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Azerbaycan Saytlari

 »  Home  »  Endodontic Articles 1  »  Quantitative analysis of substance P, neurokinin A and calcitonin gene-related peptide in pulp tissue from painful and healthy human teeth
Quantitative analysis of substance P, neurokinin A and calcitonin gene-related peptide in pulp tissue from painful and healthy human teeth
Introduction - Materials and methods.



Introduction.
Odontogenic pain is usually caused by either noxious physical stimuli or by the release of inflammatory mediators that stimulate receptors located on terminal endings of nociceptive afferent nerve fibres (Hargreaves & Dubner 1992). Nociceptive fibres are distributed throughout the body (Kumazawa 1996) and are particularly prevalent in the trigeminal nerve which innervates dental pulps and periapical tissues (Hargreaves 1998). However, the exact role of chemical mediators in the pathogenesis of toothache remains unclear.
The diagnosis of pulpal pain can be difficult, because the site of the pain often cannot be precisely located (Dummer et al . 1980). This is due, in part, to the fact that there are two types of afferent nerve fibres within the pulp which are activated in different ways (Närhi et al . 1996). Myelinated A-fibres, stimulated by cold, heat or drilling cause fast, well localized pain, whereas unmyelinated C-fibres, activated by stimuli which cause damage to pulp tissue produce dull, poorly localized pain (Närhi et al . 1992). It is now known that these C-fibres respond to chemical mediators released as a result of tissue damage (Ngassapa 1996).
Recently, the subgroup of sensory nerves containing substance P (SP), neurokinin A (NKA) and calcitonin gene-related peptide (CGRP) has been the subject of much research (Silverman & Kruger 1987, Olgart et al. 1993, Kerezoudis et al . 1994, Buck et al . 1999). SP, NKA and CGRP are abundant in the pulp and periodontium (Kim 1990, Ohkubo et al . 1993) and are the only pulpal peptides to date which have been shown to originate from the trigeminal ganglion (Gazelius et al . 1987, Wakisaka 1990). The peptides contained in these neurones are found in unmyelinated C-fibres and probably some Adelta fibres. On excitation, these nerves release neuropeptides, producing an inflammatory response which is often called neurogenic inflammation (Györfi et al . 1993). SP, NKA and CGRP released by sensory fibres exert profound effects on blood flow, inflammatory and immune responses, and connective tissue cells and thus may be significant in many inflammatory conditions including pulpal pain (Gazelius et al . 1987, Wakisaka 1990, Olgart 1996, Pashley 1996).
There is good evidence to show that peptides are important in neurogenic inflammation but their role in pulpal pain remains unclear. Therefore, the aim of this study was to compare the levels of SP, NKA and CGRP in human dental pulp tissue from painful and non-painful teeth.

Materials and methods.

Patient selection.
The study was approved by the local Research Ethics Committee of Queen’s University Belfast and all subjects ( n = 66) gave their written informed consent. The pain group consisted of 46 subjects (28 male and 18 female patients; mean age 32.3, range 18–71 years) complaining of toothache, who attended a dental hospital for treatment of their dental pain. The control group comprised 20 subjects (10 male and 10 female patients; mean age 13.9, range 11–16 years), who required extraction of healthy teeth for orthodontic reasons.

Inclusion and exclusion criteria.
The inclusion criteria for the pain group were that the subjects were adults (18 years or older), diagnosed as having acute irreversible pulpitis with no evidence of periapical inflammation and no previous treatment for the symptomatic tooth.
Exclusion criteria were conditions requiring antibiotic prophylaxis or additional treatment procedures, debilitating disease, chronic pain, diabetes, haematological disorders or a history of taking centrally acting drugs (e.g. tricyclic antidepressants), known to interfere with the release of neuropeptides, within the previous 6 months.

History and examination.
Demographic details, the history of the pain, the medical history (including full details of any analgesic medication) and the patient’s smoking habits were recorded. Routine clinical and radiographic examinations were performed prior to treatment. Pain levels were assessed using a visual analogue scale (VAS). Each patient quantified the intensity of pain by placing a mark on a 10-cm VAS running from ‘no pain’ to ‘unbearable pain’.

Collection of pulp tissue from endodontically treated painful teeth.
Local anaesthesia (2% lignocaine with 1 : 80 000 adrenaline) was administered and an access cavity was prepared under rubber dam isolation. Pulp tissue was extirpated using a barbed broach or Hedström-file, placed into a preweighed Eppendorf tube, immediately frozen in liquid nitrogen and stored at –70 C (Gazelius et al . 1987). The root canals were thoroughly irrigated with 1% sodium hypochlorite, dried with paper points and dressed with either calcium hydroxide ( n = 15) or Ledermix paste (Lederle Laboratories, Gosport, UK) ( n = 25). The access cavity was then sealed with a cotton pellet and glass ionomer cement until the next visit.

Collection of pulp tissue from painful and non-painful extracted teeth.
Six patients choose to have extraction of the painful teeth instead of root canal treatment. All extractions of painful and non-painful teeth were performed under local anaesthesia (2% lignocaine with 1 : 80 000 adrenaline) with no complications. All extractions were completed in less than 2.5 min. Immediately after extraction, each tooth was split in a vice fitted with a cutting edge and the pulp tissue was removed using a barbed broach or Hedströmfile within 1–2 min. The pulp was then placed in a preweighed Eppendorf tube, immediately frozen in liquid nitrogen and stored at –70 C.

Extraction of neuropeptides from pulp tissue.
The extraction methodology was based on the results obtained from a previous study (Awawdeh et al . 1999). Briefly, the Eppendorf tube was weighed and the pulp weight was calculated. The required amount of 0.5 mol L 1 acetic acid was measured in a ratio of 8 mLg –1 tissue and brought to boiling in a water bath. The pulp tissue, whilst still in a partly frozen condition, was immersed in the acetic acid for 10 min. The specimen was then centrifuged (2200 g , 20 min, 4 C) and the supernatant was collected and stored at –70 C.

Concentrations of SP-Ir, NKA-Ir and CGRP-Ir in painful and non-painful pulp tissue
Table 1. Concentrations of SP-Ir, NKA-Ir and CGRP-Ir in painful and non-painful pulp tissue.

Radioimmunoassay (RIA).
The substance P-immunoreactivity (SP-Ir), neurokinin A-immunoreactivity (NKA-Ir), and calcitonin gene-related peptide-immunoreactivity (CGRP-Ir) were determined by radioimmunoassay (RIA) using antisera SP 152(2), NKA 570(3) (Wellcome Research Laboratories, Queen’s University, Belfast, UK) and CGRP RAS6012 (Peninsula Laboratories, St. Helens, UK). All radioactive tracers were supplied by Amersham International (Little Chalfont, UK).
The assay system consisted of a total volume of 300 L, comprising 100 L antiserum, 100 L standard (9.5–625 pg mL –1 ) or 100 L pulp tissue extracts and 100 L of radioactive tracer [I 125 ].
The sensitivity and cross-reactivity of antisera SP 152 (2) and NKA 570 (3) have previously been reported (Maule et al . 1989). The sensitivity of antisera CGRP RAS6012 was 0.1 pg mL –1 and the cross reactivity has been reported previously (Tools for Protein and Peptide Research, Product Catalogue, Peninsula Laboratories, St. Helens, UK).
The SP-Ir assay used sodium phosphate buffer (0.04 mol L 1 , pH 7.4), containing 0.02% (w/v) thiomersal, 0.14 mol L 1 sodium chloride and 0.2% (w/v) bovine serum albumin (radioimmunoassay grade) in all dilutions of samples, antiserum, radioactive label and standards. Standards and extracted pulp tissue samples were incubated with antiserum 152(2) (final dilution of 1 : 600 000) for 24 h at 4 C. The I 125 -SP radioactive tracer (IM175, Amersham International, labelled using Bolton/Hunter reagent) was added, and the specimens incubated for a further 24 h at 4 C.
The NKA-Ir assay protocol was similar to that for SP-Ir, except that antiserum NKA 570(3) was used at a final dilution of 1 : 126 000 and the specimens incubated with radioactive tracer for 48 h.
The CGRP-Ir assay used sodium phosphate buffer (0.4 mol L 1 , pH 7.4) containing 0.02% (w/v) thiomersal, 0.05 mol L 1 sodium chloride, 0.1% (w/v) bovine serum albumin, and 0.1% (v/v) triton in all dilutions of the pulp tissue extracts, antiserum, radioactive label and standards. The antiserum RAS6012 was used at a final dilution of 1 : 900 000 and the specimens were incubated with radioactive tracer for 48 h.
A similar separation procedure was used for all three neuropeptides. The bound and free counts were separated by the addition of 1 mL of 0.5% (w/v) dextran-coated charcoal pellets in 0.4 mol L 1 sodium phosphate buffer, pH 7.4, containing 10% (v/v) horse serum (Gibco, Grand Island Biological Co, NY, USA). The specimens were then centrifuged (2000 g , 25 min, 4 C) and the charcoal pellets counted on a Cobra II Gamma Counting System (Packard Acanberra Company, Meriden, CT, USA).
All samples were assayed in duplicate and the mean values calculated. Individual controls for apparent binding in the absence of antibody were included in all assays. Radioimmunoassay results were expressed as nanogram of immunoreactive peptide per gram (ng g 1 ) of wet tissue.

Statistical analysis.
The concentrations of SP-Ir, NKA-Ir and CGRP-Ir were highly variable (Table 1) and a square root transformation was performed to produce normal distributions prior to analysis using parametric tests. The level of statistical significance was set at P < 0.05. Independent-Student’s t -tests were used to analyse differences between the neuropeptide levels in painful and non-painful teeth. Pearson’s product moment correlation coefficients were calculated for the three neuropeptides studied.
Stepwise regression analysis was used to determine whether age, gender, smoking, analgesic medication prior to treatment, tooth type (anterior, posterior), tooth location (maxillary, mandibular) or the presence of pulpal pain were independent factors affecting the levels of SP-Ir, NKA-Ir and CGRP-Ir.