Immunohistochemical Localization of Substance P in Pulp Tissue After In-Office Bleaching Technique in Rats

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Immunohistochemical Localization of Substance P in Pulp Tissue After In-Office Bleaching Technique in Rats Introduction
The dental bleaching is a treatment used to restore the definite natural color of teeth or to accomplish a whiter color and represents a prevalent treatment in dentistry.The dental bleaching is oxidizing agent, mainly constituting from hydrogen peroxide (H2O2).It removes the coloring substances by diffusion through 1 -60 1 ) 2 2 ) (20 2 ( 10 …. of Substance Immunohistochemical Localization 161 the dental enamel and dentin until reaching the pulp ) 1(.The penetration of H2O2 into dental pulp initiating cellular damage, inflammatory response and vascular permeability causing increase in the pressure inside the pulp resulting in activation of sensory nerve fibers ) 2 ( .Neurogenic inflammation is speculative to be mediated by releasing many neuropeptides from sensory nerves upon its activation at the site of inflammation )3(.Substance P (SP) it is the first neuropeptide to be recognized in the dental tissue and represents the most important neuropeptide among the others.Its main function is to induce vasodilation, increasing pulpal blood flow permitting rapid and large entrance of inflammatory cells and mediators diffuse into the inflammatory site and contributing to the transmission and keep of noxious stimuli and inflammation ) 4,5(.The chief pathway for SP mediated inflammatory reactions is mast cells, which undergo degranulation in response to SP, and releasing of large amounts of pro-inflammatory cytokines and vasoactive mediators; therefore it represents a fundamental mediator of inflammatory progressions and pain in the dental pulp )6 ( .Different methods have been used to study the effects of tooth bleaching on dental pulp up to this point, but nothing is known about the neuropeptide that is released after bleaching.PCR (polymerase chain reaction) (7), radioimmunoassay testing (8), and ELISA (enzyme-linked immunosorbent assay) (9) were among the precise methods used to assess these neuropeptides.These results, however, do not show where these neuropeptides are located in tissue, so for the present study, immunohistochemistry analysis was used to conduct a quantitative inquiry and pinpoint SP neuropeptide's position in the tooth pulp following the bleaching operation.

Materials and Methods:
All experimental procedures were carried out according to the ethical principles of animal research of College of Dentistry, University of Baghdad.Twenty four male Wistar albino rats, each weighing between 250 and 350 gm, were aged between 3 and 4 months.At the University of Baghdad's College of Pharmacy, the animals were housed in an animal house.The rats were kept in groups of six per cage in polycarbonate cages covered in wood shavings, kept in typical laboratory conditions at 25°C, fed regular rat chow pellet meal, and given free access to tap water.The procedure was accomplished in a standard sterilized condition.In order to achieve the bleaching treatment, each animal was weighted to evaluate the dosage of general anesthesia, the general anesthesia was made by Intramuscular injection of xylazine 2% (0.4 mg/kg B.W.), plus ketamine HCL 10% (15 mg/kg-B.W.) to prevent the movement of the rat through the bleaching session.The rats were randomly divided into two equal groups, the first group (group A):The right maxillary molars in each rat were treated with single in-office bleaching session using 40% of hydrogen peroxide(Opalescence Boost, UT, USA) (2×20min) and the left maxillary molars kept without treatment used as (negative control).The second group ( group B): The right maxillary molars in each rat were treated with single in-office bleaching session using 30% of hydrogen peroxide(Philips Dash, USA) (3×15min) and the left maxillary molars kept without treatment (negative control).Then each group was divided into two sub groups (6 rats for each) consistent with the time of scarification (1st day and 7th day) after the bleaching session.At the end of each interval, the animals were sacrificed with over dose of ketamine and posterior maxillary segments with three molar teeth were used as specimens.All tissue specimens were fixed in 10% neutral formalin and processed in a standard paraffin blocks after complete decalcification.Each paraffin-embedded block from all the studied samples were prepared in a 4μm thickness serial sections and mounted on glass slides for routine H&E staining.For the immunohistochemistry localisation of SP, additional 4 μm thick slices were placed on positively charged slides..The procedure of the IHC assay was carried out in accordance with the manufacturer

Results:
Regarding the immunohistochemical findings the control group showed few DPCs (fibroblast, endothelial cells and nerve cells) positively expressed for SP with normal organized pulp tissue at 1st day and 7th days Fig. )1, 2).Most pulp tissues subjected to 40% bleaching revealed at 1st day showed high expression of SP in DPCs surround the necrotic areas (Fig. 3 and 4).at 7th day there was less expression of SP in DPCs like fibroblasts, inflammatory cells, and odontoblast-like cells with more organized pulp tissue (Fig. 5).Concerning IHC expression in the pulp tissues chamber in 1st day after the bleaching with 30% H2O2 there was high expression of SP in DPCs including fibroblast, inflammatory cells, and endothelial cells (Fig. 6).while at 7th day less expression of SP was observed in DPCs and almost normal pulp tissue in both periods Fig. (7).As regards the results of statistical analysis revealed significant group difference in dental pulp cells that positively expressed for SP at 1st day duration and non-significant group difference at 7th day duration also higher mean value of the number of the pulp cells that had positive reaction to SP was recorded in 40% bleached group compared with other groups at both periods Table (1).Concerning multiple group comparisons by uses Games-Howell post hoc test of positively expressed DPCs for SP at 1 day duration, the result of this test showed significant difference between control and 30% bleached group Table (2).About the duration difference by Paired sample T-test for DPCs positively expressed for SP in each group.The result showed significant difference between 1day and 7days duration in 30% bleached group and non-significant difference regarding other groups Table (3).

Discussion:
Tooth bleaching generates several adverse effects including neurogenic inflammation and pain sensation within the tooth, in which SP neuropeptide plays an important role in developing these conditions (3).Neurogenic inflammation has been defined as the involvement of the nervous system in local inflammatory reactions.This process is described by the stimulation of peripheral neurons and these neurons act as environmental sensors which express numerous receptors that make these cells have the capacity to identify and stimulated by harmful thermal, mechanical and chemical stimuli consequently, they release SP, which is stored in the synaptic vesicle of these neurons, changed the vascular processes by vasodilatation, increased vascular permeability and assist in the accumulation of leukocytes (6).The SP is found in normal healthy pulp since it is released at a constant level from small vesicles stored in the sensory nerves ending in order to preserve the balance of vascularity to maintain blood flow and nutrient supply to the pulp constantly as well as regulate pressure in the interstitial pulp (10).This clarifies the present study's  (11).Concerning the 40% bleached group (group A) at 1 day duration, due to the presence of necrotic areas in most specimens, it was only possible to observe the positive expression of SP in the cells that remain in the pulp chamber also this explain the non-significant difference when compared with control grou .These finding agreed with recent studies carried by Gallinari et al. (2019) (12) and Hadi and AL-Ghaban (2020) (13)which their results revealed necrosis in 1st day after bleaching procedure.On the other hand, the present study revealed advanced number of DPCs that positively expressed for SP was found in the other specimens in the same group and this reflecting the higher mean value of positively expressed DPCs for SP when compared to other groups.Regarding the 30% bleached group (group B) at 1st day duration, the immunohistochemical finding revealed high number of DPCs that positively expressed for SP in most specimen with significant difference when compared to the control group.This result was in agreement with recent study carried by da Silva et al. (2020) (3).At 7th day duration after bleaching with 40% hydrogen peroxide the immunohistochemical finding revealed decrease in the mean value of the number of DPCs that positively expressed for SP but still higher than control and 30% bleached group, because the inflammation was still present in most specimens.Generally, the inflammatory process produced after tooth bleaching with a high concentration of H2O2 frequently lasts almost 2 weeks.Throughout this time, releases of proinflammatory cytokine continue, These changes result in SP release for extended periods (11).Regarding the 30% bleached group, the immunohistochemical results after 7th day showed a significant reduction in the number of DPCs that positively expressed for SP with significant difference compared with 1st day duration since the pulp on 7th day exhibited almost similar expression that resembled the control group.Therefore the present study showed decrease in SP expression within time and these findings agreed with Ismiyatin et al. study (2018) (14) who found significant a decline in SP expression in pulp at 72 hours duration when compared to 24 hours duration.

Conclusion:
It is important to focus that the adverse effect of the in-office bleaching agent on the dental pulp was proportional to the concentrations of hydrogen peroxide , therefore the higher the concentration of hydrogen peroxides in bleaching agent the higher penetration and higher inflammatory response, pressure and irritation of sensory nerve fibers subsequent in more SP releasing, and since the inflammatory response after bleaching was reversible there was a declined in SP expression with time.

Table ( 1
): Descriptive statistics and one way ANOVA test for DPCs positively expressed to Substance P antibody between groups in both periods.

Table ( 2
): Multiple group difference by Games-Howell post hoc test for immunohistochemical results in the pulp chamber at 1 st day duration.

Table ( 3
): Duration difference by Paired sample T-test for DPCs positively expressed for Substance P in each group.