Sivakami Rethnam Haug

Modulation of Dental Inflammation by the Sympathetic Nervous System

Recent findings have indicated that immune responses are subjected to modulation by the sympathetic nervous system (SNS). Moreover, the findings show that the SNS inhibits the production of pro-inflammatory cytokines, while stimulating the production of anti-inflammatory cytokines. The present review is an attempt to summarize the current results on how the SNS affects inflammation in dental tissues. In dental tissues, it has been found that the SNS is significant for recruitment of inflammatory cells such as CD 43+ granulocytes. Sympathetic nerves appear to have an inhibitory effect on osteoclasts, odontoclasts, and on IL-1α production. The SNS stimulates reparative dentin production, since reparative dentin formation was reduced after sympathectomy. Sprouting of sympathetic nerve fibers occurs in chronically inflamed dental pulp, and neural imbalance caused by unilateral sympathectomy recruits immunoglobulin-producing cells to the dental pulp. In conclusion, this article presents evidence in support of interactions between the sympathetic nervous system and dental inflammation.

Effects of sympathectomy on experimentally induced pulpal inflammation and periapical lesions in rats.

The role of sympathetic nerves in bone physiology is largely unknown. Recent studies have shown a correlation between sympathectomy and bone remodeling. The present experiments were aimed to study the effects of unilateral sympathectomy on bilateral experimentally induced pulpitis and periapical lesions in the rat maxilla and mandible. Adult male Sprague-Dawley rats were used. Experimental rats (n=11) had the right superior cervical ganglion surgically removed (SCGx) and control rats (n=5) had sham surgery. Pulpal inflammation and periapical bone lesions in the maxilla and mandible were created 14 days later in both experimental and control rats by exposing the dental pulp in the first and second molars and leaving them open to the oral microflora. The rats were perfused 20 days thereafter and the jaws processed for immunohistochemistry with neuropeptide Y (NPY) and ED1 as primary antibodies. Sympathectomy resulted in an almost complete loss of NPY-immunoreactive (IR) fibers in the right SCGx jaws. In the non-sympathectomized (non-SCGx) left side and in the control rats, sprouting of NPY-IR fiber was observed in the inflamed pulp tissue adjacent to reparative dentin formation and in the apical periodontal ligament of the partially necrotic first molars. Significantly more ED1-IR osteoclasts were found in the resorptive lacunae lining the periphery of the periapical lesions on the SCGx side compared with the non-SCGx side (P<0.04) and the controls (P<0.03). The size of the periapical lesions were larger on the SCGx side compared with the non-SCGx side (P<0.03) in the mandible, but not in the maxilla. We conclude that inflammation causes sprouting of NPY-IR nerve fibers and that unilateral removal of the SCG increases both the area of the periapical lesions and the number of osteoclasts in the inflamed region.

Sympathectomy decreases size and invasiveness of tongue cancer in rats.

The sympathetic nervous system plays a role in carcinogenesis wherein locally released sympathetic neurotransmitters affect proliferation, angiogenesis, vessel permeability, lymphocyte traffic and cytokine production. The present in vivo study was designed to investigate whether surgical sympathectomy, both unilateral and bilateral, had an effect on tumor growth, interstitial fluid pressure (IFP) and lymphatics in rat tongue cancer. We used 4-nitroquinoline-1-oxide (4-NQO) in drinking water for 19 weeks to induce tongue cancer in 20 Dark Agouti rats. After 11 weeks, one group underwent unilateral sympathectomy and another underwent bilateral sympathectomy, while the third group underwent sham surgery. By 19 weeks, tumors in the bilaterally sympathectomized (BL-SCGx) rats were significantly smaller (P<0.05), more diffuse in appearance and less invasive (P<0.05) compared with the large exophytic tumors in the sham-operated rats. The relative lymphatic area was significantly decreased (P<0.05) in tumors in the BL-SCGx rats compared with the sham group. Interestingly, the tumors in rats that underwent unilateral or bilateral sympathectomy had a significantly lower (P<0.05) IFP than those in sham rats. Lack of tyrosine hydroxylase (TH) immunoreactive nerves and few neuropeptide Y (NPY) positive fibers indicate absence of sympathetic nerve fibers in the bilateral sympathectomized group. The peritumoral lymph vessel area was correlated with the tumor size (P<0.001), depth of invasion (P<0.001), weight of rats (P<0.005) and IFP (P<0.05). In conclusion, the present study presents evidence that deprivation of sympathetic nerves decreases tumor growth in rat tongue, probably caused by decreasing IFP and lymph vessel area.

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.

High interstitial fluid pressure in rat tongue cancer is related to increased lymph vessel area, tumor size, invasiveness and decreased body weight

BACKGROUND Interstitial fluid pressure (IFP) in most tumors is high, and this high pressure has been correlated with poor prognosis. Measurements of IFP in normal tongue and in tongue cancer are lacking. Recent research suggests the existence of a relationship between increased peritumoral lymph vessels (PTLV) and survival, and a correlation of increased lymphatic vessel density with an unfavorable prognosis has been reported. MATERIALS AND METHODS In the present study, tongue squamous cell carcinoma (SCC) was induced by adding the carcinogen 4-nitroquinoline oxide in drinking water for 19 weeks. The IFP was measured by micropuncture and immunohistochemistry was used to visualize lymph vessels. RESULTS In normal tongue, IFP averaged 3.1 +/- 0.3 mmHg. The IFP, both in the tumor (29.1 +/- 2.9 mmHg) and 0.5 cm anterior to it (15.4 +/- 2.1 mmHg) was consistently increased (P < 0.005) with values ranging from 10 to 40 mmHg. The highest IFP values were measured in rats with large tumors (P < 0.05) and low body weight (P < 0.001), suggesting that IFP increases with cancer progression. Lymphatic vessel area (%), as determined with the lymphatic specific marker LYVE-1 antibody, was significantly increased in the peritumoral area when compared to intratumoral and control mucosa (P < 0.05). There was a significant positive correlation between IFP, PTLV area, tumor size and invasiveness. CONCLUSIONS Our data show that IFP is increased in tongue cancer. Corresponding changes in PTLV area, invasiveness, tumor area and IFP suggest that the increased pressure is caused by defective lymph drainage and solid stress generated by tumor cells growing in a low compliant environment.

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.

Sympathectomy suppresses tumor growth and alters gene-expression profiles in rat tongue cancer

Sympathetic nerves are known to affect carcinogenesis. Recently we found that sympathetic denervation decreases the size of rat tongue tumors. To identify genes involved in rat tongue carcinogenesis and to study the effect of sympathetic nerves on these genes, we compared gene-expression profiles in normal rat tongue (control) and in tumor-induced tongues with (SCGx) and without (Sham) bilateral sympathectomy. Significance analysis of microarrays revealed 280 genes (168 up-regulated, 112 down-regulated) that showed at least a twofold differential expression between Sham and SCGx tumors (false discovery rate < 5%). These included genes associated with cell adhesion, signaling, structure, proliferation, metabolism, angiogenesis, development, and immunity. Hierarchical clustering demonstrated that controls and sympathectomized tumors grouped together, while Sham tumors grouped separately. We identified 34 genes, known to be involved in carcinogenesis, that were not differentially expressed between sympathectomized tumors and control tongues, but which showed a significant change in expression in Sham tumors. Microarray results of 12 of these genes were confirmed by quantitative reverse transcription-polymerase chain reaction. In conclusion, sympathectomy significantly altered the gene-expression profile and inhibited tumor growth. The expression of several cancer genes were increased more than threefold in Sham tumors, but unaltered in the sympathectomized tumors when compared with controls, indicating that these genes may be of significance in rat tongue carcinogenesis.

Effect of electrical tooth stimulation on blood flow and immunocompetent cells in rat dental pulp after sympathectomy.

Previous experiments show that nerves have effect on the emigration of immunocompetent cells during acute neurogenic inflammation. The present study aims to determine whether the sympathetic or sensory nerves are responsible for emigration of CD43+ and I‐A antigen‐expressing cells in the dental pulp after electrical tooth stimulation. Wistar rats were used. Experimental rats (n = 6) had the right superior cervical ganglion removed (SCGx), whereas control rats (n = 6) had sham surgery. Fourteen days later, electrical stimulation of the right maxillary 1st molar was performed in both groups for 20–25 s every 5th min for a total period of 4 h. Changes in pulpal blood flow (PBF) were recorded with a laser Doppler flowmeter. All rats were transcardiacally perfused and processed for immunohistochemistry using antibodies against neuropeptides and immune cells. Intermittent electrical stimulation consistently increased PBF and depleted sympathetic and sensory neuropeptides in the dental pulp. The increase in PBF gradually decreased and approached control values at the end of the 4 h stimulation period. A significant increase in the number of I‐A antigen‐expressing dendritic cells was found in both the SCGx (P < 0.001) and control rats (P < 0.007). In contrast, tooth stimulation did not increase the number of CD43+ cells in the SCGx rats compared to the unstimulated contralateral control molar. Significantly more CD43+ PMN cells (P < 0.01) were found in the control rats after stimulation. It is concluded that stimulation of sympathetic nerves causes recruitment of CD43+ PMN cells, whereas stimulation of sensory nerves causes emigration of I‐A antigen‐expressing dendritic cells in the dental pulp.

Immunoglobulin producing cells in the rat dental pulp after unilateral sympathectomy

Recent studies show that sympathetic nerves participate in immunomodulation. We investigated the effects of unilateral sympathectomy on recruitment of cells expressing kappa and lambda (kappa and lambda) light chains in the rat dental pulp. Superior cervical ganglion was removed in experimental rats (n=10) while control rats (n=8) received sham surgery. Following perfusion 18 days later, mandibular jaws were processed for immunohistochemistry and electron microscopy. Sympathectomy results in recruitment of cells expressing kappa and lambda light chains into the dental pulp (P=0.005). Electron microscopy revealed these cells to be mainly plasma cells and Mott cells. We conclude that neural imbalance caused by unilateral sympathectomy recruits immunoglobulin producing cells in the dental pulp. Our results are in agreement with a model of immune regulation in which the sympathetic nervous system exerts a tonic regulatory effect over lymphocyte proliferation and migration.