Ellen Berggreen

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.

The Role of Sensory Neuropeptides and Nitric Oxide on Pulpal Blood Flow and Tissue Pressure in the Ferret

A study was designed to investigate the effects of close intra-arterial infusion of antagonists to the sensory neuropeptides calcitonin gene-related peptide and substance P, as well as the effect of the nitric oxide synthesis inhibitor L-NAME on pulpal blood flow and interstitial fluid pressure during resting conditions and after electrical tooth stimulation. The micropuncture technique was used to measure tissue pressure and laser-Doppler flowmetry for blood flow recordings in ferret canine teeth. Close intra-arterial infusion of antagonists to calcitonin gene-related peptide and substance P significantly reduced resting blood flow (p < 0.05) and interstitial fluid pressure (p < 0.005) by unchanged systemic arterial pressure, while L-NAME administration caused a significant rise in interstitial fluid pressure (p < 0.05) and systemic arterial pressure (p < 0.005), with a concomitant fall in resting blood flow (p < 0.005). Tooth stimulation after calcitonin gene-related peptide antagonist infusion gave no significant change in blood flow or interstitial fluid pressure, whereas substance P antagonist infusion only partly eliminated the vasodilator response. L-NAME had no effect on the vasodilation induced by tooth stimulation. It is concluded that a resting vasodilator tone due to release of calcitonin gene-related peptide, substance P, and nitric oxide exists in the ferret dental pulp. The sensory neuropeptides exert their effect predominantly on pre-capillary vessels, and nitric oxide predominantly on post-capillary vessels. The sensory neuropeptide calcitonin gene-related peptide seems to be mainly responsible for the increase in blood flow and interstitial fluid pressure during tooth stimulation, whereas there was no evidence that nitric oxide participates in the vasodilation induced by tooth stimulation.

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.

The Effect of Unilateral Sympathectomy and Cavity Preparation on Peptidergic Nerves and Cells in Rat Dental Pulp

Recent evidence suggests interactions between primary afferent nociceptors and postganglionic sympathetic efferents in the pathogenesis of inflammation. The effect of unilateral removal of the superior cervical ganglion on the innervation pattern of nerve fibers immunoreactive (IR) to calcitonin gene-related peptide (CGRP), substance P (SP), and neuropeptide Y (NPY), as well as the occurrence of immune cells in the injured and uninjured rat molar pulp, was investigated. Light microscopic immunocytochemistry demonstrated that the molar pulps contralateral to the sympathectomy contained a NPY-IR nerve fiber network more dense and heavily stained than unoperated control rats. The NPY-IR fibers showed, however, no sprouting after deep cavity preparation. There was no compensatory increase in CGRP- and SP-IR nerve fibers in the dental pulp after unilateral sympathectomy, although a significant increase in cells IR to CGRP and SP was found in the ipsilateral trigeminal ganglion. Unilateral sympathectomy induced a significant increase in immune cell density both in the inflamed and in the uninflamed dental pulp bilaterally. Our results demonstrate, for the first time, a trophic effect of the sympathetic nerves on immune cells in the dental pulp, indicating that an imbalance of sympathetic nerves may induce inflammation and pain in teeth.

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.

Effect of the sensory neuropeptide antagonists h-CGRP(8–37) and SR 140.33 on pulpal and gingival blood flow in ferrets

In a previous study, it was concluded that the neuropeptides calcitonin gene-related peptide (CGRP) and substance P are released during resting conditions in the (exposed) ferret dental pulp, contributing to a basal vasodilator tone in the pulpal vessels. In order to exclude the possibility that the method used elicited axon reflexes, which might be responsible for neuropeptide release, the present study was designed without pulp exposure. Non-invasive laser-Doppler flowmetry was used to measure the effects of intra-arterial infusions of the antagonists h-CGRP((8-37)) and SR 140.33 (neurokinin 1-receptor antagonist) on pulpal and gingival blood flow before, during and after electrical tooth stimulation. Infusions of h-CGRP((8-37)) reduced the basal blood flow in the pulp by 31.4+/-5.2% (p<0.001) and in the gingiva by 22.6+/-4.8% (p<0.05). A further significant decrease in basal blood flow was measured in both pulp and gingiva following SR 140.33 administration. The reduction in blood flow was 16.9+/-1.9% (p<0.005) in the pulp and 19. 3+/-5.6% (p<0.05) in the gingiva. The systemic arterial pressure remained unchanged both during and after the periods of infusion. Tooth stimulation before the antagonist infusion significantly increased the pulpal blood flow by 71.9+/-15.3% (p<0.005). Infusion of h-CGRP((8-37)) greatly reduced this electrically induced vasodilatation, indicating that CGRP is the principal factor responsible for the vasodilatation observed after tooth stimulation. This study confirms the previous finding that a resting vasodilator tone due to the release of CGRP and SP exists in the ferret dental pulp. It is concluded that spontaneous, basal release of the neuropeptides CGRP and substance P exists both in dental pulp and gingiva in the ferret.

IL-17 Receptor A Signaling Is Protective in Infection-Stimulated Periapical Bone Destruction

IL-17 is a pleiotropic cytokine produced by Th17 T cells that induces a myriad of proinflammatory mediators. However, different models of inflammation report opposite functional roles of IL-17 signal in terms of its effects on bone destruction. In this study we determined the role of IL-17RA signal in bone resorption stimulated by dentoalveolar infections. Infrabony resorptive lesions were induced by surgical pulp exposure and microbial infection of mouse molar teeth. IL-17 was strongly induced in periapical tissues in wild-type (WT) mice by 7 d after the infection but was not expressed in uninfected mice. Dentoalveolar infections of IL-17RA knockout (KO) mice demonstrated significantly increased bone destruction and more abscess formation in the apical area compared with WT mice. Infected IL-17RA KO mice exhibited significantly increased neutrophils and macrophages compared with the WT littermates at day 21, suggesting a failure of transition from acute to chronic inflammation in the IL-17RA KO mice. The expression of IL-1 (both α and β isoforms) and MIP2 were significantly upregulated in the IL-17RA KO compared with WT mice at day 21 postinfection. The development of periapical lesions in IL-17RA KO mice was significantly attenuated by neutralization of IL-1β and MIP2. Taken together, these results demonstrate that IL-17RA signal seems to be protective against infection-induced periapical inflammation and bone destruction via suppression of neutrophil and mononuclear inflammation.

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.

Effect of denervation on healing after tooth replantation in the ferret

Studies have shown that the sensory nerves participate in inflammation and immune responses and possess trophic-facilitating wound healing in general. Tooth avulsion represents a pulpal and periodontal injury, and the mechanisms involved in the healing responses subsequent to replantation of teeth are still unclear. The objective of this study was to investigate the healing responses after denervation and replantation of teeth. Unilateral denervation was performed in 15 ferrets by axotomy of the inferior alveolar nerve, 5 days before extraction of the first lower premolars. Six weeks later the mandibles were excised and processed for histological evaluation. Immunohistochemistry was performed using antibodies against the sensory neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP), and measurements of root resorption and ankylosis were performed in four sections from each replanted tooth. After 6 weeks substantial reinnervation was observed in the jaws. Immunoreactivity in the pulp was observed in only two replanted teeth on the denervated side, compared with four on the innervated side. Total pulp necrosis appeared in 10 replanted teeth on the denervated side and in 5 on the innervated, indicating that sensory nerves promote survival of the pulp after replantation. SP-immunoreactive (IR) fibers were more frequently observed in the resorptive lacunae than CGRP-IR fibers. However, resorptive areas lacking IR fibers were frequently found along the root surface. Root resorption averaged 0.062 - 0.029 mm2 on the innervated side compared to 0.016 - 0.0043 mm2 on the denervated (P < 0.02). Ankylosis was observed in four of the replanted teeth on the innervated side (169.3 - 49.7 µm) and in six on the denervated side (332.56 - 193.2 µm) (P = 1). It is concluded that the sensory nerves promote root resorption after pulpoperiodontal injuries but have less influence on the osteoblastic activity expressed by ankylosis.

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.