Rok Gašperšič

Anti-NGF Treatment Reduces Bone Resorption in Periodontitis

Periodontitis is characterized by periodontal tissue destruction, including the alveolar bone. One of its critical components is the release of pro-inflammatory neuropeptides from sensory nerve endings innervating the periodontium. Since nerve growth factor (NGF) has been reported to up-regulate neuropeptides in sensory neurons, we hypothesized that it would be increased in ligature-induced periodontitis in rats, and that systemic NGF neutralization would reduce the periodontitis-associated alveolar bone resorption. Real-time PCR analysis disclosed a statistically significant time-dependent up-regulation of NGF mRNA in gingiva during 2 weeks of periodontitis. Interestingly, NGF up-regulation was also detected in the contralateral gingiva. In addition, immunohistochemistry of trigeminal ganglion neurons innervating the gingivomucosa demonstrated increased expression of TrkA receptor for NGF. Systemic anti-NGF treatment during periodontitis significantly reduced interleukin-1β expression in gingiva and bilateral alveolar bone resorption. This suggests that NGF promotes periodontal inflammation and implicates a possible use of anti-NGF treatment in periodontitis.

Muscle activity-resistant acetylcholine receptor accumulation is induced in places of former motor endplates in ectopically innervated regenerating rat muscles

Expression of acetylcholine receptors (AChRs) in the extrajunctional muscle regions, but not in the neuromuscular junctions, is repressed by propagated electric activity in muscle fibers. During regeneration, subsynaptic‐like specializations accumulating AChRs are induced in new myotubes by agrin attached to the synaptic basal lamina at the places of former motor endplates even in the absence of innervation. We examined whether AChRs still accumulated at these places when the regenerating muscles were ectopically innervated and the former synaptic places became extrajunctional. Rat soleus muscles were injured by bupivacaine and ischemia to produce complete myofiber degeneration. The soleus muscle nerve was permanently severed and the muscle was ectopically innervated by the peroneal nerve a few millimeters away from the former junctional region. After 4 weeks of regeneration, the muscles contracted upon nerve stimulation, showed little atrophy and the cross‐section areas of their fibers were completely above the range in non‐innervated regenerating muscles, indicating successful innervation. Subsynaptic‐like specializations in the former junctional region still accumulated AChRs (and acetylcholinesterase) although no motor nerve endings were observed in their vicinity and the cross‐section area of their fibers clearly demonstrated that they were ectopically innervated. We conclude that the expression of AChRs at the places of the former neuromuscular junctions in the ectopically innervated regenerated soleus muscles is activity‐independent.

EFP Delphi study on the trends in Periodontology and Periodontics in Europe for the year 2025

AIM The aim was to assess the potential trends in Periodontology and Periodontics in Europe that might be anticipated by the year 2025, using the Delphi method. MATERIAL AND METHODS The expert opinion of 120 experts was sought through the use of an open-ended questionnaire, developed by an advisory group, containing 40 questions concerning the various trends in periodontology. RESULTS The experts (113 responders) expect a stabilization of the prevalence of periodontitis, both for the chronic as well as the aggressive cases, but an increase in implant-related diseases up to the year 2025. Concurrently, the importance of implants is seen to be increasing. They foresee an increased demand for postgraduate periodontology and implantology training. This is mirrored in an increase in publications for implant dentistry and increase in demand and need for training. Concerning the patients, better-informed individuals seeking more routine check-ups are expected. CONCLUSION A continued need for specialized periodontists, but also well trained dental practitioners is foreseen for next decade in Europe. Apart from periodontology they will be increasingly exposed to and trained in implant dentistry.

Expression of TrkA receptor for neurotrophins in trigeminal neurons innervating the rat gingivomucosal tissue

The purpose of this study was to characterize and evaluate the expression of TrkA receptor in trigeminal ganglion (TG) neurons that innervate the rat gingivomucosal tissue. A retrograde nerve tracer Fluorogold (FG) was injected into the gingiva (group 1) or applied into the gingival sulcus (group 2) of the first right maxillary molar to identify the neurons in TG that innervate the gingivomucosa. After 10 days TG were dissected and FG fluorescence in neurons was observed under UV light microscope. To draw a comparison, approximately 1000 neurons per ganglion from the entire TG (group 3) and approximately 350 neurons per ganglion from the maxillary region in TG (group 4), were analyzed. Expression of TrkA receptor in TG neurons was investigated by immunohistochemistry. About 70% of neurons in groups 1 and 2 contained TrkA receptor, which was statistically significantly more than in groups 3 (41%) and 4 (38%). FG-labeled TrkA-immunopositive neurons were predominantly small or medium-sized (less than 1200microm(2)). However, the neurons innervating the rat gingivomucosa were on average larger than the neurons in the entire TG or in the maxillary region. In conclusion, the majority of neurons in TG that innervate the rat gingivomucosa are small or medium-sized, contain TrkA receptor and are most probably nociceptive.

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.

Dental pulp and gingivomucosa in rats are innervated by two morphologically and neurochemically different populations of nociceptors

OBJECTIVES Difference in phenotypes of sensory neurons innervating dental pulp or gingivomucosa may be responsible for intense pain sensations in pulpitis in contrast to relatively painless chronic periodontitis. Therefore, we classified these neurons according to their size and two neurochemical characteristics of nociceptors, their TrkA expression and isolectin IB4 binding. DESIGN In rats (n=6) fluorescent tracers Fluorogold and TrueBlue were simultaneously applied into the standard-sized tooth cavity and nearby gingival sulcus, respectively. After the fluorescence on paraffin trigeminal ganglia (TG) sections was identified and photographed, immunohistochemistry for TrkA expression and IB4 binding was performed on the same sections. RESULTS The average sizes of TG neurons projecting to the gingivomucosa and dental pulp were 894±441μm(2) and 1012±381μm(2), respectively. The proportions of small-sized gingival and pulpal neurons were 14% and 5%, respectively (p<0.05). The proportions of TrkA-positive neurons among all gingival or pulpal neurons were 76% and 86%, respectively (p<0.05). Among all gingival or pulpal neurons the proportions of IB4-positive neurons were 46% and 3% (p<0.001), respectively, and the majority of them were small-medium sized. CONCLUSIONS Dental pulp and gingivomucosa are richly innervated by nociceptive TrkA-expressing neurons. However, while great majority of pulpal neurons are larger NGF-dependent A-fibre nociceptors without affinity to bind IB4, almost half of the gingival neurons are smaller IB4 binding C-fibre nociceptors. The difference in phenotype of sensory neurons might partially explain the different sensitivity of both tissues during normal and pathological conditions.

A Cytolethal Distending Toxin Variant from Aggregatibacter actinomycetemcomitans with an Aberrant CdtB That Lacks the Conserved Catalytic Histidine 160

The periodontopathogen Aggregatibacter actinomycetemcomitans synthesizes several virulence factors, including cytolethal distending toxin (CDT). The active CDT holoenzyme is an AB-type tripartite genotoxin that affects eukaryotic cells. Subunits CdtA and CdtC (B-components) allow binding and intracellular translocation of the active CdtB (A-component), which elicits nuclear DNA damage. Different strains of A. actinomycetemcomitans have diverse virulence genotypes, which results in varied pathogenic potential and disease progression. Here, we identified an A. actinomycetemcomitans strain isolated from two patients with advance chronic periodontitis that has a regular cdtABC operon, which, however, codes for a unique, shorter, variant of the CdtB subunit. We describe the characteristics of this CdtBΔ116–188, which lacks the intact nuclear localisation signal and the catalytic histidine 160. We show that the A. actinomycetemcomitans DO15 isolate secretes CdtBΔ116–188, and that this subunit cannot form a holotoxin and is also not genotoxic if expressed ectopically in HeLa cells. Furthermore, the A. actinomycetemcomitans DO15 isolate is not toxic, nor does it induce cellular distention upon infection of co-cultivated HeLa cells. Biological significance of this deletion in the cdtB remains to be explained.