WITHDRAWN: Catecholamines in healthy and inflamed pulp of primary teeth

doi:10.21203/rs.3.rs-4124784/v1

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WITHDRAWN: Catecholamines in healthy and inflamed pulp of primary teeth

https://doi.org/10.21203/rs.3.rs-4124784/v1

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Introduction: The dental pulp, traditionally recognized for its roles in dentinogenesis and pain perception, is a dynamic macrocosm. Recent research has unveiled a complex network of neurotransmitters, with catecholamines, including dopamine, adrenaline, and noradrenaline, emerging as a particularly understudied component.

Objective: To elucidate the role of catecholamines in both inflamed and non-inflamed pulp of primary teeth. Documented scientific evidence suggests higher catecholamine levels in inflamed dental pulp, which significantly contribute to maintaining overall pulp health.

Methods: Twenty children aged 6 to 8 were selected and categorized into healthy and inflamed pulp groups. After thorough clinical examination and radiographic evaluation, the teeth with deep carious lesions indicative of irreversible pulp changes were subjected to cold and heat tests. Only those teeth positively responding to heat and cold tests were finally included. The concentration of catecholamines was calculated based on the peak area found on HPLC graphs and the retention time of each catecholamine compared with the already established standard curves for pure drugs.

Results: Statistical analysis was completed using SPSS for Windows- version 17.0. Independent t-test and Mann- Whitney U test were used for inter-group comparisons. The mean concentrations of Norepinephrine, epinephrine, and dopamine in the inflamed pulp surpassed those in the healthy pulp. However, no significant differences in catecholamine concentrations between boys and girls were observed.

Conclusion: Norepinephrine, epinephrine and dopamine concentrations were notably elevated in inflamed pulp compared to healthy pulp. Heightened catecholamine concentration is attributed to increased nerve stimulation, suggesting robust synthesis during stressful conditions like inflammation.

Catecholamines

Inflammation

Pain

Primary teeth

Pulpitis

The dental pulp is a highly vascularized tissue with many tiny blood vessels comprising arterioles, capillaries, venules, and highly innervated autonomic and sensory fibres. The primary and permanent teeth are highly vascularized; however, the primary teeth never attain the extent of neural development that occurs in permanent teeth [1, 2]. Over the past century, much research has been conducted on the intense innervation of the dental pulp, a peripheral end-organ lacking collateral nerves [3].

The condition of the dental pulp largely depends on the state of the dentin or enamel; any injury to it affects the pulp [4]. It has been established that pulp is a delicate vascular structure that can survive a series of insults by thermal stimuli, i.e. heat or cold, and attack by toxins of microorganisms that result in tooth decay. Depending on the type of irritant and its potency, this can lead to either degeneration of pulp tissue or to more minor changes, which can be repaired due to the regenerative capacity of the pulp.

Pulp inflammation is a dynamic and complex process involving several neural, vascular and immunological reactions [5, 6]. Numerous molecules secreted by nerve endings, including excitatory amino acids, neuropeptides, and classical neurotransmitters, are responsible for the regulatory activities during pulpal inflammation. Pulpitis is one of the most common oral diseases and is often associated with severe dental pain. In pulpitis, dental nerve fibres transmit pain impulses and regulate inflammatory responses [3, 7, 8]. The amino acids which are present in abundance in the human dental pulp play a crucial role in the defence mechanism against insult.

There are five established biogenic amine neurotransmitters: Dopamine, Norepinephrine, Epinephrine, histamine and serotonin. Catecholamines, namely Epinephrine, Norepinephrine and Dopamine, are essential to neuroendocrine regulation. They are biosynthesized from the amino acid L-tyrosine [9]. Catecholamines are present in human dental pulp and have a significant clinical implication as they play an essential role in controlling intra-pulmonal pressure via acting as a mediator of vasoconstriction [10].

Vasoconstriction is brought on by stimulation of the α and β- adrenoreceptors that innervate the sympathetic blood vessels of the dental pulp [11]. The vascular muscle contraction, which results in vasoconstriction, is caused by the α receptors. However, there has been no indication of β-adrenergic vasodilation in human tooth pulp [12].

After being released from storage, catecholamines attach to particular receptors on cell membranes known as adrenoreceptors [11]. The pace at which humans synthesize catecholamines rises dramatically in response to unusual stressors such as childbirth, burns, colds, hypoxia, immobilization, or physical activity. Individuals experiencing pain-related pulpal inflammation may also exhibit elevated pulp catecholamine production [10].

Catecholamines can be quantified using High-Performance Liquid Chromatography (HPLC). It was suggested that HPLC with electrochemical detection is sufficiently sensitive for analyzing the concentrations of Norepinephrine, Epinephrine, and Dopamine in small amounts of plasma (0.2 to 2.0 ml) [13]. Hence, the present study was designed to quantify catecholamines using high-performance liquid chromatography in healthy, inflamed human primary teeth pulp.

Ethic statement:

This study was designed as a human observational case control study. All parents or guardians of the participants provided written informed consent for this institutionally approved study. Approval for this study was obtained from the Maharishi Markandeshwar University. Approval to conduct this study was given by the Institution Ethics Committee.

A total of 20 children in the age group of 6 to 8 years were selected from the outpatient department of Pediatric and Preventive Dentistry, Maharishi Markandeshwar University, after confining the case definitions, inclusion and exclusion criteria for each respective Group. The detailed plan and purpose of the study were explained, and informed written consent was obtained from the accompanying parent/guardian of each child. The teeth to be included in the study were categorized into the following groups:

Group I - Primary Teeth with Healthy Pulp (n = 10)
Group II - Primary Teeth with Inflamed Pulp (n = 10)

The teeth were selected into a particular group based on the following case definitions:

Primary tooth- healthy pulp (Group I): A retained primary tooth with no associated pain or evidence of dental caries and not more than one-third root resorption.
Primary tooth- inflamed pulp (Group II): A deep carious lesion in the primary tooth with history, clinical and radiographic findings indicative of irreversible pulpal changes along with a positive response to pulp sensibility tests (heat or cold) but without any calcific changes on the radiograph.

Exclusion criteria for both Group I and II

History of trauma with the associated tooth.
Presence of any systemic disease
Any ongoing anti-inflammatory or systemic medication.

After thorough clinical examination and radiographic evaluation, the teeth with deep carious lesions indicative of irreversible pulp changes were subjected to cold and heat tests. The teeth were air-dried and subjected to a heat test using a gutta-percha stick. Then, an ice stick was used on the middle 1/3rd of the labial surface of the tooth to be tested and the contralateral tooth for comparative evaluation. Only those teeth positively responding to heat and cold tests were finally included.

These children were assigned and coded with a sequential number as per the Group in which they were categorized, i.e., numbers 1 to 10 for Group I and 11 to 20 for Group II, respectively.

In each Group, surface anaesthesia (lignocaine jelly 2%) was applied at the injection site. This was followed by parenteral administration of local anaesthesia without Epinephrine to prevent the introduction of exogenous Epinephrine. Maxillary teeth were anaesthetized by local infiltration, and mandibular teeth were anaesthetized by mandibular nerve block.

The required procedure was performed after ensuring adequate subjective symptoms of the anaesthetic effect.

Healthy pulp: Group I

Teeth were extracted as traumatically as possible by only one operator and immediately stored in an isotonic saline solution. Pulpal tissue was then obtained with appropriately sized endodontic broaches within 5 minutes of extraction by cutting off the crown with the help of a straight diamond bur attached to a high-speed handpiece with a water spray.

Then, extirpation of pulp tissue was performed judiciously using endodontic broaches of an appropriate size to remove the pulp intact and avoid tearing.

Inflamed pulp: Group II

The access cavity was prepared, and the coronal pulp was removed using a spoon excavator. Radicular pulp tissue was extracted judiciously using endodontic broaches to remove the pulp intact and avoid tearing.

Pulp Sample Collection and Analysis

Tissue Handling:

For all four groups, pulp tissue was placed in a pre-weighed Eppendorf tube immediately after extirpation. It was again weighed using a high-precision electronic balance to determine the exact weight of pulp tissue.

Then, 0.5 ml of 0.1 M perchloric acid and 0.5 ml of 1mM sodium-metabisulfite were added to each sample. The samples were labelled with the same number that had already been assigned to the corresponding patient. Samples were then stored at -80˚C.

Extraction of catecholamines:

The technique of Opacka-Juffry et al. [13] was strictly followed to extract catecholamines from the dental pulp.

HPLC analysis:

Chromatographic conditions

Chromatographic separation was performed in HPLC (Shimadzu Corporation, LC2010C HT, Kyoto, Japan) equipped with column Luna 5u C18 (2) 100A (250 x 4.6 mm), Phenomenex, USA. Detection was made at 280 nm with an Ultraviolet (UV) detector. The sample cooler temperature was maintained at five °C, and the column temperature was maintained at 25°C. The system was run at a flow rate of 1 ml/ minute.

Preparation of mobile phase

The mobile phase was phosphate buffer (pH 4.5, 50mM concentration) acetonitrile (95:5 v/v). The 50nM phosphate buffer was prepared by dissolving potassium dihydrogen phosphate (3.5 gm) in 450 ml freshly prepared de-ionized distilled water, and to this solution, 54.0 mg of 1-heptane sulphonic acid sodium salt was added, as previously explained by Yu and coworkers [14]. After it was dissolved entirely, the dilution was made to 500 ml to obtain a concentration of 50 mM and the pH was adjusted to 4.5 using orthophosphoric acid. The mobile phase was filtered through a 0.45 µm membrane filter, degassed through an ultrasonicator, and gradient delivered at a 1 ml/minute flow rate.

Preparation of standard solutions

The sequential concentrations of 1, 2 and 3µg/ml of standard pure drugs of Norepinephrine, Epinephrine and Dopamine were prepared in distilled water and then filtered with a syringe filter in separate microtubes. 20 µl volume of each sample was injected, and the effluent was monitored at 280 nm.

Each HPLC analysis of standard compounds or extracted samples was run for 15 minutes. The same mobile phase was used to run the catecholamines extracted from each pulp sample in HPLC. The chromatographic data were collected and integrated using LC Solution 2010 software with a computerized data station. The standard drugs were run to estimate the retention time and calculate the concentration of catecholamines, i.e., Norepinephrine, Epinephrine, and Dopamine.

The concentration of individual catecholamines (i.e. Norepinephrine, Epinephrine and Dopamine) was calculated based on the peak area obtained on HPLC graphs and the retention time of each catecholamine compared with the already established standard curves for pure drugs. The calculations were made by using the proportion of peaks and areas with the following formula with standard purity taken as 100%:

Concentration of catecholamines (µg/g)=

\(\frac{\text{A}\text{r}\text{e}\text{a} \text{o}\text{f} \text{s}\text{a}\text{m}\text{p}\text{l}\text{e}}{ \text{a}\text{r}\text{e}\text{a} \text{o}\text{f} \text{s}\text{t}\text{a}\text{n}\text{d}\text{a}\text{r}\text{d}}\) × \(\frac{\text{a}\text{m}\text{o}\text{u}\text{n}\text{t} \text{o}\text{f} \text{s}\text{t}\text{a}\text{n}\text{d}\text{a}\text{r}\text{d} \left(\text{m}\text{g}\right)}{ \text{a}\text{m}\text{o}\text{u}\text{n}\text{t} \text{o}\text{f} \text{s}\text{a}\text{m}\text{p}\text{l}\text{e} \left(\text{m}\text{g}\right)}\) × \(\frac{\text{d}\text{i}\text{l}\text{u}\text{t}\text{i}\text{o}\text{n} \text{f}\text{a}\text{c}\text{t}\text{o}\text{r} \text{o}\text{f} \text{s}\text{a}\text{m}\text{p}\text{l}\text{e} }{\text{d}\text{i}\text{l}\text{u}\text{t}\text{i}\text{o}\text{n} \text{f}\text{a}\text{c}\text{t}\text{o}\text{r} \text{o}\text{f} \text{s}\text{t}\text{a}\text{n}\text{d}\text{a}\text{r}\text{d}}\) × \(\begin{array}{c}\text{S}\text{t}\text{a}\text{n}\text{d}\text{a}\text{r}\text{d}\\ \text{p}\text{u}\text{r}\text{i}\text{t}\text{y}\end{array}\)

Statistical analysis:

Statistical analysis was completed using SPSS(Statistical Package for Social Studies) for Windows, version 17.0. Independent t-tests and the Mann- Whitney U test were used for inter-group comparisons.

A total of 20 children in the age group of 6 to 8 years were selected based on the informed written consent obtained from the accompanying guardian/parent. The teeth were assigned into a particular group based on the state of the pulp, i.e. healthy pulp Group I and Primary tooth- inflamed pulp Group II. The eligible children were categorized out of the examined children, i.e., numbers 1 to 10 for Group I and 11 to 20 for Group II, respectively. These 20 children were analyzed and completed the study protocol without dropouts. The study participants were of Indian origins and were local to that region. Two observers examined them clinically and radiographically, and the same age range was used for both groups to remove any confounding factors.

The mean retention time of catecholamines, i.e. Norepinephrine, Epinephrine and Dopamine, was 5.941 (± 0.044) minutes, 8.531 (± 0.070) minutes and 12.939 (± 0.486) minutes, respectively, on HPLC measured by taking sequential concentrations of their standard pure drugs is depicted by graphs in Fig. 1,2, and 3 respectively.

Figure 4 graph depicts the concentration of Norepinephrine (µg/g of pulpal tissue) measured by HPLC in Group I and Group II. Figure 5 graph depicts the concentration of Epinephrine (µg/g of pulpal tissue) measured by HPLC in Group I. Figure 6 graph depicts the concentration of Dopamine (µg/g of pulpal tissue) measured by HPLC in Group I.

The mean concentration of Norepinephrine, Epinephrine and Dopamine for Group II (inflamed pulp) was greater than that of Group I (healthy pulp). The difference in the mean concentration of Norepinephrine, Epinephrine and Dopamine between Groups I and II, when analyzed statistically by Mann Whitney U test, was significant, as shown in Table 1.

Table 1

Concentration of catecholamines (µg/g of pulpal tissue) measured by HPLC technique in dental pulp in Group I (Healthy pulp) and Group II (Inflamed pulp) using Mann Whitney U test
Mean Conc. (± SD) Groups	Norepinephrine (µg/g)	Epinephrine (µg/g)	Dopamine (µg/g)
Group I	0.193638 (± 0.0831)	0.152766 (± 0.1067)	0.157398 (± 0.1099)
Group II	1.213302 (± 0.3283)	1.144935 (± 1.3431)	1.308048 (± 0.5090)
Significance	z= -3.674 p < 0.001***	z= -3.024 p = 0.0025**	z= -3.511 p < 0.001***

Table 2 shows the influence of gender on variation in the concentration of catecholamines in dental pulp amongst children. The difference in concentration of Norepinephrine, Epinephrine, and Dopamine amongst boys and girls of Group I and Group II was statistically insignificant when analyzed using an Independent t-test.

Table 2

The concentration of catecholamines in dental pulp varies according to the gender of children in Group I (Healthy pulp) and Group II (Inflamed pulp) using Independent t-test analysis.
Catecholamine	Groups	Mean conc. (± SD) (µg/g of pulpal tissue)		Significance
Catecholamine	Groups	Boys	Girls	Significance
Norepinephrine	Group I	0.210482 (± 0.1041)	1.767944 (± 0.0631)	t = 0.619 df = 8 p = 0.553
Norepinephrine	Group II	1.060188 (± 0.1911)	1.335793 (± 0.3821)	t= -1.305 df = 7 p = 0.233
Epinephrine	Group I	0.192894 (± 0.1394)	0.112637 (± 0.0463)	t = 1.222 df = 5 p = 0.278
Epinephrine	Group II	1.711628 (± 1.7641)	0.578242 (± 0.3798)	t = 1.404 df = 8 p = 0.198
Dopamine	Group I	0.198743 (± 0.1436)	0.116053 (± 0.0477)	t = 1.222 df = 5 p = 0.278
Dopamine	Group II	1.433368 (± 0.3325)	1.151399 (± 0.6962)	t = 0.808 df = 7 p = 0.446

Catecholamines are hormones that also function as neurotransmitters. The body produces them in the brain, nerve tissues, and adrenal glands. The adrenal glands are above the kidneys [11]. In 1995, Schachman and coworkers were the first to determine the quantity of catecholamines in uninflamed dental pulp [15]. Catecholamine levels found to be present in the pulp during inflammation were more significant than the baseline level established in uninflamed pulp tissues [10]. Thus, the current clinical study aimed to quantify catecholamines in both healthy and inflamed primary human teeth pulp using high-performance liquid chromatography

The age range was selected as 6 to 8 years to study exclusively the primary teeth. Boys and girls were included proportionately in each Group to determine if gender correlated with the concentration of catecholamines in dental pulp.

Because of the relatively low levels of catecholamines and their metabolites in various physiological fluids, extreme care must be exercised during sample collection, storage and processing to avoid contamination of samples and losses due to decomposition. Thus, all glassware handling the samples must be carefully washed and acid-cleaned. Due to the known instability of catecholamines in primary media, the sample must be acidified before storage, so 0.1M perchloric acid was added to each sample. In addition, 1mM sodium metabisulphite as an anti-oxidant was added to maintain the compounds in their reduced form. This is particularly important for electrochemical detection since catecholamines are generally monitored in the oxidative mode. The samples were stored at a temperature of -80˚C before their analysis was finally done to minimize the loss of catecholamines from the sample. This was done in compliance with the recommendations for the storage of samples to estimate catecholamines [13, 16, 17]. The other similar studies also stored the pulp samples at -80˚C till the HPLC analysis was done [10, 15].

Extraction of catecholamines from pulp samples-

The technique of Opacka-Juffry and coworkers [13] was strictly followed to extract catecholamines from dental pulp. Similar methods for extracting catecholamines from pulp samples were used in similar studies estimating catecholamines from dental pulp [10, 15].

Opacka-Juffry [10] stated that the susceptible HPLC system, in combination with this improved solvent extraction technique, offers a reliable method for determining Epinephrine and other catecholamines in very small samples.

Catecholamines: Norepinephrine, Epinephrine and Dopamine-

The low concentration of catecholamines and the presence of contamination hamper the measurement. Otherwise, the individual variability of the quantity of catecholamines in human dental pulp may also play an important role. The detection of catecholamines was performed using the high-performance liquid chromatography (HPLC) technique. As stated in earlier studies, this method is precise, accurate, and sensitive due to the electrochemical detection of catecholamine content [10, 17, 18].

The mean retention time of standard pure drugs of Norepinephrine, Epinephrine and Dopamine obtained from the standardization curves were 5.9 minutes, 8.5 minutes and 12.9 minutes, respectively. In the study by Walton and coworkers [15] the retention time of Norepinephrine, Epinephrine and Dopamine were 8.5, 9.5 and 12 minutes, respectively. In another study done by Wang and coworkers [19], the retention time was 7, 11.5 and 8.5 minutes for Norepinephrine, Epinephrine and Dopamine, respectively. These studies' retention time differed from the present study's retention time because the mobile phase and the column diameter used differed from that used in the present study. The column length and the mobile phase used directly influence the retention time.

The values for the mean concentration of Norepinephrine, Epinephrine and Dopamine in dental pulp for Group I was 0.193638 µg/g (± 0.0831), 0.152766 µg/g (± 0.1067) and 0.157398 µg/g (± 0.1099) of pulpal tissue respectively and for Group II was 1.213302 µg/g (± 0.3283), 1.144935 µg/g (± 1.3431) and 1.308048 µg/g (± 0.5090) of pulpal tissue respectively. In reviewing the current literature, it was found that the present study was the first in which human pulp tissue of primary teeth was analyzed and quantitated for the presence of catecholamines.

The mean concentrations of Norepinephrine, Epinephrine and Dopamine were higher in inflamed pulp when compared with healthy pulp. These differences were found to be statistically significant between Group I and II for Norepinephrine (p < 0.001), epinephrine (p = 0.002) and dopamine (p < 0.001). These findings agree with the work of Nup and coworkers, who demonstrated that the catecholamine levels found to be present in the pulp during inflammation were more significant than the baseline level established in the uninflamed pulp tissue of permanent human teeth (Norepinephrine and Epinephrine, p < 0.05; Dopamine, p < 0.01) [10]. This significant rise in the concentration of catecholamines in inflamed dental pulp compared to healthy dental pulp can be attributed to nerve stimulation increasing the synthesis of catecholamines. Hence, in stress situations, the production of catecholamines is greatly enhanced. Patients who undergo pain-associated pulpal inflammation may also produce increased catecholamines in the pulp [15].

Influence of gender on catecholamine concentration-

The difference in the mean concentration of all the catecholamines, i.e. Norepinephrine, Epinephrine and Dopamine, amongst boys and girls was not found to be statistically significant (p > 0.05).

Therefore, no influence of gender was found on the concentration of catecholamines, i.e. Norepinephrine, Epinephrine and Dopamine in dental pulp in the present study. In the previous studies conducted to estimate the concentration of catecholamines in dental pulp, the influence of gender on the concentration of catecholamines was not analyzed. Also, no other relevant literature was found in this context.

The values for the mean concentration of Norepinephrine, Epinephrine and Dopamine measured by HPLC technique in dental pulp for Group I was 0.193638 µg/g (± 0.0831), 0.152766 µg/g (± 0.1067) and 0.157398 µg/g (± 0.1099) of pulpal tissue respectively and for Group II was 1.213302 µg/g (± 0.3283), 1.144935 µg/g (± 1.3431) and 1.308048 µg/g (± 0.5090) of pulpal tissue respectively.

The mean concentrations of Norepinephrine, Epinephrine and Dopamine were higher in inflamed pulp than the baseline levels established in healthy pulp. These differences were found to be statistically significant between Group I and II for Norepinephrine (p < 0.001), epinephrine (p = 0.002) and dopamine (p < 0.001). This significant rise in the concentration of catecholamines can be attributed to nerve stimulation increasing the synthesis of catecholamines. Hence, in stressful situations like inflammation, the production of catecholamines is greatly enhanced.

Acknowledgements:

We would like to thank all those who participated in the study groups.

Author contributions:

Dr. Satyawan G Damle: contributed to conception, design, data acquisition, analysis and interpretation, drafted manuscript and critically revised manuscript

Dr Nilika Chopal: Contributed to the conception, design, data acquisition, analysis and interpretation, drafting of the manuscript, and critically revised manuscript.

Dr. Gaurav Kamboj: contributed to conception, design, data acquisition, analysis and interpretation, drafted manuscript and critically revised manuscript

Dr. Abhishek Dhindsa: contributed to conception, design data acquisition, analysis and interpretation, drafted manuscript and critically revised manuscript

Dr. Dhanashree. Sakhare: contributed to conception, design data acquisition, analysis and interpretation, drafted manuscript and critically revised manuscript

Dr. Shreya Dasgupta: contributed to design data analysis, drafted and critically revised manuscript.

All authors gave their final approval and agreed to be accountable for all aspects of the work.

Ethics declarations:

Ethics approval and consent to participate

All parents/ guardians of the participants signed an ‘informed consent’ before the study participation. This study was designed as a human observational case control study. All parents or guardians of the participants provided written informed consent for this institutionally approved study. Approval for this study was obtained from the Maharishi Markandeshwar University. Approval to conduct this study was given by the Institution Ethics Committee.

Consent for publication:

No individual data is contained in this study.

Competing interests:

The authors declare that they have no competing interests.

Data availability:

The dataset generated during the current study is available in the tables attached. The datasets generated and/or analysed during the current study are not publicly available due to protected health information but de-identified data are available from the corresponding author on reasonable request.

Funding:

None

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WITHDRAWN: Catecholamines in healthy and inflamed pulp of primary teeth

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