Evaluation of the expression of nerve fiber markers in healthy and inflamed dental pulp

Abstract The diagnosis of irreversible pulpitis (IP) depends on clinical data, especially the chief complaint of the patient, visual inspection, response to the application of stimuli, and radiographic examination. The characterization of nerve fibers (NF) in IP may contribute to better interpret painful symptoms, but has been barely explored. This study sought to characterize the density and integrity of NF in 16 samples of IP and in five healthy pulps (HP) using S-100 and PGP 9.5 markers. Immunohistochemistry was performed to determine the density/mm2 of S-100+ and PGP 9.5+ in NF. The amount of degenerated NF was obtained by subtracting the total NF density from the amount of intact NF. Associations between NF density and integrity and symptomatology were calculated. All samples were positive for S-100 and PGP 9.5. Compared to HP samples (38.20/mm2), IP samples had a lower density of intact NF (6.24/mm2). A significantly higher density of degenerated NF was found in IP samples with spontaneous pain (39.59/mm2) compared to those with provoked pain (23.96/mm2) (p = 0.02). No association was observed between intensity of the inflammatory infiltrate and NF density and integrity (p > 0.05). The findings of this study suggest that pulpitis may involve different stages of degeneration and may be more advanced in cases with spontaneous pain. The symptoms reported by affected individuals do not appear to depend on the intensity of the inflammatory infiltrate, but rather on the integrity of NF.


Introduction
The dental pulp is a complex connective tissue that, together with the periodontal ligament, is highly innervated with trigeminal sensory neurons and a high nociceptor density. 1 The pulpodentine complex is densely innervated and pain occurs when this tissue is attacked or inflamed. 2,3The sensory innervation of the dental pulp occurs through axons originating from trigeminal nodules, whose nerve fibers (NF) are mainly type A and C. [4][5][6][7] Type A myelinic fibers are responsible for rapid responses to acute painful stimuli and are located in the outermost portion of the coronary pulp.Type C unmyelinated fibers are responsible with a slower transmission of painful stimuli in the presence of aggression. 4,5,7,8ental caries is the main aggression that affects pulp tissue, and its progression triggers an inflammatory process that alters the physiology and integrity of the NF, influencing the pain threshold of the affected individual. 9,10ain assessment based on the response to dental pulp sensibility tests is currently the clinical tool most commonly used to determine pulp status and severity of inflammation and to suggest the diagnosis. 3,11However, a systematic review has revealed that the characterization of pain based only on clinical tests does not appear to be entirely reliable for making an endodontic diagnosis. 124][15][16] This is due to the fact the histopathological data of the inflamed pulp are not available during the clinical examination and that the association between histology and painful symptoms is not clear. 12,13,16,17egarding pulp changes, irreversible pulpitis (IP) occurs more frequently in decayed posterior teeth or in extensive restorations with pulp involvement in female individuals. 12lthough it is not the only criterion for the definition of IP, provoked pain that persists after the stimulus is removed or spontaneous pain, in addition to a positive response to dental pulp sensibility tests, is the main clinical finding. 5,13,14,18revious studies have demonstrated a possible association between pain and changes in the dental pulp of IP; however, this link has not yet been fully elucidated. 129][20][21] The S-100 protein, expressed mainly by Schwann cells, has been used to investigate NF density in healthy and inflamed human pulp, 18,22 and NF integrity is possibly impaired in an inflamed pulp with the evolution of inflammation.In addition, the protein gene product 9.5 (PGP 9.5) is a neuron-specific protein with opposite biologic role, functioning as a ubiquitin carboxyl-terminal hydrolase and ligase in which it is expressed in axons and used to map patients with neurodegenerative conditions. 23,24portantly, PGP 9.5 has also been used to quantify the neural protein recovered from pulpal tissue. 20he hypothesis is that the early loss or absence of expression of this protein appears to be associated with nerve degeneration processes. 23,24Thus, PGP 9.5 expression can be a promising marker for the identification of NF in inflamed dental pulp.
According to Diogenes, 25 innervation is a key component of the pulpodentine complex, since it modulates vascular, immunological, and dentinogenic responses to injuries in addition to having a sophisticated sensory function.Given the high frequency of IP, the diagnosis and treatment of which are based mainly on clinical data, including painful symptoms, 14,19 and the scarcity of investigations that have documented the association between symptoms and histopathological features of pulp innervation, 21,26 the microscopic characterization of pulpal NF status may contribute to a better interpretation of the pain experienced by the patient.In view of the above considerations, the purpose of the present study was to analyze the density and integrity of NF in samples of inflamed human dental pulp with a clinical diagnosis of IP and in healthy pulp (HP) based on the immunoexpression of the S-100 and PGP 9.5 markers.

Methodology Study design and dental pulp samples
A total of 21 huma n teet h ext racted for endodontic, periodontal, or surgical reasons in the Dental Urgency Service of the School of Dentistry, Universidade Federal de Goiás, Goiânia, Brazil were selected for this study as a convenience sample, as used by other authors in similar researches. 17,20ixteen of these teeth had a clinical diagnosis of inflamed pulp based on the following criteria: spontaneous symptomatology, positive response to a pulp sensibility test (1,1,1,2-tetrafluoroethane spray; Endo-Ice, Maquira, Paraná, Brazil), and absence of pulp exposure associated with dental caries.Five teeth, intact impacted third molars with indication for extraction, had the dental pulp removed and used as controls.The patients had no history of systemic diseases, nor had they used any medications (e.g., anti-inflammatory drugs) during the last three months.Also, the tooth couldn't have undergone previous restorative treatment or have clinicoradiographic findings of periapical disease. 17The study was approved by the local Ethics Committee (#102/2007).Patients' identities remained anonymous according to the Declaration of Helsinki.
All 16 teeth with inflamed pulp were properly dried and isolated before the dental pulp sensibility test.Of these teeth, two were extracted due to associated significant periodontal bone loss and fixed in 10% formaldehyde.The other teeth were treated following the protocol: anesthesia, absolute isolation of the operative field, asepsis with 1% sodium hypochlorite, and coronary opening.After determining the working length, the dental pulp was removed with Hedström files and gently placed on filter paper for fixation in 10% formaldehyde to aid in specimen positioning during histological processing.The root canal was then prepared and filled using a lateral condensation technique with gutta-percha points and an endodontic sealer.

Clinicopathologic data
Demographic data (age and sex), affected tooth, symptomatology, i.e., reported spontaneous or provoked pain that lingered, pain on palpation (absent or present), pulp test results (negative or positive), and horizontal percussion test (negative or positive) were obtained for each patient.The extracted teeth included in the study were decalcified for 90 days in ethylenediamine tetra acetic acid (EDTA).Fivemicrometers-thick sections of paraffin-embedded material obtained from each case were stained with hematoxylin and eosin (H&E).The degree of deposition of collagen fibers, the intensity of the inflammatory infiltrate, areas of exudate, vascular congestion, and state of organization of the odontoblastic layer were evaluated.Cases with intense collagen deposition were excluded from the study.

Immunohistochemistry
In each case, 3-μm-thick sections mounted o n p o l a r i z e d s l i d e s w e r e s u b j e c t e d t o immunohistochemical analysis by the polymer-based detection method.Immunohistochemical analyses were performed using monoclonal antibodies against anti-S-100 (clone N1573; Dako; 1:1000) and anti-PGP 9.5 (clone Z5116, Dako; 1:300).For the deparaffinization, rehydration and antigen-retrieval steps, the sections were submitted to TRILOGY™ Concentrate (Cell Marque; 1:100) at 96ºC in a digital water bath (DeLeo) for 30 minutes.Next, the sections were treated with the Novolink™ Max Polymer Detection System (Novocastra, Leica Biosystems Gmb) and the reactions were developed with 3.3′-diaminobenzidine (DAB; Dako).S-100 + or PGP 9.5 + NF was considered as an internal positive control, while negative controls consisted of replacement of the primary antibody with 1% bovine serum albumin in the buffer solution.

Microscopic evaluation
H&E-stained slides were subjected to quantitative analysis of the inflammatory infiltrate in 10 alternating microscopic fields at ×40 magnification.The results of the histological analysis were defined according to the method of Bruno et al. 17 and Vaz et al. 27 , i.e., a) without inflammation, when most of the fields evaluated (> 7) did not have inflammatory cells; b) mild inflammation, when most fields (> 7) had < 35% of inflammatory cells; and c) intense inflammation, when most fields (> 7) had ≥ 35% of inflammatory cells.All teeth had dental caries.The presence of exudate areas, disorganization of the odontoblastic layer, and congested vessels was also investigated.
S -10 0 + and PGP 9.5 + NF were eva luated quantitatively (NF + by density/mm 2 ).The assessment was performed on the entire pulp tissue (i.e., coronary and root area).All sections were evaluated by two examiners (E.F.M. and D.A.C.A.) in a blinded fashion.Disagreements were jointly reviewed in order to reach a consensus.Briefly, NF was quantified in each case (S-100 + and PGP 9.5 + ) and mean density in mm 2 was obtained using the area determined by an integration reticle (4740680000000-Netzmikrometer ×12.5;Carl Zeiss, Göttingen, Niedersachsen, Germany) connected to a light microscope (AxioScope; Carl Zeiss, Niedersachsen, Germany).The quantification was performed in 10 alternating fields at ×40 magnification.At this magnification, the area of one field corresponds to 0.0961 mm 2 .Then, the amount of NF PGP 9.5 (intact) was subtracted from the value of S-100 (intact and degenerated) of each case in order to obtain the number of degenerated NF.

Data analysis
Data were analyzed using the GraphPad Prism software version 7.00 (GraphPad Software, San Diego, USA).The Shapiro-Wilk test revealed a nonnormal distribution of the data.A non-parametric Mann-Whitney test was used for comparative analysis of NF density and inflammatory infiltrate.The level of significance was set at < 0.05 in all analyses.

Results
A total of 16 teeth with a diagnosis of IP were selected, 11 (68.8%)maxillary molars and five (31.2%) maxillary incisors.The teeth were extracted due to endodontic (n = 4), periodontal (n = 4), or surgical (n = 8) reasons.The mean age of the individuals was 31.2 (± 11.6) years (range: 17-65 years).All of them had a positive pulp sensibility test to cold and a closed cavity due to carious lesion, 10 (62.5%) of them reported spontaneous pain, and six (37.5%) reported provoked pain.For the HP group, five maxillary molars of individuals with a mean age of 28.3 (± 6.3) years, most of them men (n =4 ; 81.6%), were included.
In all cases of IP, mild collagen deposition, areas of exudate, vessel congestion, disorganization or degeneration of the odontoblastic layer, and a predominantly mononuclear inflammatory infiltrate were observed (Figure 1A and B).Thirteen (81.3%) cases had a mild inflammatory infiltrate, while three (18.7%)cases exhibited an intense inflammatory filtrate.The five control teeth (intact teeth) had normal microscopic pulp architecture with normally aligned odontoblasts and absence of inflammatory cells, dilated vessels, or fibrosis (Figure 1C and D).All samples were positive for S-100 and PGP 9.5.The brown labeling of these proteins was evident in the nuclear and cytoplasmic region of the NF (Figure 2).Regarding the clinical characteristics (age, sex, tooth, referred pain, pain on palpation, horizontal percussion test) of IP and their association with immunoexpression of S-100 + and PGP 9.5 + in the analysis of the density and integrity of the NF, no significant association was observed (p > 0.05).IP samples showed a lower density of intact NF regarding the expression of PGP 9.5 + (6.24/mm 2 ) compared to control samples (38.20/mm 2 ) (p = 0.01) (Figure 3A).In addition, a significantly higher density of degenerated NF was found in IP samples with spontaneous pain (39.59/mm 2 ) than in samples with provoked pain (23.96/mm 2 ) (p = 0.02) (Figure 3B).No association was observed between the density and integrity of NF and the intensity of the inflammatory infiltrate (p > 0.05).

Discussion
Pain is the only sensation induced in response to activation of pulp sensory nerves, regardless of the type of stimulus applied to the tooth.The groups of pulp nerve fibers may explain changes in the quality of symptoms in pulp inflammation.The type and duration of symptoms in teeth with pulp inflammation are of diagnostic value and can give some indication of the type of pulp disease. 12,14t should be stressed that the poor correlation between clinical symptoms and histopathological changes in pulpitis and the determination of the type and extent of inflammatory changes based on symptomatology is inaccurate. 2,3,28On this basis, in the inflamed dental pulp, the terminals of the afferent primary nociceptors detect the presence of mediators with receptors that are synthesized in the cell body of the afferent fiber and then transported to the periphery.If the mediator reaches a sufficient concentration in the inflamed tissue to activate the receptor, the nociceptive neuron can be activated, i.e., the membrane would be conducted to the central nervous system or sensitized. 3,25Thus, attention has been closely paid to toothache, described as pain originating from innervated dental tissue or tissues immediately adjacent to the teeth, since it is a common experience and is considered to be a public health concern. 29In view of the high occurrence of IP, 14,19 it is essential to understand the role of neural markers in order to elucidate and mitigate the painful symptoms that often occur in affected individuals.This study revealed a similar density of NF by immunoexpression of S-100 + for IP and HP, as well as a reduction of intact NF by PGN 9.5 + in the IP samples, as illustrated in Figure 4. Also, a higher density of degenerated NF was demonstrated in IP samples associated with spontaneous pain.These findings suggest that the evolution of inflammation may alter the axonal integrity of dental pulp NF, promoting pain conditions regardless of the presence of a stimulus.Accordingly, previous studies have used S-100 protein to identify NF in inflamed and healthy pulp. 18,21A study has detected a higher density of NF by immunoexpression of S-100B + in inflamed pulp associated with spontaneous pain than in HP samples.Although the quantification methods were not described, the authors argued that inflammation may induce NF ramifications by increasing their quantity. 18Likewise, a higher density of S-100 + structures was reported in inflamed pulps than in the control group.The authors postulated that this high density was associated with the expression of this protein in the NF, macrophages, and dendritic cells during inflammation. 21Conversely, our study carried out a comparative evaluation of NF by the expression of S-100 + in IP and HP by quantifying in a standardized manner only those structures with NF morphology that were degenerated or intact.PGP 9.5 has been employed to assess axonal integrity, with reduced expression in NF altered by neurodegenerative diseases 23,24 and leprosy. 30,31evertheless, the use of this marker has also been considered to identify NF in inflamed pulp. 20,32,33The expression of PGP 9.5 + in intact NF of inflamed pulp and HP was demonstrated by an immunofluorescence assay. 33Additionally, a study revealed a similar expression of PGP 9.5 + in inflamed healthy pulp by using a western blot assay.Certainly, this similarity occurred because the evaluation of PGP 9.5 expression was performed in nerve structures and cells with neural receptors. 20Rodd and Boissonade 32 evaluated the double expression of PGP 9.5 and substance P by indirect immunofluorescence in symptomatic and asymptomatic pulp.The authors, however, found a greater expression of PGP 9.5/substance P + in NF samples associated with pain.This is in line with our findings, which revealed a relationship between the expression of PGP 9.5, the state of integrity of pulp NF and the symptomatology, i.e., provoked or spontaneous pain.
Herein, the difference in PGP 9.5 + and S-100 + expression in intact NF resulted in the amount of degenerated NF, whose density was higher in samples with spontaneous pain.Furthermore, no association was detected between the density of degenerated NF and the intensity of the inflammatory process.In fact, the evolution of inflammation may lead to different stages of NF degeneration, particularly in type C fibers that are more resistant to aggression. 5,9,10n addition to inflammatory cells, vascular events and neuromodulatory mediators secreted in the presence of an aggression increase the volume of pulp tissue, promoting NF compression and axonal degeneration. 34,35However, the increase in inflammatory mediators may modify the sensory ion channels and depolarize the plasma membrane of the NF.In principle, this change may increase the susceptibility of the NF to triggering action potentials in the absence of any stimulus. 36lthough spontaneous pain is poorly understood, possibly the most advanced stage of degeneration of pulp NF is one of the factors responsible for the excitability of these structures.Considering the link between higher density of degenerated NF and spontaneous pain, the report of this symptom may be another important clinical parameter for the therapeutic decision of pulpectomy; however, these findings should be further explored in a large survey.
Similar to the data reported in the literature, 14,36 our IP samples were mainly from decayed molars.Our results highlight the fact that understanding the pathophysiology of pulp status can mitigate the burden on the public health system.Nonetheless, we acknowledge that this study reports preliminary results based on a limited convenience sample, a fact that may be inherent to screening of oral health services, which explains the difficulty in obtaining IP samples.Future studies should standardize patients by age group to address the hypothesis that age may influence the alteration of NF density in patients with IP.

Conclusion
In summary, a lower density of NF was found in teeth diagnosed with IP than in those diagnosed with HP, whereas the density of degenerated NF was higher in cases with spontaneous pain.These data suggest that the pulp tissue may be in different degeneration stages in cases of IP, being more advanced in cases with spontaneous pain.Thus, the referred pain can certainly contribute to the therapeutic decision of the clinician and endodontist regarding pulpectomy.

Figure 1 .
Figure 1.(A, B) Morphological characteristics detected in irreversible pulpitis samples.There are areas of disorganization in the odontoblastic layer, congested vessels, and intense, predominantly mononuclear inflammatory infiltrate.(C, D) Morphological characteristics observed in healthy pulps samples.Preserved odontoblastic layer, possible calcifications, and no signs of inflammation are present (hematoxylin and eosin, ×20 and ×40, respectively).

Figure 4 .Figure 3 .
Figure 4. Schematic figure illustrating the density of nerve fibers (NF).S-100 + similarities for irreversible pulpitis (IP) and healthy pulp, while there is a pronounced reduction of intact NF by PGN 9.5 + in IP.