Research ReportBiphasic cytoarchitecture and functional changes in the BBB induced by chronic inflammatory pain
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
References (61)
- et al.
Loss of the tight junction proteins occludin and zonula occludens-1 from cerebral vascular endothelium during neutrophil-induced blood–brain barrier breakdown in vivo
Neuroscience
(1998) - et al.
Hypoxia/aglycemia alters expression of occludin and actin in brain endothelial cells
Biochem. Biophys. Res. Commun.
(2005) - et al.
To stick or not to stick: the new leukocyte homing paradigm
Curr. Opin. Cell Biol.
(1996) - et al.
Tight junctions, from tight intercellular seals to sophisticated protein complexes involved in drug delivery, pathogens interaction and cell proliferation
Adv. Drug Deliv. Rev.
(2005) - et al.
Effect of lambda-carrageenan-induced inflammatory pain on brain uptake of codeine and antinociception
Brain Res.
(2004) - et al.
Viability of microvascular endothelial cells to direct exposure of formalin, lambda-carrageenan, and complete Freund's adjuvant
Eur. J. Pharmacol.
(2002) - et al.
Structure of the conserved cytoplasmic C-terminal domain of occludin: identification of the ZO-1 binding surface
J. Mol. Biol.
(2005) - et al.
The JAM family of proteins
Adv. Drug Delivery Rev.
(2005) - et al.
Mammalian tight junctions in the regulation of epithelial differentiation and proliferation
Curr. Opin. Cell Biol.
(2005) - et al.
Protein kinase C{alpha}-RhoA cross-talk in CCL2-induced alterations in brain endothelial permeability
J. Biol. Chem.
(2006)
Establishment and characterization of cultured epithelial cells lacking expression of ZO-1
J. Biol. Chem.
Molecular anatomy of intercellular junctions in brain endothelial and epithelial barriers: electron microscopist's view
Brain Res. Rev.
Antibody array analysis of peripheral and blood cytokine levels in rats after masseter inflammation
Neurosci. Lett.
Pain and the blood–brain barrier: obstacles to drug delivery
Adv. Drug Deliv. Rev.
Dynamics of CNS barriers: evolution, differentiation, and modulation
Cell. Mol. Neurobiol.
Effects of lipopolysaccharide on the blood–brain barrier permeability in prolonged nitric oxide blockade-induced hypertensive rats
Int. J. Neurosci.
Contribution of claudin-5 to barrier properties in tight junctions of epithelial cells
Cell Tissue Res.
Multiple domains of occludin are involved in the regulation of paracellular permeability
J. Cell. Biochem.
Endothelial cell-to-cell junctions: molecular organization and role in vascular homeostasis
Physiol. Rev.
Chronic inflammatory pain leads to increased blood–brain barrier permeability and tight junction protein alterations
Am J. Physiol.: Heart Circ. Physiol.
Role of claudin interactions in airway tight junctional permeability
Am. J. Physiol.: Lung Cell. Mol. Physiol.
Immunogold localization of tight junctional proteins in normal and osmotically-affected rat blood–brain barrier
J. Mol. Histol.
Preparation of a claudin-targeting molecule using a C-terminal fragment of Clostridium perfringens enterotoxin
J. Pharmacol. Exp. Ther.
Inflammation, pain, and chronic disease: an integrative approach to treatment and prevention
Altern. Ther. Health Med.
Redistribution and phosphorylation of occludin during opening and resealing of tight junctions in cultured epithelial cells
J. Membr. Biol.
Helicobacter pylori activates myosin light-chain kinase to disrupt claudin-4 and claudin-5 and increase epithelial permeability
Infect. Immun.
Attaching and effacing pathogen-induced tight junction disruption in vivo
Cell. Microbiol.
The blood–brain barrier/neurovascular unit in health and disease
Pharmacol. Rev.
Inflammatory pain alters blood–brain barrier permeability and tight junctional protein expression
Am. J. Physiol.: Heart Circ. Physiol.
Blood–brain barrier tight junctions are altered during a 72-h exposure to lambda-carrageenan-induced inflammatory pain
Am. J. Physiol.: Heart Circ. Physiol.
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