Chronic inflammatory pain leads to increased blood-brain barrier permeability and tight junction protein alterations

Abstract

The blood-brain barrier (BBB) maintains brain homeostasis by limiting entry of substances to the central nervous system through interaction of transmembrane and intracellular proteins that make up endothelial cell tight junctions (TJs). Recently it was shown that the BBB can be modulated by disease pathologies including inflammatory pain. This study examined the effects of chronic inflammatory pain on the functional and molecular integrity of the BBB. Inflammatory pain was induced by injection of complete Freund's adjuvant (CFA) into the right plantar hindpaw in female Sprague-Dawley rats under halothane anesthesia; control animals were injected with saline. Edema and hyperalgesia were assessed by plethysmography and infrared paw-withdrawal latency. At 72 h postinjection, significant edema formation and hyperalgesia were noted in the CFA-treated rats. Examination of permeability of the BBB by in situ perfusion of [14C]sucrose while rats were under pentobarbital anesthesia demonstrated that CFA treatment significantly increased brain sucrose uptake. Western blot analysis of BBB TJ proteins showed no change in expression of zonula occludens-1 (an accessory protein) or actin (a cytoskeletal protein) with CFA treatment. Expression of the transmembrane TJ proteins occludin and claudin-3 and -5 significantly changed with CFA treatment with a 60% decrease in occludin, a 450% increase in claudin-3, and a 615% increase in claudin-5 expression. This study demonstrates that during chronic inflammatory pain, alterations in BBB function are associated with changes in specific transmembrane TJ proteins.

Fig. 1

Edema established with injection of complete Freund’s adjuvant (CFA). Percent change in paw volume at 24, 48, and 72 h postinjection of saline or CFA. CFA caused significant edema at each time measured in only the ipsilateral paw compared with saline; *P < 0.001. For comparison purposes, the contralateral paw of the CFA-injected animal as well as both paws of the saline-injected animal were also measured and demonstrated no volume change; n ≥ 20 rats/treatment.

Fig. 2

Hyperalgesia established with CFA. Hyperalgesia was measured as paw-removal latency (in seconds) from infrared heat source. At 24, 48, and 72 h postinjection, CFA caused a significant decrease in removal latency compared with saline; *P < 0.001; n ≥ 20 rats/treatment.

Fig. 3

Multiple time [¹⁴C]sucrose uptake. In this study, [¹⁴C]sucrose was used as a marker of blood-brain barrier paracellular permeability. Values are mean brain radioactivity (R_br) ± SE; n = 4–8 rats/time point. CFA significantly increased the rate of [¹⁴C]sucrose uptake into the brain (K_in) without significantly altering its initial vascular distribution (V_D).

Fig. 4

Tight junction (TJ) protein expression. Expression of various TJ proteins was measured 72 h post-CFA injection (n ≥ 6 rats). CFA was not found to alter the expression of the accessory TJ protein zonula occludens (ZO)-1 or the cytoskeletal support protein actin. Western blots showing protein expression demonstrate representative results from at least triplicate independent experiments. Optical density of each protein band was measured and normalized to saline control expression on each membrane. Expression of the transmembrane protein occludin was significantly decreased (*P = 0.001). Expression of both claudin-3 and -5 was significantly increased (**P < 0.001). Gels were stained with Coomassie blue as a loading control.

Fig. 5

Localization of TJ proteins. Representative micrographs of changing blood-brain barrier TJ proteins occludin and claudin-3 and -5; n ≥ 6 rats. Data represent minimally triplicate independent and separate experiments. No changes in localization were noted with claudin-3 or -5. Occludin, however, was noted to change from a continuous localization to a more punctate distribution (white arrows).