis and Alzheimer's. Exploring the underlying pathology of chronic IP, we demonstrated alterations in BBB paracellular permeability with correlating changes in tight junction (TJ) proteins: occludin and claudin-5. The present study examines the IP-induced molecular changes leading to a loss in functional BBB integrity. IP was induced by injection of Complete Freund's Adjuvant (CFA) into the plantar surface of the right hindpaw of female Sprague–Dawley rats. Inflammation and hyperalgesia were confirmed, and BBB paracellular permeability was assessed by in situ brain perfusion of [14C]sucrose (paracellular diffusion marker). The permeability of the BBB was significantly increased at 24 and 72 h post-CFA. Analysis of the TJ proteins, which control the paracellular pathway, demonstrated decreased claudin-5 expression at 24 h, and an increase at 48 and 72 h post-injection. Occludin expression was significantly decreased 72 h post-CFA. Expression of junction adhesion molecule-1 (JAM-1) increased 48 h and decreased by 72 h post-CFA. Confocal microscopy demonstrated continuous expression of both occludin and JAM-1, each co-localizing with ZO-1. The increased claudin-5 expression was not limited to the junction. These results provide evidence that chronic IP causes dramatic alterations in specific cytoarchitectural proteins and demonstrate alterations in molecular properties during CFA, resulting in significant changes in BBB paracellular permeability.

Introduction

More than 85 million people in the United States suffer from chronic pain, costing billions of dollars yearly in medical treatments, productivity losses, legal fees and compensation. Inflammation is recognized not only as a burden to the health of the U.S. population, but also as an underlying basis of many diseases. Inflammatory pain constitutes a major component in multiple sclerosis (MS), Alzheimer's dementia, meningitis, systemic lupus erythematosis, arthritis (both rheumatoid and osteo), Crohn's disease, irritable bowel syndrome, diabetic neuropathy and several types of cancer (Edwards, 2005, Matter et al., 2005, Sharma et al., 2004, Wieseler-Frank et al., 2005, Wolka et al., 2003). Therapeutic agents used to treat these conditions often need to cross an endothelial or epithelial barrier. In particular, many of these drugs have sites of action within the central nervous system (CNS). The CNS, however, is protected by the presence of an endothelial barrier – the blood–brain barrier (BBB) – and therapeutic agents have limited entry, and thus limited efficacy, for treatment of these disease states.

The BBB is a metabolic and physical barrier that is strictly regulated to maintain separation of the CNS from systemic circulation. Although previously thought to be a static barrier, the BBB is a dynamic structure capable of rapid modulation to maintain homoeostasis within the CNS (Abbott, 2005). Tight regulation of the BBB is required to allow nutrient and oxygen passage, while preventing access to circulating toxins, ions, amino acids and xenobiotics. The BBB endothelial cells are characterized by a lack of fenestrations, decreased pinocytosis and the presence of tight junctional proteins, multiple transport systems and enzymatic detoxification enzymes (Hawkins and Davis, 2005, Loscher and Potschka, 2005). Each of these characteristics serves to maintain the homeostatic environment of the CNS, as well as limit the entry of many therapeutic substances into the brain (Pardridge, 1997).

Tight junctions (TJs) of endothelial cells within the brain govern the paracellular route of entry into the brain. TJs span the apical paracellular cleft, tightly linking neighboring endothelial cells together, and their presence serves to limit paracellular mediated distribution (Gonzalez-Mariscal and Nava, 2005). The TJ is formed through complex interactions of numerous transmembrane, accessory and cytoskeletal proteins. The primary seal of the TJs is formed by the transmembrane proteins occludin, JAM-1 and the claudins, with the accessory protein zonula occludens-1 (ZO-1) interacting with their C-termini, linking them to the actin cytoskeleton (Farshori and Kachar, 1999, Mitic et al., 2000). The TJs are dynamic structures modulated by physiological and pathological conditions (Hawkins and Davis, 2005, Wolka et al., 2003). Changes in expression and intracellular localization of the TJ proteins are associated with alterations in paracellular transport (Balda et al., 2000, Coyne et al., 2003, Stamatovic et al., 2005).

The contribution of individual TJ proteins on the development or progression of neurological conditions is varied. In some cases, TJ alterations and subsequent increased BBB permeability are an effect of the underlying pathology; alternatively, it is a causative and mediating event in disease development. For example, TJ disruptions and subsequent BBB perturbations are involved in the development of MS (Kirk et al., 2003, Neuwelt, 2004), while ischemic stroke and traumatic brain injury lead to BBB perturbations (Ilzecka, 1996, Morganti-Kossmann et al., 2001). There are many diseases, such as Alzheimer's disease, where the direct correlation is not yet known, but are currently being investigated (Wardlaw et al., 2003).

We have previously reported a compromise in BBB integrity and paracellular permeability in three different inflammatory hyperalgesic (pain) models-formalin, carrageenan and complete Freund's adjuvant (CFA). Significant increases in uptake of the normally impermeable marker [14C]sucrose, as well as [3H]codeine, were observed. In addition to increased codeine transport into the brain, increased antinociceptive efficacy was noted. These changes in barrier function correlated with alterations in TJ proteins (Brooks et al., 2005, Hau et al., 2004, Huber et al., 2001, Huber et al., 2002a, Huber et al., 2002b).

Chronic inflammatory hyperalgesia induced by CFA for 72 h was previously demonstrated to alter the expression of several transmembrane TJ proteins (Brooks et al., 2005). We observed a decrease in occludin expression and increased expression of the claudin proteins following CFA injection. Moreover, an increase in paracellular permeability of [14C]sucrose was demonstrated via multi-time uptake studies. There were no changes in either mean blood pressures or initial volume of distribution noted. This indicates an increase in [14C]sucrose paracellular diffusion. With hyperalgesia established for 3 days, alterations in BBB characteristics were apparent.

In the present study, we expand upon these findings by examining the functional and molecular integrity of the BBB at earlier time points than the previous report. We sought to do this in order to understand the manner and extent of molecular cytoarchitectural changes which may lead to alterations in BBB functional integrity. In particular, we examine paracellular permeability across the BBB at time points leading up to the previously demonstrated change at 72 h. Also, we describe the expression and co-localization of integral TJ proteins. In addition to the previously studied ZO-1, occludin and claudin-5 (Brooks et al., 2005), we have assessed JAM-1, a key transmembrane TJ protein which is related to immune modulation of the BBB (Mandell and Parkos, 2005). Each of these TJ proteins is integral to membrane function, and alterations in any of them may lead to a perturbation of barrier integrity (Balda et al., 2000, Coyne et al., 2003, Stamatovic et al., 2005). Understanding the molecular mechanisms that lead to alterations in brain permeability may lead to new drug delivery strategies for neuropharmaceuticals.

Section snippets

Edema formation and thermal hyperalgesia

In order to confirm CFA injection and the onset of inflammatory hyperalgesia, both hindpaw volume and thermal sensitivity were measured from 0 to 72 h. Measurements were taken for both saline and CFA treated rats, in both the injected and the contralateral hindpaw. As reported previously, volume was not changed by saline over the 72 h time course in either the injected or the contralateral hindpaws (Brooks et al., 2005). Similarly, the contralateral hindpaw of the CFA injected rats did not

Discussion

In the present study, we demonstrated that CFA causes the onset of inflammatory hyperalgesia, as characterized by increased paw volume, enhanced thermal hyperalgesia and a shift in the population of white blood cells from lymphocytes to neutrophils, with no other significant alterations in the amount of white blood cell or packed red blood cell volume. This inflammatory hyperalgesia/pain (over 72 h) led to a temporal disregulation of key transmembrane TJ proteins (i.e. occludin, JAM and

Radioisotopes, antibodies and chemicals

[14C]sucrose (specific activity of 492 mCi/mmol, 99.5% purity) was purchased through ICN Pharmaceuticals (Irvine, CA). Primary antibodies for ZO-1 (mouse or rabbit), occludin (rabbit), JAM-1 (rabbit) and claudin-5 (mouse) were purchased from Zymed (San Francisco, CA). Conjugated anti-mouse immunoglobin G-horseradish peroxidase and anti-rabbit immunoglobin G-horseradish peroxidase were obtained from Amersham Life Science Products (Springfield, IL). Complete Freund's adjuvant (CFA) and all other

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