Athanasia Bletsa

Cytokine signalling in rat pulp interstitial fluid and transcapillary fluid exchange during lipopolysaccharide-induced acute inflammation

The dental pulp consists of loose connective tissue encased in rigid dentinal walls. Because of its topography the tissue has low interstitial compliance and limited capacity to expand during fluid volume changes. Due to limitations regarding access to interstitial fluid, basic knowledge on transcapillary fluid transport parameters is lacking for this organ. The scope of this project was dual: first we aimed at establishing a method for isolation of pulp interstitial fluid (IF), and second we applied the method in rats subjected to lipopolysaccharide (LPS)‐induced endotoxaemia. The aim was to measure colloid osmotic pressure (COP) and pro‐inflammatory cytokines in the pulp IF during acute inflammation. Fluid volumes and pulpal blood flow (PBF) were measured to obtain more information about microcirculatory changes that take place in this pulpitis model. By centrifugation of incisor pulp at 239 g we were able to extract fluid representative for IF. Pulp IF had a relative high control COP (∼83% of plasma COP) and was similar to plasma COP 3 h after LPS challenge. The pulp exhibited a high content of IF (0.60 ± 0.03 ml (g wet weight)−1) and a vascular volume of 0.03 ± 0.01 ml (g w.w.)−1 No differences were observed in the distribution of fluid volumes after 1.5 and 3 h LPS exposure. PBF and systemic blood pressure dropped significantly after LPS administration. PBF remained low whereas systemic blood pressure was re‐established during the 3‐h period, implying organ dysfunction. There was a differential pattern of cytokine expression in pulp IF and serum with cytokines such as IL‐1α, IL‐1β and TNF‐α locally produced, whereas others such as IFN‐γ and IL‐6 were produced systemically and probably spilled over to the pulp IF after LPS exposure. Our findings show that pulp IF can be isolated by centrifugation and that this method is useful when studying fluid balance and extracellular signalling mechanisms in the dental pulp in normal and pathological conditions.

Cytokines are produced locally by myocytes in rat skeletal muscle during endotoxemia.

Cytokines act as chemical mediators during the inflammatory process. Measurements of cytokine levels in tissue have previously been performed in homogenized tissue, but the true concentrations in native interstitial fluid (ISF), i.e., the compartment where cytokines exert their biologically active role, have remained unknown. The role of skeletal muscle myocytes as a source for cytokines during endotoxemia was explored by collecting muscle ISF using a wick method, and the levels of 14 cytokines in ISF and plasma were related to the corresponding changes in mRNA levels to reveal any potential discrepancies between gene expression and protein release of cytokines to ISF. The majority of investigated cytokines were elevated in muscle ISF during endotoxemia, and an analysis of cytokine mRNA levels revealed consistency between gene expression and protein release. The elevated cytokine level in ISF, in addition to elevated gene expression in muscle, indicated a significant local production and release of several proinflammatory cytokines and chemokines within skeletal muscle tissue during endotoxemia. Immunohistochemistry revealed that myocytes constituted a significant source of IL-1beta and TNF-alpha production during endotoxemia, whereas the contribution from inflammatory cells i.e., leukocytes, was found to be less significant. Muscle cells apparently constitute an important source of several different cytokines during endotoxemia, governing the level in the muscle microenvironment, and are likely to contribute significantly to cytokine levels in plasma.

IL-1α and TNF-α Expression in Rat Periapical Lesions and Dental Pulp after Unilateral Sympathectomy

Objectives: Apical periodontitis is an inflammatory disease characterized by bone resorption, and sympathetic nerves are known to modulate bone resorption and bone remodeling. Higher numbers of osteoclasts and larger periapical lesions have been observed after sympathectomy in rats, but the mechanisms underlying the inhibitory effect of sympathetic nerves on osteoclasts are unknown. This study aimed to test the hypothesis that sympathetic nerves inhibit the production of the bone-resorbing pro-inflammatory cytokines IL-1α and TNF-α in rat periapical lesions. Methods: Rats were unilaterally sympathectomized and apical lesions were induced by exposing the dental pulp of molar teeth to the oral microflora. We quantified the cytokines IL-1α and TNF-α by enzyme-linked immunosorbent assay, and immunohistochemical analysis was done for qualitative localization. Pulp from intact incisor teeth was tested as a control. Results: We showed that IL-1α was increased, but not TNF-α, in the periapical lesions on the sympathectomized side. Both IL-1α and TNF-α were expressed in unexposed pulp. TNF-α was significantly decreased in the denervated incisor pulp, whereas the level of IL-1α remained unchanged. Conclusions: This study suggests that sympathetic nerves have an inhibitory effect on IL-1α in periapical lesions and a stimulatory effect on TNF-α in the intact rat pulp.

Vascular Endothelial Growth Factors and Receptors Are Up-regulated during Development of Apical Periodontitis

INTRODUCTION Apical periodontitis is a common inflammatory disease caused by persistent root canal infection and is characterized by bone resorption. Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) have been described in many pathologic and inflammatory conditions, but their involvement in the development of apical periodontitis has not been thoroughly investigated. The aim of this study was to quantify gene expression and localize VEGF-A, VEGF-C, and VEGF-D and VEGFR-2 and VEGFR-3 in a rat model of apical periodontitis. METHODS Molar pulps were unilaterally exposed to the oral cavity for 10 or 21 days. Jaw sections were used for localization of VEGFs and VEGFRs with immunohistochemistry and identification of cells with double immunofluorescence. Gene expression analysis for VEGF-A, VEGF-C, and VEGFR-3 of periapical tissues was performed with quantitative real-time polymerase chain reaction. RESULTS All investigated factors and receptors were expressed immunohistochemically in blood vessels at the periodontal ligament of control teeth and were up-regulated during lesion development. In apical lesions, macrophages and neutrophils expressed all studied factors and receptors, with macrophages being an important source of VEGF-C and VEGF-D. Osteoclasts expressed VEGFR-2 and VEGFR-3, and the latter was also identified in fibroblast-like cells in the lesions. VEGF-A and VEGFR-3 gene expression was up-regulated at days 10 and 21 (P < .05). CONCLUSIONS The current findings indicate that the VEGF family and receptors are involved in vascular remodeling and immune functions during disease development. The presence of VEGFR-2 and VEGFR-3 on osteoclasts indicates that bone resorbing activity is influenced by VEGFs.

Sensory pulpal nerve fibres and trigeminal ganglion neurons express IL-1RI: a potential mechanism for development of inflammatory hyperalgesia

AIM To localize interleukin-1 receptor type I (IL-1RI) in rat dental pulp and trigeminal ganglion (TG) and to test the hypothesis that pulpal inflammation increases neuronal expression of IL-1RI. METHODOLOGY Female Wistar rats were subjected to unilateral pulp exposures in the maxillary and mandibular first molars, whereas the contralateral jaws served as untreated controls. Seven days later the animals were transcardiacally perfused and the jaws and the TGs were removed and prepared for immunohistochemistry. Immunoreactivity for IL-1RI was examined alone (DAB) and together with calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY), CD31 or CD34 by multiple-labelling immunofluorescence. Quantification of IL-1RI-immunoreactive (-IR) cells in the maxillary and mandibular division of the ganglion was performed in parasagittal immunoreacted sections of the right and left TGs. Data were analysed with Mann-Whitney Rank Sum test (P < 0.05). RESULTS Interleukin-1 receptor type I was found on sensory (CGRP-IR) and sympathetic (NPY-IR) nerve fibres and on blood vessels (CD31- and CD34-IR) in the dental pulp. It was also localized on sensory neurons and axons in the TG. Pulpal inflammation significantly increased the expression of IL-1RI in the TG (P < 0.001). CONCLUSIONS The localization of IL-1RI on sensory nerve fibres and its up-regulation in TG neurons during pulpal inflammation may imply a direct effect of IL-1 in pulpal nociception. The presence of IL-1RI on sympathetic nerve fibres and on blood vessels may indicate a vasoactive role of the same cytokine in the pulp.

Isolation of rat trachea interstitial fluid and demonstration of local cytokine production in lipopolysaccharide-induced systemic inflammation

Access to interstitial fluid from trachea is important for understanding tracheal microcirculation and pathophysiology. We tested whether a centrifugation method could be applied to isolate this fluid in rats by exposing excised trachea to G forces up to 609 g. The ratio between the concentration of the equilibrated extracellular tracer 51Cr-labeled EDTA in fluid isolated at 239 g and plasma averaged 0.94 +/- 0.03 (n = 14), suggesting that contamination from the intracellular fluid phase was negligible. The protein pattern of the isolated fluid resembled plasma closely and had a protein concentration 83% of that in plasma. The colloid osmotic pressure in the centrifugate in controls (n = 5) was 18.8 +/- 0.6 mmHg with a corresponding pressure in plasma of 22 +/- 1.5 mmHg, whereas after overhydration (n = 5) these pressures fell to 9.8 +/- 0.4 and 11.9 +/- 0.4 mmHg, respectively. We measured inflammatory cytokine concentration in serum, interstitial fluid, and bronchoalveolar lavage fluid in LPS-induced inflammation. In control animals, low levels of IL-1 beta, IL-6, and TNF-alpha in serum, trachea interstitial fluid, and bronchoalveolar lavage fluid were detected. LPS resulted in a significantly higher concentration in IL-1 beta and IL-6 in interstitial fluid than in serum, showing a local production. To conclude, we have shown that interstitial fluid can be isolated from trachea by centrifugation and that trachea interstitial fluid has a high protein concentration and colloid osmotic pressure relative to plasma. Trachea interstitial fluid may also reflect lower airways and thus be of importance for understanding, e.g., inflammatory-induced airway obstruction.

Lymphangiogenesis Is Induced during Development of Periodontal Disease

Lymphangiogenesis, the formation of new lymphatics, is associated with chronic inflammation and tissue injury, and its role is to enhance lymphatic flow, immune cell transport, and antigen clearance. It is unknown if lymphangiogenesis takes place during periodontal disease development, and we hypothesized that growth of lymphatic vessels occurs in gingiva during development of periodontitis in mice. Inflammation was induced in gingiva with Porphyromonas gingivalis gavage, and bone resorption was verified after 42 days. Growth of lymphatic and blood vessels was measured after immunofluorescent staining with LYVE-1 and CD31. Expression of vascular endothelial growth factors and 2 inflammatory cytokines was investigated 10 days post-infection. Gingival lymphangiogenesis was found 10 days and 42 days post-infection, but proliferation of vessels was observed only in the shortest observation period. Epithelial expression of vascular growth factors (VEGF) A, C, and D was observed in gingiva, and increased numbers of immune cells expressing VEGF-C were found after infection, along with up-regulation of IL-1β and TNF-α at protein levels. We conclude that lymphangiogenesis takes place in gingiva during periodontal disease development, and that up-regulation of vascular growth factor C in recruited immune cells is likely important for the growth of lymphatic vessels.

Importance of lymph vessels in the transcapillary fluid balance in the gingiva studied in a transgenic mouse model

The gingiva is frequently challenged by oral bacterial products leading to inflammatory responses such as increased fluid filtration and edema formation. The role of initial lymphatics for transcapillary fluid balance in the gingiva is unknown and was therefore investigated in genetically engineered K14-VEGF receptor 3-Ig (K14) lymphedema mice. The mutant mice demonstrated a total lack of lymphatics in the gingiva, whereas lymphatics were found in the submucosal parts of the alveolar mucosa, although they were almost completely absent in the mucosa. In wild-type (WT) mice, lymphatic vessels were detected in mucosal and submucosal parts of the alveolar mucosa. Interstitial fluid pressure (P(if)) measured with micropipettes was increased in the gingiva of K14 mice in the normal situation (P < 0.001) and after inflammation (P < 0.01) induced by lipopolysaccharide from the oral bacteria Porphyromonas gingivalis compared with WT littermates. Fluid volume expansion caused a >75% increase in interstitial fluid volume followed by a drop in P(if) after recovery in both strains. Continuous measurements during the expansion showed an increase in P(if) followed by a decline, suggesting that compliance is increased after the disruption of the extracellular matrix during edema formation. In the alveolar mucosa, no strain differences were observed in P(if) in the normal situation or after fluid volume expansion, suggesting that lymph vessels in the mucosa are not critical for tissue fluid regulation in any situation. Our study demonstrates an important role of gingival lymphatics in transcapillary fluid balance in the steady-state condition and during acute perturbations.

Characterization of the dental lymphatic system and identification of cells immunopositive to specific lymphatic markers

The lymphatic system is important for immune barrier function and for tissue fluid balance. During inflammation, lymphangiogenesis takes place to enhance the transport of filtered fluid, proteins, and immune cells. Dental tissue is frequently exposed to inflammatory insults, but the lymphatic system and its responses to injury have not been investigated in detail using specific lymphatic markers. We aimed to study this system and to establish whether lymphangiogenesis takes place during wound healing. Immunostaining of the lymphatic endothelial hyaluronan receptor-1 (LYVE-1) and vascular endothelial growth factor receptor-3 (VEGFR-3) demonstrated initial lymphatics in the coronal molar pulp, whereas in incisors the initial lymphatics were found only in the apical part. In molars, lymphatic vessels exit the pulp through the apex and lateral canals. In interdental bone, transverse lymphatics were found, raising the possibility that an infection can be spread from the periodontal ligament to a neighbouring tooth. LYVE-1(+) and VEGFR-3(+) immune cells were found in both molar and incisor pulps, and phenotyping of the cells showed that they are of a monocytic lineage. In inflamed pulp these cells were not observed. Macrophages are suggested to contribute directly to the formation of lymphatic vessels after pulp exposure.

Vascular endothelial growth factors signalling in normal human dental pulp: a study of gene and protein expression.

In the well-vascularized dental pulp vascular endothelial growth factor A (VEGF-A) is expressed. Vascular endothelial growth factor A is a member of the VEGF family, which includes VEGFs-B, -C, and -D. The latter three have not been investigated in the pulp. Vascular endothelial growth factors C and D are the only ligands for vascular endothelial growth factor receptor (VEGFR)-3, which is usually expressed in lymphatic endothelium. They can also activate VEGFR-2, the main angiogenic receptor. We aimed to study VEGFs signalling in human dental pulp at the gene level and to identify the cellular source for protein expression using immunolabelling. All VEGFs (-A, -B, -C, and -D) were expressed in the pulp and may exert both autocrine and paracrine effects in blood vessels and immune cells found to be equipped with VEGFRs-2 and -3. Lymphatic vessel endothelial hyaluronan receptor-positive macrophages, known to be involved in angiogenesis, were found in the pulp, whereas lymphatic vessels were not detected. Twenty-six of 84 VEGF signalling genes, including VEGFR-3, were expressed at a significantly higher level in the pulp than in the control periodontal ligament. In conclusion, the normal human pulp represents a tissue with relatively high VEGF signalling involving both immune responses and vascular activity.