Resumen de Toll-Like Receptor 4 Expression in Trigeminal Neurons Is Increased During Ligature-Induced Periodontitis in Rats
Background: Periodontitis, activated by oral bacteria and orchestrated by innate immune response, is regulated by primary nociceptive neurons, which are generally considered to have small- to medium-sized perikaryons. Bacterial byproducts (e.g., lipopolysaccharides) activate primary nociceptive neurons directly through Toll-like receptors (TLRs). Therefore, this study aims to morphometrically characterize rat trigeminal neurons, which express TLR4, and to investigate the changes in the TLR4 expression in neurons during periodontal inflammation.
Methods: Trigeminal neurons innervating gingivomucosa were identified by application of the retrograde tracer hydroxystilbamidine into the gingival sulcus of the maxillary molar in 14 rats. Periodontitis was induced by ligature around the same molar in seven rats. TLR4 expression was investigated by immunohistochemistry on paraffin sections of the trigeminal ganglia (TG). Semiquantitative method was used to identify the intensity of TLR4 expression.
Results: In the control group without the ligatures, TLR4 was detected in 19% of the neurons in the maxillary region of TG and in 29% of neurons innervating gingivomucosa. Expression of TLR4 was more frequent and intensive in small- to medium-sized neurons than in large-sized neurons. One week after ligature-induced periodontitis, the percentage of TLR4-positive neurons in the maxillary region and among the neurons innervating inflamed gingivomucosa significantly increased statistically to 32% and 41%, respectively.
Conclusions: TLR4 is predominantly, but not exclusively, expressed in smaller trigeminal nociceptive neurons in the rat. Experimental periodontitis upregulates TLR4 expression in the trigeminal neurons. The hypothesis that bacterial byproducts regulate the pathogenesis of periodontitis by activation of trigeminal nociceptors through TLR4 should be explored.
Pathogenesis of periodontitis involves intricate interactions of the microbial biofilm microorganisms with the host immunoinflammatory response and subsequent alterations in bone and connective tissue homeostasis. Most of the microorganisms harboring the subgingival area express virulence factors capable of activating the innate immune response, which has been proposed to be more important in orchestration of the course of chronic periodontitis (CP) than the adaptive immune response.1 Toll-like receptors (TLRs) comprise a family of evolutionary, conserved pattern recognition receptors that act as the first line of defense in the innate immune system recognizing invariant molecular structures of pathogens.2 TLR4 functions as receptor for lipopolysaccharides (LPSs) of Gram-negative bacteria,3 including those associated with periodontitis.4,5 Notably, genetic variants of TLR4 may act as risk factors for the development of CP in humans.6,7 In addition, anti-TLR4 treatment inhibited LPS-dependent upregulation of the proinflammatory interleukin-6 in human gingival fibroblasts.4 TLRs are also localized on several cells that are not primarily involved in innate immune response,4,8,9 among them cells of the peripheral neural system,10 including trigeminal sensory neurons and their nociceptive nerve endings innervating tooth pulp.11 In addition, Wadachi and Hargreaves11 reported that TLR4 could also be located on trigeminal non-nociceptors, which are generally considered to be primary sensory neurons with large cell bodies. The percentage of trigeminal sensory neurons expressing TLR4 and their morphometry has not been determined yet. In addition, it is not known whether the primary sensory neurons projecting to the gingivomucosa also express TLR4.
Several studies on animal models have shown that neurogenic mechanisms related to primary nociceptive neuron activation regulate the progression of local inflammatory processes, including periodontitis.12-14 During stimulation, these neurons release various vasoactive neuropeptides, such as calcitonin gene-related peptide (CGRP) and substance P. It has been shown that synthesis of these neuropeptides in trigeminal ganglion (TG) neurons and their release from gingival nerve endings are increased during periodontal inflammation.13-16 During periodontitis, bacteria or bacterial byproducts may activate neurons indirectly via paracrine action by evoking the release of certain soluble factors (e.g., nerve growth factor, prostaglandins, cytokines, etc.) in the infected gingivomucosal tissue.17,18 In addition, in vitro experiments showed that certain bacterial byproducts (i.e., LPS) activate primary nociceptors, including capsaicin-sensitive small-sized trigeminal nociceptors, directly through TLR4.19-21 In this respect, it should be noted that constitutive expression of TLR4 in gingival tissue is upregulated in periodontitis.4,22 Therefore, the primary objective of this study is to determine whether the sensory neurons innervating the gingivomucosa also express TLR4 and to find out whether experimentally induced periodontal inflammation would increase the expression of TLR4 in these neurons. In addition, the authors of the present study hypothesize that TLR4 is mainly expressed in small- and medium-sized trigeminal neurons that are considered to be predominantly nociceptive.
