Expression Levels Of Tight Junction Proteins Research Articles

Enhancement of blood-brain barrier permeability and reduction of tight junction protein expression are modulated by chemokines/cytokines induced by rabies virus infection.

Infection with laboratory-attenuated rabies virus (RABV) enhances blood-brain barrier (BBB) permeability, which has been demonstrated to be an important factor for host survival, since it allows immune effectors to enter the central nervous system (CNS) and clear RABV. To probe the mechanism by which RABV infection enhances BBB permeability, the expression of tight junction (TJ) proteins in the CNS was investigated following intracranial inoculation with laboratory-attenuated or wild-type (wt) RABV. BBB permeability was significantly enhanced in mice infected with laboratory-attenuated, but not wt, RABV. The expression levels of TJ proteins (claudin-5, occludin, and zonula occludens-1) were decreased in mice infected with laboratory-attenuated, but not wt, RABV, suggesting that enhancement of BBB permeability is associated with the reduction of TJ protein expression in RABV infection. RABV neither infects the brain microvascular endothelial cells (BMECs) nor modulates the expression of TJ proteins in BMECs. However, brain extracts prepared from mice infected with laboratory-attenuated, but not wt, RABV reduced TJ protein expression in BMECs. It was found that brain extracts from mice infected with laboratory-attenuated RABV contained significantly higher levels of inflammatory chemokines/cytokines than those from mice infected with wt RABV. Pathway analysis indicates that gamma interferon (IFN-γ) is located in the center of the cytokine network in the RABV-infected mouse brain, and neutralization of IFN-γ reduced both the disruption of BBB permeability in vivo and the downregulation of TJ protein expression in vitro. These findings indicate that the enhancement of BBB permeability and the reduction of TJ protein expression are due not to RABV infection per se but to virus-induced inflammatory chemokines/cytokines. Previous studies have shown that infection with only laboratory-attenuated, not wild-type, rabies virus (RABV) enhances blood-brain barrier (BBB) permeability, allowing immune effectors to enter the central nervous system (CNS) and clear RABV from the CNS. This study investigated the mechanism by which RABV infection enhances BBB permeability. It was found that RABV infection enhances BBB permeability by downregulation of tight junction (TJ) protein expression in the brain microvasculature. It was further found that it is not RABV infection per se but the chemokines/cytokines induced by RABV infection that downregulate the expression of TJ proteins and enhance BBB permeability. Blocking some of these cytokines, such as IFN-γ, ameliorated both the disruption of BBB permeability and the downregulation of TJ protein expression. These studies may provide a foundation for developing therapeutics for clinical rabies, such as medication that could be used to enhance BBB permeability.

Effects of selective hypothermia on blood-brain barrier integrity and tight junction protein expression levels after intracerebral hemorrhage in rats

Hypothermia has neuroprotective effects on global cerebral ischemic injuries. However, its efficacy after intracerebral hemorrhage (ICH) is inconclusive. In this study, bacterial collagenase was used to induce ICH stroke in male Wistar rats. We assessed the effects of normothermia and 4 h of local hypothermia (~33.2°C) initiated 1, 6, or 12 h after collagenase infusion on hemorrhage volume and neurological outcomes. Following early cooling initiated after 1 h, blood-brain barrier (BBB) disruption and brain water content were tested. Furthermore, the expression levels of tight junction (TJ) proteins (claudin 5 and occludin) and the proinflammatory cytokines interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α) were determined using Western blotting, real-time quantitative PCR, and immunohistochemical staining at 1 and 3 d after ICH. Early local hypothermia tends to reduce hemorrhagic volume and neurological deficits, but the difference is not statistically significant compared with other groups. However, early hypothermia significantly reduces BBB disruption, edema formation, the expression levels of IL-1β and TNF-α, and the loss of TJ proteins. Together, these data suggest that local hypothermia is an effective treatment for edema formation and BBB disruption via the upregulation of TJ proteins and the suppression of TNF-α and IL-1β.

Increases in Bioavailability of Poorly Absorbed Drug by Acylcarnitine

We examined the effect of acylcarnitines on the in situ bioavailability of lucifer yellow (LY) from the loops of small and large intestines of rats. The area under the blood concentration of LY versus time curve (AUC) from the jejunum was significantly increased by the treatments of the loop with 100 µM lauroylcarnitine (LC) or 100 µM palmitoylcarnitine (PC) (fourfold and 17-fold, respectively). No marked change in the expression of claudin-4 protein was observed by the treatments. On the contrary, the expression of P-glycoprotein (P-gp) was decreased by the treatment, more greatly by PC than by LC, suggesting that increases in the bioavailability of LY by LC and PC are associated with the decreased expression of P-gp in jejunum. The increase in the bioavailability was also observed for colon by the treatment of LC, but not that of PC. LC decreased the expression of claudin-4 protein, whereas PC decreased the expression of P-gp in colon. Therefore, LC and PC appear to have different impact on the intestinal transporters depending on the site (i.e., jejunum and colon).

The Effects of Electromagnetic Pulse on the Protein Levels of Tight Junction Associated-Proteins in the Cerebral Cortex, Hippocampus, Heart, Lung, and Testis of Rats

Objective To investigate changes in the expression of tight junction (TJ) proteins in the cerebral cortex, hippocampus, heart, lung, and testes of rats after exposure to electromagnetic pulse (EMP). Methods Eighteen adult male Sprague-Dawley rats were divided into sham and exposure groups. The exposure groups received EMP at 200 kV/m for 200 pulses with a repetition rate of 1 Hz. The expression of TJ proteins (ZO-1, occludin, actin) in the several organs was examined by western blotting. Results ZO-1 levels in the cerebral cortex decreased 1 h and 3 h after EMP exposure compared with sham group ( P<0.05). No significant difference was observed for occludin and actin. ZO-1 levels in the hippocampus increased 1 h and 3 h post-exposure ( P<0.05), and occludin decreased after 3 h ( P<0.05); however, actin was unaffected. ZO-1 levels in the heart increased 3 h post-exposure ( P<0.05), occludin decreased 3 h post-exposure ( P<0.05), and actin increased 1 h and 3 h post-exposure ( P<0.05). ZO-1, occludin and actin levels in the lung decreased compared with those in the sham group ( P<0.05). ZO-1 and occludin levels in the testes decreased 1 h and 3 h post-exposure ( P<0.05), but actin showed no significant change. Conclusion Exposure to EMP altered the expression levels of TJ proteins, particularly ZO-1, in the organs of adult male rats, which may induce changes in barrier structure and function.

Interleukin-6, desmosome and tight junction protein expression levels in reflux esophagitis-affected mucosa

To investigate the correlation between the expression levels of interleukin (IL)-6 and proteins in tight junctions (TJs) in the esophageal mucosa of rats modeling different types of reflux esophagitis (RE), and the ability of aluminum phosphate to protect against RE-induced mucosal damage via these proteins. Male SPF Wistar rats aged 56 d were divided randomly into acid RE, alkaline RE, mixed RE, and control groups. Various surgical procedures were performed to establish rat models of acid RE. At 14 d after the procedure, some of the rats started aluminum phosphate treatment. Transmission electron microscopy (TEM) was used to observe the morphological features of TJs and desmosomes in the esophageal epithelium. Immunohistochemical methods and Western blotting were used to measure expression of claudin 1, occludin, ZO-1, JAM-1, DSG-1 and IL-6; reverse transcription polymerase chain reaction (RT-PCR) was used to measure expression of mRNA of claudin 1, occludin, ZO-1, JAM-1, DSG-1 and IL-6. At day 14 after the procedures, an RE model was established in all subsequently sacrificed rats of groups A, B and C. By both gross and microscopic observation, the mucosa was damaged and thickened as the disease progressed. With TEM observation, a widened intercellular space was noticed, with significantly fewer desmosomes. Immunohistochemistry showed significantly higher levels of all proteins in all RE models compared to control rats at 3 d after operation (65.5% +/- 25.6% vs 20.5% +/- 2.1%, P < 0.05, respectively). At 14 d after operation, along with continuing hyperplasia in the basal layer, the expression of TJ proteins in individual cells gradually decreased (12.4% +/- 2.1% vs 20.5% +/- 2.1%, P < 0.05, respectively). Western blottings and RT-PCR showed a directly proportional increase in IL-6 levels in relation to TJ proteins, as compared to controls (0.878 +/- 0.024 vs 0.205 +/- 0.021 and 0.898 +/- 0.022 vs 0.205 +/- 0.021, P < 0.05, respectively). Upon treatment with aluminum phosphate, however, these protein levels were restored to normal gradually over 30-60 d in rats with acid RE (30.4% +/- 2.1% vs 20.5% +/- 2.1%, P > 0.05, treated vs untreated, respectively). These levels increased in the rat with alkaline RE, and this increase was accompanied by continued hyperplasia in comparison with controls (85.5% +/- 25.6% vs 20.5% +/- 2.1%, P < 0.05, respectively). Furthermore, the expression of TJ proteins was not correlated significantly with that of IL-6 in this group. These findings indicate that TJ proteins are highly expressed as an early molecular event involved in RE development, and that IL-6 is an inflammatory factor in this process.

Caffeine protects against MPTP‐induced blood‐brain barrier dysfunction in mouse striatum

The blood-brain barrier (BBB) is important physiologically. Pathologically, BBB disruption has been implicated in a wide spectrum of neurological disorders including Parkinson's disease (PD). Recent studies indicate that caffeine is protective against PD, but by poorly understood mechanisms. Using a MPTP neurotoxin model of PD we tested the hypothesis that the protective actions of caffeine were because of, at least in part, preventing MPTP-induced BBB dysfunction. FVB mice were pre-treated with caffeine (10 mg/kg, i.p.) or saline for 7 days prior to initiation of neurotoxin treatments; during the 7 days of neurotoxin treatment, caffeine or saline continued to be administered 10 min before each dose of MPTP (20 mg/kg, i.p.). Striatum (and for some studies hippocampus and cerebral cortex as well) were evaluated for BBB leakage, tight junction protein expression levels, integrity of dopaminergic neurons, and activation of astrocytes and microglia using immunostaining, immunoblotting and real-time PCR techniques. We found that caffeine blocked MPTP-induced decreases in numbers of tyrosine hydroxylase-positive dopaminergic neurons, increases in leakage of Evan's blue dye and FITC-albumin in striatum but not in cerebral cortex or hippocampus, decreases in levels of the tight junction proteins occludin and ZO-1, and increases in reactive gliosis. Our results suggest that caffeine might protect against PD and PD-like features in animal models, in part, by stabilizing the BBB.

Caffeine blocks disruption of blood brain barrier in a rabbit model of Alzheimer's disease.

High levels of serum cholesterol and disruptions of the blood brain barrier (BBB) have all been implicated as underlying mechanisms in the pathogenesis of Alzheimer's disease. Results from studies conducted in animals and humans suggest that caffeine might be protective against Alzheimer's disease but by poorly understood mechanisms. Using rabbits fed a cholesterol-enriched diet, we tested our hypothesis that chronic ingestion of caffeine protects against high cholesterol diet-induced disruptions of the BBB. New Zealand rabbits were fed a 2% cholesterol-enriched diet, and 3 mg caffeine was administered daily in drinking water for 12 weeks. Total cholesterol and caffeine concentrations from blood were measured. Olfactory bulbs (and for some studies hippocampus and cerebral cortex as well) were evaluated for BBB leakage, BBB tight junction protein expression levels, activation of astrocytes, and microglia density using histological, immunostaining and immunoblotting techniques. We found that caffeine blocked high cholesterol diet-induced increases in extravasation of IgG and fibrinogen, increases in leakage of Evan's blue dye, decreases in levels of the tight junction proteins occludin and ZO-1, increases in astrocytes activation and microglia density where IgG extravasation was present. Chronic ingestion of caffeine protects against high cholesterol diet-induced increases in disruptions of the BBB, and caffeine and drugs similar to caffeine might be useful in the treatment of Alzheimer's disease.