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Am J Physiol Heart Circ Physiol 283: H1531-H1537, 2002. First published June 13, 2002; doi:10.1152/ajpheart.00027.2002
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Vol. 283, Issue 4, H1531-H1537, October 2002

Blood-brain barrier tight junctions are altered during a 72-h exposure to lambda -carrageenan-induced inflammatory pain

J. D. Huber, V. S. Hau, L. Borg, C. R. Campos, R. D. Egleton, and T. P. Davis

Department of Pharmacology, University of Arizona College of Medicine, Tucson, Arizona 85724


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we examined the effect of lambda -carrageenan-induced inflammatory pain on the functional and structural properties of the rat blood-brain barrier (BBB) over a 72-h time period. Systemic inflammation was induced by an intraplantar injection of 3% lambda -carrageenan into the right hind paw of female Sprague-Dawley rats. In situ brain perfusion and Western blot analyses were performed at 1, 3, 6, 12, 24, 48, and 72 h. In situ brain perfusion showed lambda -carrageenan significantly increased brain uptake of [14C]sucrose at 1, 3, 6, and 48 h (139 ± 9%, 166 ± 19%, 138 ± 13%, and 146 ± 7% compared with control, respectively). Capillary depletion analysis insured the increased brain uptake was due to increased BBB permeability and not vascular trapping. Western blot analyses for zonula occludens-1 (ZO-1) and occludin were performed on isolated cerebral microvessels. ZO-1 expression was significantly increased at 1, 3, and 6 h and returned to control expression levels by 12 h. Total occludin expression was significantly reduced at 1, 3, 6, 12, and 48 h. This investigation demonstrated that lambda -carrageenan-induced inflammatory pain elicits a biphasic increase in BBB permeability with the first phase occurring from 1-6 h and the second phase occuring at 48 h. Furthermore, changes in BBB function are correlated with altered tight junctional protein expression of occludin and ZO-1. Changes in the structure of tight junctions may have important clinical ramifications concerning central nervous system homeostasis and therapeutic drug delivery.

inflammation; ZO-1; ZO-2; membrane-associated guanylate kinase; occludin; immunoprecipitation


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

COMPOSITION OF BRAIN EXTRACELLULAR FLUID must be controlled within a precise physiological range, independent of fluctuations within the systemic circulation, to maintain an optimal environment for neuronal function. Several pathological states such as human immunodeficiency virus-1 encephalitis (12), multiple sclerosis (36), hypoxia/aglycemia (1), cerebral malaria (8), and bacterial meningitis (27) have been shown to induce increased permeability of the blood-brain barrier (BBB), leading to perturbations in homeostasis. Breaches in the BBB have been associated with alterations in ionic and nutritional balance of the central nervous system (CNS), leading to impaired neuronal function, altered delivery of therapeutic agents, and, in severe cases, serum protein extravasation and edema formation (21).

Positioned at endothelial cells of cerebral microvessels, the BBB is characterized by specific transporters, lack of fenestrations, and tight junctions (15). Because of the presence of tight junctions and low transcytotic activity, the BBB is a very selective barrier between the CNS and systemic circulation that limits entry of molecules according to size, charge, hydrophobicity, and utilization of selective transport mechanisms. Tight junctions form a rate-limiting barrier to paracellular diffusion of substances, keeping the microenvironments of the systemic circulation and the brain distinct.

Structurally, tight junctions form a continuous network of parallel, interconnected, intramembrane fibril networks that circumscribe the apexes of endothelial cells (38, 43). Studies of tight junctions from different tissues with varying membrane electrical resistances show a correlation between increasing organization of cytoplasmic fibrils and decreasing permeability of the membrane (9, 10). BBB tight junctions are composed of an intricate combination of transmembrane and cytoplasmic proteins linked to an actin-based cytoskeleton that allow tight junctions to form an impermeant seal while remaining capable of rapid modulation and regulation.

Tight junctional strands are primarily comprised of two distinct, transmembrane proteins: claudins and occludin. Claudins form dimers that bind homotypically to adjacent endothelial cells to form the "seal" of the tight junction (19). Occludin has recently been shown to function as a dynamic regulatory protein whose presence in the membrane is correlated with increased electrical resistance across the membrane and decreased paracellular permeability. (6, 30). When observed using immunofreeze fracture microscopy, occludin is concentrated within tight junctional fibrils (18) with a detergent-extractable pool found along the basolateral surface that is not embedded in the membrane (37). This intracellular pool of occludin may serve as a reservoir for the dynamic regulation of tight junctional complexity (11, 37).

Several accessory proteins are necessary to form, maintain, and regulate tight junctions, including zonula occludens (ZO-1, ZO-2, and ZO-3), cingulin, AF6, and 7H6.

ZO proteins are members of a family of membrane-associated guanylate kinase-like homologues, which play important roles in signal transduction, structural support, and site recognition (20, 26, 49). ZO-1 is the most characterized of these accessory proteins and has binding sites for occludin, claudin, ZO-2, ZO-3, cingulin, and actin, which enables it to maintain and regulate tight junctional structure (16, 44).

Although current research is rapidly characterizing the structure of tight junctions under normal physiological conditions, much less is known about tight junctional regulation under pathophysiological conditions. However, several recent studies (24, 29, 32, 42) have clearly shown that occludin and ZO-1 are important regulatory proteins in maintaining BBB tight junctional integrity during pathological insult.

In a previous study, we showed that peripheral inflammatory pain increased BBB permeability and altered tight junctional protein expression at peak inflammation using the formalin-, lambda -carrageenan-, and complete Freund's adjuvant-induced pain models (24). We have also shown that lambda -carrageenan, when administered directly into the peripheral circulation (intravenously), had no effect on BBB functional and structural integrity. The purpose of this study was to further investigate the effects of lambda -carrageenan-induced inflammatory pain on the functional and structural integrity of BBB tight junctions over a time course from 0 to 72 h and to evaluate the correlation between increased BBB permeability and alterations in occludin and ZO-1 protein expression.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Radioisotopes/antibodies/chemicals. [14C]sucrose was obtained from ICN Pharmaceuticals (specific activity: 492 mCi/mmol, >99.5% purity; Irvine, CA). Primary antibodies (anti-ZO-1, anti-ZO-2, and anti-occludin) were obtained from Zymed (San Francisco, CA). Conjugated anti-rabbit IgG- and anti-mouse IgG-horseradish peroxidase were purchased from Amersham Life Science Products (Springfield, IL). Anti-actin and all other chemicals, unless otherwise stated, were purchased from Sigma (St. Louis, MO).

Animals/treatments. Female Sprague-Dawley rats (Harlan Sprague Dawley; Indianapolis, IN) weighing 250-300 g were housed under standard 12:12-h light-dark conditions and received food ad libitum. All protocols involving animals were approved by the University of Arizona Institutional Animal Care and Use Committee and abide by National Institutes of Health guidelines. Rats were anesthetized with pentobarbital sodium (60 mg/kg ip), subsequently injected (100 µl sc) with 3% lambda -carrageenan into the plantar surface of the right hind paw, and placed into two groups. The first group of animals underwent a 20-min in situ brain perfusion at 1, 3, 6, 12, 24, 48, or 72 h postinjection. Brains from the animals in the second group were harvested, and the protein isolated was used for Western blot analyses (at the same time points as above). Control animals were injected (100 µl sc) with 0.9% saline into the plantar surface of the right hind paw. Naïve controls showed no significant difference in BBB alterations compared with the saline-treated controls and are therefore not included in this study. Pentobarbital sodium was used in this study to insure no interference with N-methyl-D-aspartate receptor activity.

In situ brain perfusion. Rats were anesthetized as above and heparinized (10,000 U/kg). Body temperature was maintained using a heating pad. The common carotid artery was exposed and cannulated with silicone tubing connected to a perfusion circuit. Perfusate consisted of a modified mammalian Ringer solution [containing 117 mM NaCl, 4.7 mM KCl, 0.8 mM MgSO4, 24.8 mM NaHCO3, 1.2 mM KH2PO4, 2.5 mM CaCl2, 10 mM D-glucose, 10 g/l dextran (mol wt 70,000), and 1 g/l bovine serum albumin (type V); pH 7.4] (35). The addition of Evans blue (55 mg/l) to the Ringer solution provided a control for BBB integrity. The perfusate was aerated with 95% O2-5% CO2 and warmed to 37°C. The ipsilateral jugular vein was sectioned to allow drainage. Once the desired perfusion pressure and flow rate were achieved (85-95 mmHg and 3.1 ml/min, respectively), the contralateral carotid artery was cannulated and perfused. Radiolabeled sucrose was infused using a slow-drive syringe pump (0.5 ml · min-1 · hemisphere-1; model 22, Harvard Apparatus; South Natick, MA) into the inflow of the perfusate. After a 20-min brain perfusion, the animal was decapitated, and the brain was removed. The choroid plexi and meninges were excised, and the cerebral hemispheres were sectioned and homogenized. Perfusate containing the radiolabeled marker was collected from each carotid cannula at the termination of the perfusion to serve as a reference.

Cerebral hemispheres (~500 mg) and 100 µl perfusate were prepared for liquid scintillation counting by adding 1 ml tissue solubilizer (TS-2, Research Products International; Mount Pleasant, IL). After 2 days of solubilization, 100 µl of 30% glacial acetic acid were added to eliminate chemiluminescence. Four milliliters of Budget Solve Liquid Scintillation Cocktail (Research Products International) were added, and samples were measured for radioactive counts (model LS 5000 TD Counter, Beckman Instruments; Fullerton, CA).

Capillary depletion. Measurement of the vascular component to total brain uptake was performed using capillary depletion (45). After a 20-min in situ brain perfusion, the brain was perfused for 20 s without [14C]sucrose. The brain was removed, and the choroid plexi and meninges were excised. Brain tissue (50 mg wet wt) was homogenized (Polytron homogenizer, Brinkman Instruments; Westbury, NY) in 1.5 ml capillary depletion buffer [containing 10 mM 4-(2-hydroxyethyl)-piperaxineethane sulfonic acid, 141 mM NaCl, 4 mM KCl, 2.8 mM CaCl2, 1 mM MgSO4, 1 mM NaH2PO4, and 10 mM D-glucose; pH 7.4] and kept on ice. Two milliliters of ice-cold 26% clinical grade dextran were added, and homogenization was repeated. Aliquots of homogenate were centrifuged at 5,400 g for 15 min. Capillary-depleted supernatant was separated from the vascular pellet. Homogenization procedures were performed within 2 min of euthanizing the animal. The homogenate, supernatant, and pellet were taken for radioactive counting. The amount of [14C]sucrose in the brain homogenate, supernatant, and pellet was expressed as the percent ratio of tissue (Cbrain; in disintegrations · min-1 · g-1 of disintegrations · min-1 · ml-1) to perfusate activities (Cperfusate; in disintegrations · min-1 · ml-1) and expressed as Rbrain
R<SUB>brain</SUB> = (C<SUB>brain</SUB>/C<SUB>perfusate</SUB>) × 100

Microvessel isolation. At each time point after inflammatory insult, rats were anesthetized with pentobarbital sodium and decapitated, and the brains were removed. The meninges and choroid plexi were excised, and the cerebral hemispheres were homogenized in 4 ml microvessel isolation buffer [containing 103 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 15 mM HEPES, 2.5 mM NaHCO3, 10 mM D-glucose, 1 mM sodium pyruvate, and 10 g/l dextran (mol wt 64,000); pH 7.4] with protease inhibitor cocktail (0.2 mM phenylmethylsulfonyl fluoride, 1 mM benzamide, 1 mM NaVO4, 10 mM NaF, 10 mM sodium pyrophosphate, and 10 µg/ml aprotinin and leupeptin). Four milliliters of ice-cold 26% dextran were added, and the homogenates were vortexed. Homogenates were centrifuged at 5,600 g for 10 min, and the supernatant was aspirated. Pellets were resuspended in 10-ml microvessel isolation buffer and passed through a 100-µm filter (Falcon, Becton-Dickinson; Franklin, NJ). The filtered homogenates were centrifuged at 3,000 g. Protein was extracted from the pellets using 6 M urea lysis buffer [containing 6 M urea, 0.1% Triton X-100, 10 mM Tris (pH 8.0), 1 mM dithiothreitol, 5 mM MgCl2, 5 mM EGTA, and 150 mM NaCl] with protease inhibitor cocktail. Protein concentrations were determined by bicinchoninic acid protein assay (Pierce, Rockford, IL) with bovine serum albumin as the standard.

Immunoprecipitation and immunoblotting. Isolated microvessel homogenates were analyzed for expression of occludin and ZO-1. Immunoprecipitation studies were performed to determine ZO-1 and occludin interactions with other tight junctional and cytoskeletal proteins. In brief, 100 µg total protein was diluted 10-fold with lysis buffer without urea, combined with 5 µg anti-occludin or anti-ZO-1, and incubated overnight at 4°C. The next day, 50 µl of rec-protein G Sepharose beads (Zymed; San Francisco, CA) were added. Samples were incubated for 4 h at 4°C, pelleted, washed twice with 1 M urea buffer, and washed once with 10 mM Tris (pH 8.0). Samples were resuspended in Laemmli sample buffer and heated to 96°C for 10 min before electrophoresis.

Microvessel samples (20 µg) and immunoprecipitants were resolved on 4-12% Tris-glycine gels (Novex; San Diego, CA) for 90 min at 125 V and transferred to a polyvinylidene difluoride (PVDF) membrane for 30 min at 240 mA. Gelcode blue (Pierce) was used to stain gels and ensure proper protein loading. PVDF membranes were blocked in Tris-buffered saline (TBS) (141 mM NaCl, 10 mM Tris base, and 0.1% Tween 20) with 5% nonfat milk for 4 h. Blots were incubated in primary antibody at room temperature for 2 h, rinsed with TBS with 5% nonfat milk for 1 h, and incubated with secondary antibody for 1 h. Blots were developed using enhanced chemiluminescence (ECL+; Amersham Life Science Products) and analyzed using Scion image.

Statistical analysis. Statistical significance (alpha  = 0.05) for differences in Rbrain and protein expression of occludin, ZO-1, and immunoprecipitants was determined by one-way ANOVA followed by Newman-Keuls post hoc test. Two-way ANOVA followed by Tukey's honestly significant difference post hoc analysis was performed to determine statistical significance (alpha  = 0.05) and interaction in the capillary depletion studies. Data are expressed as means ± SE.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In situ brain perfusion. The effects of lambda -carrageenan-induced inflammation on basal permeability across an intact BBB were assessed from 0 to 72 h using in situ perfusion of the brain with [14C]sucrose, a membrane-impermeant marker. Visual inspection of the brain immediately after in situ perfusion showed no influx of Evans blue albumin into the brain parenchyma.

Figure 1 shows Rbrain at time points ranging from 0 to 72 h. The control (0 h) Rbrain value of 1.8 ± 0.2, representative of vascular space volume, was converted to a vascular space of 17.9 µl/g brain tissue. Results demonstrate a biphasic response with a significantly higher distribution of sucrose in the brain at 1, 3, 6, and 48 h [139 ± 9%, 166 ± 19%, 138 ± 13%, and 146 ± 7% compared with control, respectively]. Capillary depletion data after the 20-min in situ brain perfusions showed the amount of [14C]sucrose trapped in the pellet at the various time points was not significantly different from control (Table 1). Furthermore, the study revealed the percent amount of [14C]sucrose associated with actual entry into the brain parenchyma was not statistically different from the homogenate and the amount of radioactivity associated with the pellet was significantly lower (P < 0.01) than that associated with the homogenate (Table 1).


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  		Fig. 1.   Time course of [14C]sucrose blood-brain barrier (BBB) permeability after lambda -carrageenan-induced inflammatory pain using a 20-min in situ brain perfusion. Results indicate a significantly higher distribution of sucrose in the cerebral hemispheres at 1, 3, 6, and 48 h compared with the control (0 h). Rbrain, ratio of radioactivity found in brain parenchyma compared with radioactivity found in perfusate media. Each bar represents the mean ± SE; n = 6. Statistical significance was determined using one-way ANOVA, followed by Newman-Keuls post hoc test. *P < 0.05 and **P < 0.01 versus control.


                              
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  		Table 1.   Capillary depletion studies after a 20-min in situ brain perfusion

Immunoprecipitation and immunoblotting of tight junction proteins. Western blot analyses indicated an alteration in expression of tight junctional proteins after lambda -carrageenan induced-inflammatory pain. Figure 2 shows changes in occludin expression after 0- to 72-h treatments. Total occludin expression was significantly reduced at 1, 3, 6, 12, and 48 h [62 ± 7%, 39 ± 10%, 26 ± 3%, 63 ± 8%, and 38 ± 6% of control (0 h), respectively]. Figure 2 also shows occludin migrates as two bands, referred to as alpha  and beta  (3). During the initial phase of inflammation (0-6 h), the decrease in occludin expression was primarily due to a decrease in the beta -band. The second phase (12-72 h) showed a decrease in the alpha -band with a concomitant increase in beta -band expression; an exception was at 48 h, where both alpha - and beta -bands decreased. Table 2 illustrates the percent difference in occludin expression in the alpha - and beta -bands from 1 to 72 h compared with the control (0 h).


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  		Fig. 2.   Western blot analyses indicate alterations in occludin expression after lambda -carrageenan-induced inflammatory pain. Total occludin expression was significantly reduced at 1, 3, 6, 12, and 48 h (62 ± 7%, 39 ± 10%, 26 ± 3%, 63 ± 8%, and 38 ± 6%, respectively) compared with the control (0 h). Inset, representative blot image showing occludin migrating as two distinct bands, referred to as alpha  and beta . Statistical significance was determined using one-way ANOVA, followed by Newman-Keuls post hoc test. *P < 0.05 and **P < 0.01 versus control.


                              
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  		Table 2.   Percent relative decrease in occludin expression in the alpha - and beta -bands compared with controls after induction of lambda -carrageenan inflammatory pain

Figure 3 shows the alterations in ZO-1 expression during 0-72 h of lambda -carrageenan-induced inflammatory pain. ZO-1 was significantly increased (P < 0.01) at 1, 3, and 6 h [377 ± 76%, 235 ± 17%, and 217 ± 25% of control (0 h), respectively] and returned to control expression levels by 12 h.


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  		Fig. 3.   Western blot analyses indicate alterations in zonula occludens (ZO)-1 expression after lambda -carrageenan-induced inflammatory pain. ZO-1 expression was significantly increased at 1, 3, and 6 h (377 ± 76%, 235 ± 17%, and 217 ± 25%, respectively) compared with the control (0 h). Inset, representative blot image showing ZO-1 migrating at 220 kDa. Statistical significance was determined using one-way ANOVA, followed by Newman-Keuls post hoc test. *P < 0.05 and **P < 0.01 versus control.

Table 3 depicts the coimmunoprecipitation of associated tight junctional and cytoskeletal proteins with occludin and ZO-1. Occludin, ZO-2, and actin precipitated with ZO-1. ZO-1 association with ZO-2 increased during the period from 1 to 24 h, whereas ZO-1 association with actin (1-24 h) and occludin (1-6 h) decreased. Results also indicate that ZO-2 did not show a significant change in association with occludin and actin did not immunoprecipitate to any detectable amount with occludin (Table 2).

                              
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  		Table 3.   Percent change in protein expression compared with control of proteins immunoprecipitated with ZO-1 or occludin


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In a previous study, we showed that peripheral inflammatory pain models increased BBB permeability and altered tight junctions at peak inflammation using the formalin-, lambda -carrageenan-, and complete Freund's adjuvant-induced pain models (24). This study further investigated the effects of lambda -carrageenan-induced inflammatory pain on the functional and structural integrity of BBB tight junctions over the time course (0-72 h) of inflammation. The lambda -carrageenan-induced pain model was chosen due to its onset of action and duration of effects compared with both the formalin and CFA-induced pain models. The effects of lambda -carrageenan-induced inflammation on basal permeability across an intact BBB were assessed using in situ brain perfusion with [14C]sucrose. The control vascular space volume of 17.9 µl/g brain tissue was similar to that in our previous study (17.1 µl/g brain tissue) (24) and consistent with other studies using vascular space markers (7, 22, 48). lambda -Carrageenan-induced inflammation elicited a biphasic increase in BBB permeability at 1-6 h and at 48 h. The increase seen in this study from 1 to 6 h was consistent with our previous findings at 3 h (24) and demonstrates the maximal increase in BBB permeability coincides with maximal inflammatory response (33, 50).

Previous studies (13, 28, 34) investigating changes in BBB permeability have found evidence of increased vesicular transport. Therefore, to investigate the possible contribution of increased vesicular activity to the increased association of sucrose with the brain, capillary depletion studies were conducted. These studies showed the amount of sucrose trapped in the vascular pellet at each time point was not statistically different from the control and the amount of radioactivity associated within the pellet was not significantly different among time points compared with the control. These results suggest the increased BBB permeability observed in this study was not due to changes in vascular volume (i.e., changes in cerebral blood flow, vasoconstriction/dilatation) or increased vascular trapping (i.e., increased endocytotic activity), thereby indicating that lambda -carrageenan-induced inflammatory pain significant increased BBB permeability, most likely via increased paracellular diffusion between brain microvascular endothelial cells.

To investigate this point, we examined the expression of tight junctional proteins, ZO-1 and occludin, to determine whether the increase in BBB paracellular permeability was correlated with alterations in tight junctional structural integrity. In the current study, we investigated ZO-1 expression over the time course (0-72 h) of lambda -carrageenan-induced inflammatory pain. ZO-1 expression was increased during the first phase of the inflammatory process (1-6 h) and returned to basal levels by 12 h (Fig. 3). ZO-1 expression is not altered at 48 h, although BBB permeability increased. Previous studies have shown that ZO-1 is phosphorylated on tyrosine and serine/threonine residues (5, 40, 41), but the effect of phosphorylation on tight junctional physiology remains unclear. Several studies (3, 4, 47) have shown that tyrosine phosphorylation of ZO-1 increases paracellular permeability. However, other studies indicate tyrosine phosphorylation of ZO-1 is important for tight junction assembly and establishment of barrier resistance (31, 46). These differing studies reflect the complexity of formation and maintenance of tight junctions and may be a result of diverse signaling pathways.

To further evaluate the possible role of tight junctional proteins in reorganization of the tight junction after an inflammatory insult, we immunoprecipitated with antibodies to ZO-1 and occludin and probed for associated proteins. Table 3 shows the association between ZO-1 and actin significantly decreased at 3, 6, 12, and 24 h but was significantly increased at 48 and 72 h. In contrast, the association between ZO-1 and ZO-2 was significantly increased at 1, 3, 6, 12, and 24 h and decreased at 48 and 72 h. These findings are very interesting and bring up many questions regarding the dynamics of tight junction reorganization. As BBB permeability increases, ZO-1 appears to be less tightly associated with actin and more tightly associated with ZO-2, perhaps indicating a disruption between the tight junction scaffold and the cytoskeleton.

Occludin plays a dynamic, functional role in regulating tight junction integrity during lambda -carrageenan-induced inflammation. Numerous phosphorylation sites allow occludin to rapidly respond to environmental stimuli (3, 23, 39, 46). Our data demonstrated time-dependent changes in occludin expression from 0 to 72 h, with statistically significant reductions in occludin expression at the same time as increased BBB permeability was observed (i.e., 1, 3, 6, and 48 h). As has been previously shown, reductions in occludin expression decrease paracellular permeability, resulting in an increased flux between BBB endothelial cells (14, 47).

Phosphorylation of occludin regulates tight junction function by redistributing occludin from the cytoplasm to the lateral surface of the plasma membrane (2, 17). Phosphorylation of occludin occurs at both tyrosine and serine/threonine sites and correlates with permeability changes in existing tight junctions and assembly of new tight junctions (16, 45). Two migrating bands recognized by anti-occludin antibodies, referred to as alpha  and beta  by Antonetti et al. (3), demonstrated evidence for a change in occludin posttranslational modification. The alpha -band migrates at 60 kDa, and the beta -band migrates at 62 kDa. In this study, occludin migrated most strongly in the beta -band, which has been characterized as posttranslationally modified (3). As Table 2 depicts, the loss of occludin expression occurs primarily in the beta -band at 1, 3, 6, and 48 h. During the latter portion of the time course, most of the decreased expression occurred in the alpha -band. During periods of increased BBB permeability, occludin expression in the beta -band decreased, whereas during periods showing improved BBB function, occludin expression decreased primarily from the alpha -band, suggesting that occludin may redistribute from the alpha -band to the beta -band during reassembly of barrier function.

Although the increase in BBB permeability was relatively small (~66% increase over the 20-min period at 3 h) in magnitude, the implications of these increases are physiologically significant. Generally, we would not expect a several-fold increase in BBB permeability, as seen after osmotic disruption (~300% increase in BBB permeability after 1.6 mM mannitol infusion), following a peripheral inflammatory insult. If this were the case, the BBB would become compromised after every inflammation or infection. Rather, our primary concern centers on disruption of CNS homeostasis and proper neuronal function. Several CNS pathologies, including human immunodeficiency virus-1 encephalitis (12), multiple sclerosis (36), hypoxia/aglycemia (1), cerebral malaria (8), epilepsy (25), and bacterial meningitis (27), have shown a correlation between increased BBB permeability and altered CNS homeostasis and neuronal function. Furthermore, there are numerous therapeutic agents used in the management of illnesses with a peripheral pain component with molecular masses similar to that of sucrose (342 Da), such as morphine (285 Da), codeine (300 Da), acetaminophen (150 Da), methotrexate (454 Da), fluoxetine (320 Da), amitripyline (278 Da), and cyclobenzaprine (276 Da), whose transport into the CNS may be different than seen in healthy individuals. However, caution must be taken in extrapolating these findings to larger therapeutic agents, because the exact size of the BBB opening is not yet known.

In summary, we show that the lambda -carrageenan-induced inflammatory pain model elicited a biphasic increase in BBB permeability, with an initial phase occurring from 1 to 6 h and a second phase at 48 h. Furthermore, changes in BBB permeability correlate with changes in the tight junction occludin expression and modified protein-protein interactions between ZO-1 and occludin, ZO-2, and actin. The exact mechanisms by which these changes occur are still unknown; however, evidence clearly supports the idea that changes in tight junctional organization play a role in increased BBB paracellular permeability. These findings suggest that the lambda -carrageenan-induced inflammatory pain model produces alterations in BBB function that may affect CNS homeostasis and have important clinical ramifications concerning therapeutic drug delivery and drug dosing regimens during pain. Future studies will focus on regional differences in BBB perturbations and begin elucidating the central and peripheral components responsible for the changes observed in this study.


    ACKNOWLEDGEMENTS

This study was funded by National Institutes of Health Grants NS-42652, NS-39592, and DA-06037.


    FOOTNOTES

Address for reprint requests and other correspondence: T. P. Davis, Dept. of Pharmacology, Univ. of Arizona College of Medicine, Tucson, AZ 85724 (E-mail: davistp{at}u.arizona.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

June 21, 2002;10.1152/ajpheart.00027.2002

Received 18 February 2002; accepted in final form 12 June 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Abbruscato, TJ, and Davis TP. Combination of hypoxia/aglycemia compromises in vitro blood-brain barrier integrity. J Pharmacol Exp Ther 289: 668-675, 1999[Abstract/Free Full Text].

2.   Andreeva, AY, Krause E, Muller EC, Blasig IE, and Utepbergenov DI. Protein kinase C regulates the phosphorylation and cellular localization of occludin. J Biol Chem 276: 38480-38486, 2001[Abstract/Free Full Text].

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Am J Physiol Heart Circ Physiol 283(4):H1531-H1537
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