Evaluation Of the Salivary Levels Of Nerve Growth Factor In Symptomatic Irreversible Pulpitis: An In Vivo Study

Keywords

Pulpitis; Nerve Growth Factor; Saliva; Pain; Nociception.

Introduction

Ever since the advent of dentistry, the primary reason for a patient seeking dental treatment has been pain. Thus, elimination of pain takes precedence and is highly important in the successful management of a patient. Pain is multifactorial, multidimensional and a complex phenomenon and a dental practitioner must be well versed with the various techniques of relieving the patient of it before, during and after a dental procedure. Various inflammatorybiomarkersand proteins are elevated during dentin-pulp complex pathologies. [1] Nerve Growth Factor which belongs to the family of Neurotrophic factors, is a protein whose levels have been proven to be elevated during inflammation and peripheralnerve injury. [2]

Growth factors are proteins that play a role in regulating cellular processes like cell proliferations, maturation and differentiation and thus are govern the growth of specific tissues. [3] Neurotrophic factors are comprised of three families of growth factors namely; Nerve Growth Factors also known as Neurotrophins, glial cell line derived neurotrophic factor and certain heterogenous molecules which belong to the family of cytokines. [4] The Neurotrophin family includes the Nerve Growth Factor, Brain Derived neurotrophic Factor, NT-3, NT-4 and NT-6. [5, 6]

Nerve Growth Factor (NGF) was discovered in 1952 by Levi- Montalcini and was the first member of the Neurotrophin family. [7] It plays a role in maintaining the phenotype and the survival of specific peripheral and central neurons during their phase of development and maturation. It has been shown that certain types sensory neurons involved in nociception require NGF for survival in utero and for their normal development during the initial post-natal phase where as in adulthood NGF is mainly involved in inflammation and hyperalgesia. [8] Nerve Growth Factor signalling is an active process which is involved in nociceptive and neuropathic pain. Nociceptive pain involves activation of nociceptors by a noxious stimulus. [9] Dental pulp contains the A? (myelinated) and C (unmyelinated) fibres which governs the pulpal pain in response to a noxious stimulus. [10]

Previously our team had conducted numerous clinical studies[11, 14] , case reports [15], in vitro studies [16, 19], surveys [20, 21] and reviews [22, 25] in various aspects of endodontics and conservative dentistry over the past five years from which the idea of the present study has stemmed. Thus, the present study was conducted to evaluate the levels of Nerve Growth Factor in saliva of patients suffering from symptomatic irreversible pulpitis.

Aim and Objectives

The aim of the present study was to evaluate the salivary level of Nerve Growth Factor in condition of symptomatic irreversible pulpitis.

The following were the objectives of the study:

To determine and compare the levels of salivary NGF in patients diagnosed with symptomatic irreversible pulpitis and symptom free healthy individuals.

To determine the significance of salivary NGF as a diagnostic marker for pain in symptomatic irreversible pulpitis. To evaluate the pharmacological implication of the involvement of neurotrophic factors in eliciting pulpal pain.

Materials and Methods

The present study comprised of two groups with a sample size of 10 per group. Group A comprised of patients in whom a single tooth was diagnosed with symptomatic irreversible pulpitis and Group B was the control group of symptom free healthy individuals. Patients with multiple decayed or multiple pulpally involved teeth, patients with periodontitis and gingivitis and patients with any other systemic condition were excluded from the study. The patients were informed about the study and asked to sign a consent form.

The salivary samples were collected between 9 am in the morning and 12 noon by asking the patients to pool their saliva and then spit it in the given sterile containers. The patients were abstained from eating or drinking two hours prior to the collection of saliva. The samples were then analysed for the level of Nerve Growth Factor.

Human NGF ELISA Kit was purchased from Thermo scientific, USA. Addition of 100µL of each standard and saliva was done in to appropriate pre coated wells. The wells were covered and incubate for 2.5 hours at room temperature or overnight at 4ºC with gentle shaking. The solution was discarded after which each well was filled with Wash Buffer (300µL) using a multi-channel Pipette and washed 4 times with 1X Wash Buffer. To each well, 100µL of 1X prepared biotinylated antibody specific to human nerve growth factor was added and Incubate for 1 hour at room temperature with gentle shaking. Next, 100µL of prepared Streptavidin- HRP solution was added to each well and incubate for 45 minutes at room temperature with gentle shaking followed by addition of 100µL of TMB Substrate to each well. It was incubated for 30 minutes at room temperature in the dark with gentle shaking. The plates were evaluated within 30 minutes of stopping the reaction. The absorbance was measured on an ELISA plate reader set at 450nm.

The data obtained from the experiment was expressed as Mean +/- standard deviation. For statistical analysis, data was subjected to one-way analysis of variance (ANOVA) followed by Student’s t-test. The Dunnett post hoc analysis was performed to compare each experimental test group’s mean result to a control group’s mean result. A level of P<0.01 was taken as significant. The statistical analysis was done using the SPSS statistical package (version).

Results and Discussion

The results of the present study demonstrate a significantly higher level of Nerve Group Factor (*P<0.01) in the salivary samples of the patients diagnosed with symptomatic irreversible pulpitis (8.33 ± 0.60*) as compared to the control group (4.03 ± 0.47*) (Figure 1).

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Figure 1:

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Figure 1: Levels of nerve growth factor in symptomatic irreversible pulpitis. The X axis represents the two groups under the study and the y axis represents the levels of nerve growth factor as detected in the saliva.

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Saliva is considered as the reflection of the body’s state of health and disease and a variety of analytes from systemic sources reach the oral cavity through various pathways and are found in saliva. [26] This is a novel study where the levels of salivary NGF has been evaluated in patients diagnosed with symptomatic irreversible pulpitis. According to the results of the present study, the levels of salivary NGF were significantly higher in patients with symptomatic irreversible pulpitis as compared to healthy individuals.

Nerve Growth Factor acts by binding to two types of surface receptors: Neurotrophin receptors p75 for which is has low affinity and tropomyosin-related kinase A (trkA) receptor for which it has high affinity. [27] The trkA receptor is selectively expressed on the peripheral terminals of A-delta nerve fibres and unmyelinated Cfibers. [27, 28] According to the“neurotrophic factor hypothesis” and the classical neurotrophic model, the target tissues synthesize and released and release NGF during embryonic development which promotes the growth, differentiation, and survival of neurons in a dose dependent manner. [29] During embryogenesis, the sensory neurons of the dorsal root ganglia (DRG) shows a higg expression of TrkA; however, postnatally there is a shift of NGF–trkA signalling from promoting the growth of neuron and its survival to regulating peripheral nervous system’s sensitivity to a noxious stimulus. [30]

Nociceptors which are located in peripheral tissues are activated in response to noxious stimuli thereby causing nociceptive pain. Any stimulus (eg, chemical, thermal, or mechanical) that either damages or threatens to cause damage to normal tissues is a noxious stimuli. Following noxious stimuli (eg, injury and inflammation), NGF is produced and released by peripheral tissues secondary to the release of inflammatory cytokines, such as interleukin-1 and tumor necrosis factoralpha. Effects on pain signalling is modulated by the binding of NGF to the trkA receptors on multiple targets. [28] Once the NGF–trkA complex is formed, it gets internalized and is transported retrogradely to DRG cell bodies where it modulates and /or increases the expression of a variety of cell surface receptors involved in nociception. [16] The binding of NGF to the trkA receptors located on mast cells causes an additional effect on pain processing. This process is proinflammatory and a positive feedback loop is generated by eliciting the release of inflammatory mediators such as histamine, serotonin or 5-hydroxytryptamine (5-HT), protons, as well as NGF itself. [28, 31] Thus, NGF signalling plays two roles, it increase the expression of nociceptive receptors located peripherally and pronociceptive neurotransmitters located centrally, and in response to inflammation, it also sensitizes adjacent nociceptive neurons.

A study conducted Woodnut et.al evaluated the expression of neurotrophin receptors and NGF in nonneuronal cells of normal and injured tooth pulp. The study showed an upregulation of NGF in injured pulp and its accumulation in surviving odontoblast cells. [32]

A study conducted by Mitsiadis et.al showed a weak expression of NGF, p75NTR and dental pulp fibroblasts and odontoblasts of intact functional teeth, while a strong expression of NGF and p75NTR molecules was seen in nerve fibres that innervated the dental pulp. An upregulation of NGF and TrkA was seen in carious and injured teeth in odontoblasts surrounding the injury sites. This indicated a correlation between NGF signalling and dental tissue repair events. [33]

Non-steroidal anti-inflammatory (NSAIDs) are routinely used for the management and control of pain. [34] The discovery of Nerve Growth Factor has led to the exploration of newer pharmacological approaches targeting the NGF pathway for effective pain management. These approaches mainly aim at sequestration of NGF, prevention of binding of NGF to trkA receptor and inhibition of trkA function. Nerve Growth Factor sequestration involves the use of NGF antibody. [2] The prevention of binding of the factor to its receptor was done using mouse monoclonal anti-trkA, MNAC13. It was capable of inducing analgesia in models of inflammatory and neuropathic pain. A synergistic was observed when it was used in combination with low-dose opioids. However, analogous species were not introduced in to clinical trials due to lack of equivalent humanized antibody. [35] k252a is a small-molecule protein kinase inhibitor that inhibits the activation of the entire tropomyosin receptor kinase family (trkA, trkB, and trkC). However, due to lack of specificity, no human trials were ever been initiated. [36]

Conclusion

The activity of NGF and its interaction with trkA in nociceptive and inflammatory pain has been well established. The sequestration of NGF and inhibition of trkA signaling have demonstrated a consistent analgesic effect in the preclinical models of inflammatory and visceral pain.

The present study demonstrates an elevation of the levels of nerve Growth Factor in saliva during pulpitis suggesting its role in inflammation of pulp and dental pain. This could serve as a non-invasive prognostic marker for pulpitis and could form the basis for the development of new pharmacological approaches for effective management of dental pain.

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