Role of Hyperplasia of Gingival Lymphatics in Periodontal Inflammation.

Lymphatic vessels are important for maintenance of tissue fluid homeostasis and afferent antigen transport. In chronic inflammation, lymphangiogenesis takes place and is characterized by lymphatic endothelial cell proliferation and lymphatic hyperplasia. Vascular endothelial growth factor C (VEGFC) is the main known lymphangiogenic growth factor, and its expression is increased in periodontitis, a common chronic infectious disease that results in tissue destruction and alveolar bone loss. The role of lymphangiogenesis during development of periodontitis is unknown. Here, we test if transgenic overexpression of epithelial VEGFC in a murine model is followed by hyperplasia of lymphatic vessels in oral mucosa and if the lymphatic drainage capacity is altered. We also test if lymphatic hyperplasia protects against periodontal disease development. Transgenic keratin 14 (K14)-VEGFC mice had significant hyperplasia of lymphatics in oral mucosa, including gingiva, without changes in blood vessel vasculature. The basal lymph flow was normal but slightly lower than in wild-type mice when oral mucosa was challenged with lipopolysaccharide from Porphyromonas gingivalis. Under normal conditions, K14-VEGFC mice exhibited an increased number of neutrophils in gingiva, demonstrated enhanced phagocyte recruitment in the cervical lymph nodes, and had more alveolar bone when compared with their wild-type littermates. After induction of periodontitis, no strain differences were observed in the periodontal tissues with respect to granulocyte recruitment, bone resorption, angiogenesis, cytokines, and bone-related protein expressions or in draining lymph node immune cell proportions and vascularization. We conclude that overexpression of VEGFC results in hyperplastic lymphatics, which do not enhance lymphatic drainage capacity but facilitate phagocyte transport to draining lymph nodes. Hyperplasia of lymphatics does not protect against development of ligature-induced periodontitis.

Lymphangiogenesis and lymphatic function in periodontal disease.

Lymphatic vessels return extravasated fluid, proteins, and cells back into the circulation and are important in immune cell trafficking. In the gingiva, lymphatic vessels are located in the lamina propria and travel over the external surface of the alveolar bone. The gingival lymphatics are important for fluid drainage, since lack of lymphatics has been shown to increase interstitial fluid pressure and fluid volume. Maintenance of gingival lymphatic vessels requires continuous signaling by the growth factors VEGF-C and -D via their receptor VEGFR-3. The growth factors are expressed in the gingival epithelium and also in immune cells in the lamina propria. VEGF-C seems to be crucial for lymphangiogenesis induced during periodontal disease development. The lymphatic vessels protect against periodontitis in mice, probably by clearing bacteria and bacterial products and by promoting humoral immune responses. Down-regulation of CCL21, a ligand important for dendritic cell migration, has been demonstrated in lymphatics from patients with periodontitis. High enzymatic activity in the gingiva of these patients may also contribute to impaired lymphatic function, due to the loss of structural components in the interstitium influencing lymphatic function. So far, knowledge is limited in this field because of the dearth of studies on the role of lymphatic vessels in periodontal disease.

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-1ß 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.

Differential expression of neuropeptide Y Y1 receptors during pulpal inflammation.

AIM: To study the pattern of neuropeptide Y (NPY) Y1 receptor (Y1R) localization in the normal dental pulp and during different stages of pulpal inflammation. The hypothesis was that the expression of Y1R varies during different stages of pulpitis. METHODOLOGY: Pulp exposure injury was made on first molar teeth of Sprague-Dawley rats. Animals were killed 3, 7, 14, 21, 28 and 46 days after pulp exposure. Jaws were removed, decalcified and processed for immunohistochemistry for identification and localization of Y1R. Double labelling was performed using antibodies for laminin, CD43 and CD4 with Y1R. RT-PCR was performed to verify gene expression of Y1R in dental pulp and trigeminal ganglion. RESULTS: RT-PCR revealed the presence of NPY-Y1R in the dental pulp, trigeminal ganglion and gingiva. With immunohistochemistry, in control rats, Y1R was mainly located in capillaries and small vessels in the dental pulp as well as in the odontoblastic layer. No YIR was noted on immune cells in normal pulp. In pulpitis, immune cells such as CD43+ granulocytes and CD4+ lymphocytes expressed Y1R. As pulpitis progressed, there was a significant decrease in number of blood vessels expressing Y1R in the odontoblast layer when compared to control rat pulp. CONCLUSIONS: This study gives evidence that Y1R is a modulator of pulpal inflammation.

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.

Edema in oral mucosa after LPS or cytokine exposure.

Lowering of interstitial fluid pressure (P(if)) is an important factor that explains the rapid edema formation in acute inflammation in loose connective tissues. Lipopolysaccharide (LPS) and the pro-inflammatory cytokines interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) are pathogenetic in gingivitis. To test if these substances induce lowering of P(if) in rat oral mucosa, we measured P(if) with a micropuncture technique. IL-1beta and TNF-alpha caused lowering of P(if), whereas LPS induced an immediate increase in P(if), followed by lowering after 40 min. Measurements of fluid volume distribution showed a significant change in interstitial fluid volume (V(i)) 1.5 hr after LPS exposure as V(i) changed from 0.41 +/- 0.02 to 0.51 +/- 0.03 mL/g wet weight (p < 0.05), confirming edema. These findings show that LPS, IL-1beta, and TNF-alpha induce lowering of P(if) in the rat oral mucosa and contribute to edema formation in LPS-induced gingivitis.

Delta (delta) opioid receptors in small and medium-sized trigeminal neurons supporting the dental pulp of rats.

The control of pain perception is a challenge in clinical dentistry, most prominent during tooth pulp inflammation. The tooth pulp is a well-defined target, and is densely supplied by a sensory trigeminal innervation. Opioids are signaling molecules that are suggested to participate in pain perception. Here we analysed the presence of delta opioid receptor (DOR) in trigeminal neurons innervating the tooth pulp of rat molars. Immunohistochemical and ultrastructural analysis revealed that DOR was identified in peripheral nerves in the molar dental pulp, both in the root and the coronal pulpal parts, with branching in the highly innervated subodontoblast layer. DOR was localised in about one third of all the trigeminal dental neurons, identified by means of retrograde neuronal transport of fluorogold (FG) from the dental pulp. Of the DOR-labeled neurons, nearly all were small and medium-sized (147.5-1,810.2 microm(2), mean 749.1 +/- 327.3 microm(2)). Confocal microscopy confirmed that DOR-immunoreactivity was distributed as granules in the neuronal cytoplasm. Approximately 70% of the DOR-immunoreactive neurons were also immunopositive for vanilloid receptor 1 (TRPV1). Ultrastructural analysis demonstrated DOR-immunoreactivity in the unmyelinated and in some of the myelinated nerve fibers in the dental pulp. These results indicate that DOR may influence the function in a subset of small and medium-sized trigeminal sensory neurons supporting the tooth, which are mainly known for their ability to mediate nociceptive stimuli. Agonists, acting on DOR, may thus have an influence on a subpopulation of nociceptive neurons supporting the rat tooth.

Blood flow and interstitial fluid pressure in the rat submandibular gland during changes in perfusion.

The submandibular gland is a cell-rich encapsulated organ with high transport of fluid through the interstitial space during salivation. We hypothesized that the gland is a low-compliant tissue, i.e., that a modest increase in fluid volume will produce a rise in interstitial fluid pressure (IFP) counteracting fluid filtration into the interstitium. To test this hypothesis, we measured IFP with micropipettes and glandular blood flow (GBF) with a laser-Doppler flowmeter during changes in perfusion. Clamping of the carotid artery or the jugular vein, or electrical stimulation of the sympathetic or parasympathetic nerve to the gland, induced changes in perfusion. Baseline IFP averaged 3.5 +/- 0.5 mm Hg. Clamping of the artery reduced IFP and GBF (-56.5 +/- 8.4% and -53.1 +/- 6.4%, respectively), whereas clamping of the vein decreased GBF (-21.6 +/- 14.3%) and increased IFP (141.2 +/- 27.4%). Sympathetic nerve stimulation reduced both parameters (-86.9 +/- 16.5% and -74.4 +/- 7.0%, respectively). In contrast, stimulation of the parasympathetic nerve elicited an increase in GBF (133.2 +/- 5.9%) and in IFP (173.3 +/- 41.4%). Thus, changes in vascular volume led to concomitant changes in IFP consistent with low tissue compliance, a phenomenon of importance for fluid volume regulation.

Role of K+ATP channels, endothelin A receptors, and effect of angiotensin II on blood flow in oral tissues.

K+(ATP) channels are involved in CGRP-mediated vasodilation and in the vasoconstriction induced by endothelin or angiotensin II. In this study, we examined the effects of a K+(ATP) channel antagonist and an ET(A) receptor antagonist on resting blood flow in the pulp and gingiva, and observed their role in the vasodilation induced by tooth stimulation. We also investigated whether receptors for angiotensin II exist in the pulp and gingiva. Blood flow was measured with laser-Doppler flowmetry. Under control conditions, the K+(ATP) channel antagonist and angiotensin II caused a significant drop in blood flow in both target tissues. Blocking of ET(A) receptor did not change basal blood flow. The vasodilation observed after tooth stimulation remained unchanged following blockade of K+(ATP) channels and ET(A) receptors. Analysis of the data shows that open K+(ATP) channels exist during resting conditions in the pulp and gingiva, but that CGRP seems to induce vasodilation mainly via mechanisms other than K+(ATP) channels. ET(A) and AT(1) receptors are found in the pulp and gingiva, but ET(A) receptors are not involved in modulation of a basal vascular tone in these tissues or in the vasodilation observed after tooth stimulation.

The effect of unilateral sympathectomy and cavity preparation on peptidergic nerves and immune 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.

Regulation of extracellular volume and interstitial fluid pressure in rat bone marrow.

The volume and fluid pressure characteristics of the intact bone marrow is incompletely understood. We used microspheres and lipoproteins for measurements of intravascular volume (IVV) and EDTA for interstitial fluid volume (IFV) within the rat bone marrow. Interstitial fluid pressure (IFP) was determined with micropipettes connected to a servo-controlled counter-pressure system. Both the microspheres and the lipoproteins yielded estimates of IVV of approximately 1 ml/100 g. After a brief reactive hyperemia, IVV increased to 2.5 ml/100 g, whereas IFV decreased with approximately 1.5 ml/100 g, so that total extracellular volume did not change. Baseline bone marrow IFP was 9.7 mmHg. The hyperemia led to a transient twofold increase in IFP, whereas a marked blood loss decreased IFP by almost one-half. These novel data suggest that extracellular volume and IFP within the bone marrow can be measured with tracer methods and the micropuncture technique. The responses of IVV, IFV, and IFP during changes in blood flow to the bone marrow suggest a tight regulation and are thus compatible with those for a low-compliant tissue.

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 microm) and in six on the denervated side (332.56 +/- 193.2 microm) (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.

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.

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.

Interstitial fluid pressure in normal and inflamed pulp.

Tissue pressure is the hydrostatic pressure in the interstitial fluid which surrounds the pulpal cells. This pressure outside the vessels is normally considerably lower than the blood pressure inside the vessels. The dental pulp has a relatively low interstitial compliance due to its enclosure between rigid dentin walls. Accordingly, even a modest increase in pulpal fluid volume will raise the tissue pressure, which may compress blood vessels, leading to ischemia and necrosis. Inflammation may lead to an increase in both interstitial fluid volume and blood volume in the low-compliant pulp and thereby increase the tissue pressure. However, the increased tissue pressure may, in turn, initiate increased lymph flow and absorption of fluid into capillaries in nearby non-inflamed tissue. Both of these latter factors will transport fluid out of the affected area and subsequently out of the tooth and consequently lower the tissue pressure. Increased tissue pressure, whether caused by increased blood volume or increased capillary filtration, will promote outward flow of fluid through exposed dentin tubules and thereby help to protect the pulp against entry of harmful substances. It seems physiologically beneficial, therefore, for the pulp to have a high tissue pressure, which promptly increases when blood flow increases due to its low compliance.