asymptomatic inflammation ( ai ) ( also known as silent , low - grade , or painless inflammation ) is a concept that describes a scenario in which an etiologic agent is present without clinical evidence of harm or pain . it can be found in different types of cancer , genital tract diseases , cerebral infarction , or diabetic conditions . the oral environment is also vulnerable to suffer ai under certain pathologies , including chronic apical periodontitis   and periodontal disease . dental treatments such as deep restorations or orthodontic movements   can cause incessant injury to dental tissues that are not identified as noxious . irreversible pulpitis can also occur asymptomatically [ 7 , 8 ] , which may progress to dental pulp necrosis without treatment . vascular , neural , cellular , and biochemical changes from inflammation can be present in the absence of pain . neuropeptides released from primary afferent neurons ( pan ) are crucial factors in provoking inflammation of neural origin or  neurogenic inflammation  . substance p ( sp ) and calcitonin gene - related peptide ( cgrp ) are capable of inducing vasodilation , plasma extravasation , immune cell chemotaxis , and pain [ 11 , 12 ] . peripherally , opioid - containing immune cells ( ocic ) play the main role due to -endorphins ( -end ) and methionine - enkephalin ( met - enk ) release . these substances induce the activation of opioid receptors ( or ) located in pan , thus causing electrical changes that modulate pain partially via the inhibition of neuropeptide release [ 15 , 16 ] . considering both sides , it is interesting to analyze these two families of peptides within dental pulp , using controlled orthodontic forces that may allow nerve response without causing clinical symptomatology . the aim of this study was to determine the expression levels of sp , cgrp , -end , and met - enk in human dental pulp following orthodontic intrusion . eight patients of both sexes who were between 12 and 16 years old were selected . inclusion criteria were as follows : being systemically healthy and indicated for the extraction of the four first premolars for orthodontic reasons ; teeth without caries , fractures , or periodontal disease ; and teeth with radiographically evident complete radicular formation . exclusion criteria were as follows : patients receiving recent anti - inflammatory , analgesic , or antibiotic treatment ; those who had root resorption ( from any cause ) , permanent or provisional restorations in the first premolars or radicular dilacerations ; smokers ; pregnant patients ; and those who have occlusal disorders . a descriptive comparative pilot study was conducted with the approval of the institutional ethics committee ( approval code number ceife-002 - 010 ) , according to the declaration of helsinki . written informed consent was explained and obtained from parents / legal guardians of each patient . intrusive orthodontic appliances were designed using standard 0.018 orthodontic brackets and double tubes ( sybron / ormco , orange , ca , usa ) , selected for the first premolars and permanent first molars , respectively . both brackets were bonded to each tooth using the adhesive system prime & bond ( 3 m unitek , monrovia , ca , usa ) and composite filtek 350 ( 3 m unitek ) , standardizing their positions with an anderson calibrator ( dentaurum gmbh , ispringen , germany ) . using number 139 orthodontic pliers ( dentaurum gmbh ) , a 1 mm loop was formed with a retentive fold on the opposite side of a stainless steel wire ( 0.018    0.025 ) . the customized wire was placed in the tube slot and adjusted to allow the loop to be bent until an acute angle was achieved . using a dynamometer ( corex , haag - streit , kowniz , switzerland ) , the angulation was standardized at 40 to allow for a 150200  g intrusive force . two premolars from each patient were randomly selected and assigned to two different groups : the asymptomatic inflammation group referred to as experimental group , which would undergo controlled intrusive force for seven days ( expg ) , and the control group ( ctrg ) , used to determine the basal levels of each substance . each patient was scheduled to obtain the samples from the control tooth before any clinical intervention took place . each control tooth was anesthetized with 4% prilocaine ( pricanest , ropsohn therapeutics , bogot , colombia ) . from that moment , the entire sampling procedure was performed within a 10-minute period . after extraction , a second assistant created a 1 mm longitudinal groove over the vestibular tooth surface using a high - speed diamond bur with copious irrigation to facilitate the mechanical fracture of the tooth and the intact acquisition of pulpal tissue , which was immediately stored in a tagged cryovial containing 1.8  ml of 4% paraformaldehyde . after its activation , each patient received instructions to call if any problem or severe discomfort was experienced and , if necessary , to take 400  mg of ibuprofen as a rescue analgesic . for the following six days , the patients were contacted by telephone to evaluate their progress along the experimental period . information on their symptoms , analgesic usage , or orthodontic device displacement was recorded . at day seven , patients were scheduled to obtain the dental pulp sample from expg , as described above . a radioimmunoassay ( ria ) was performed to quantify the amount of each substance obtained from each sample , according to previous investigations reported . sp , cgrp , -end , and met - enk release were determined by competition ria binding assays using a human sp ria - kit ( reference rk-061 - 05 ) , a human cgrp ria - kit ( reference rk-015 - 02 ) , a human -end ria - kit ( reference rk-022 - 14 ) ( phoenix peptide pharmaceuticals , burlingame , ca , usa ) , and a human met - enk ria - kit ( reference s-2119 ) ( peninsula laboratories llc , bachem group , san carlos , ca , usa ) . for each kit , 100  l of antiserum and 100  l of various neuropeptide / opioid concentrations ( 1128  pg  l ) or 100  l of dental pulp tissue extracts were incubated in polypropylene tubes at room temperature for 20 hours . then , 100  l of radioactive 125i tracer was added and left to incubate for another 24 hours . bound fractions were precipitated by the addition of 100  l of a secondary antibody ( goat anti - rabbit immunoglobulin g serum ) , 100  l normal rabbit serum , and 500  l ria buffer containing 1% polyethylene glycol 4,000 . after 2 hours of incubation at room temperature , the suspensions were spun at 3,500  rpm ( 4,000  g ) for 40 minutes at 4c to precipitate the bound fractions . the supernatants were carefully aspirated , and pellet radioactivity was read on a gamma counter ( model b5002 , packard instrument international , zurich , switzerland ) . all samples were assayed in duplicate , and the mean values were calculated . finally , scatchard analysis of the binding data was used to assess the amount of neuropeptide / opioid present in each sample . the results were analyzed by the mann - whitney u test to compare the differences among groups for continuous variables . a difference was considered to be significant if the probability of its occurring by chance alone was < 5% ( p < 0.05 ) in a two - tailed test . basal levels of both neuropeptides and endogenous opioids were observed . in the expg , sp and cgrp exhibited statistically significant different levels ( p < 0.05 ) . although none of the endogenous opioids showed statistically significant differences ( p > 0.05 ) , they all expressed increasing trends in the expg . none of the patients enrolled were eliminated from the study because no one reported severe pain episodes or the need to take the rescue analgesic treatment . all patients reported tolerable discomfort localized in the tooth assigned to expg , for the two or three days after orthodontic activation . by day seven , all of the symptoms were absent , and all of the orthodontic devices were still active . mechanisms related to neurogenic inflammation and pain modulation in ai are not fully understood . in this study , the expression of two somatosensory neuropeptides and two ops in dental pulp ai caused by orthodontic intrusion was determined . this clinical model allowed us to obtain a biphasic inflammatory transition , which began with discomfort during the acute phase and finally achieved an asymptomatic state . since the intrusion period of the experiment was seven days , it was considered not objective to make a clinical measurement of the distance . in this experiment , although the majority of available evidence is focused on the biochemistry of painful conditions , the study of ai is a promising field that may help explain how important diseases appear after the development of    anonymous  inflammation . in this study , the presence of an initial inflammatory tenderness was required to assess the beginning of the asymptomatic phase ; it was a requirement for this study that all patients report no presence of pain when the samples were obtained . previous studies have demonstrated , within dental pulp , the expression of markers of neurogenic inflammation ( substance p and cgrp ) using the model of inflammation employed in this study ; this controlled orthodontic movement was selected as a well - standardized available model for human clinical trials focused on inflammation [ 6 , 1921 ] . chemotaxis of the macrophage population induced by neuropeptides in late orthodontic inflammation   is crucial to establish the neuropeptidergic and op relationship . furthermore , nerve fiber sprouting and neurogenic inflammation are common during pulpal insult , including early force application during orthodontic tooth movement . macrophages express nk1 and cgrp1/2 receptors , which are activated by sp and cgrp , respectively , favoring their activation [ 15 , 24 ] . these cells are the main secretors of op , including -end and met - enk . interestingly , under inflammatory conditions , pan , the main source of local sp and cgrp release , exhibits an upregulation expression of or [ 13 , 27 ] , which can be targeted by macrophage analgesic products ( figure 1 ) . controversy surrounds the fact that macrophages are also capable of secreting somatostatin ( a substance able to cause analgesia ) ; thus , pain control may not be exclusively caused by op . these results demonstrate an important increase of sp and cgrp levels in expg ( p < 0.05 ) . the release of sensory neuropeptides occurs almost immediately , in a manner described as an  axonal reflex  . such a response supports the role of neurogenic inflammation of the dental pulp after low - grade orthodontic continuous movement [ 20 , 22 , 28 ] . both neuropeptides are frequently associated with pain from pulpal origin [ 2931 ] ; however , they have also been identified in asymptomatic irreversible pulpitis . the importance of the role of neuropeptides in inflammation was previously studied by determining their absence , not their presence . different denervation experiments showed how the deprivation of the sensory nerve supply can attenuate the local inflammatory response . both ops are postulated to play the leading role in endogenous antinociception   due to their action as negative regulators of neurogenic inflammation   by the inhibition of sp and cgrp release [ 14 , 15 ] . however , this study shows that , after orthodontic intrusion , low levels of op were not enough to attenuate neuropeptide expression . it is important to consider why late neurogenic inflammation can develop in the absence of pain . first , dental pulp is able to adjust slowly to low - grade inflammation , assimilating higher levels of proalgesic mediators in the absence of pain . second , experimental tooth movement is capable of activating central trigeminal pain modulation mechanisms , causing a reduction in the local recruitment of ocic to peripheral injured tissues ; thus , the net effect will be the control of pain and lower peripheral opioid peptide concentration [ 15 , 25 , 3537 ] . third , new evidence explores the dual effects of neuropeptides in relation to pain experience . sp enhances opioid release , and it is possible that its n - terminal fragment may act as a miu - opioid receptor ( mor ) agonist once its expression is upregulated during inflammation . for cgrp , the antinociceptive effect is related to the upregulation of mor when it is applied centrally   and peripherally by the local suppression of interleukin-2 production , causing an anti - inflammatory effect . finally , though both neuropeptides are strong vasodilators , this effect may be masked by low blood flow after orthodontic intrusion , thus avoiding the increase of inner pressure in a low compliance pulpal environment [ 3941 ] . all of these hypotheses must be evaluated in further studies , including more patients in order to confirm these findings , as well as new complementary experiments including pulp tissue immunostaining to evaluate neurons , peptides activity , and immune and inflammatory cells participation . also , it is necessary to include peptides measurement at different time points in order to elucidate the possible mechanism of asymptomatic inflammation . in this preliminary report , sp and cgrp were identified in dental pulp after seven days of controlled orthodontic intrusion movement , even in the absence of pain . at the same time , endogenous opioids exhibited no statistical differences compared with control levels ; however , an increase tendency was appreciable for both .
purpose . 
 this study quantified the expression of substance p ( sp ) , calcitonin gene - related peptide ( cgrp ) , -endorphins ( -end ) , and methionine - enkephalin ( met - enk ) in human dental pulp following orthodontic intrusion . methods . 
 eight patients were selected according to preestablished inclusion criteria . from each patient , two premolars ( indicated for extraction due to orthodontic reasons ) 
 were randomly assigned to two different groups : the asymptomatic inflammation group ( expg ) , which would undergo controlled intrusive force for seven days , and the control group ( ctrg ) , which was used to determine the basal levels of each substance . 
 once extracted , dental pulp tissue was prepared to determine the expression levels of both neuropeptides and endogenous opioids by radioimmunoassay ( ria ) . 
 results . 
 all samples from the ctrg exhibited basal levels of both neuropeptides and endogenous opioids . by day seven , all patients were asymptomatic , even when all orthodontic - intrusive devices were still active . in the expg , 
 the sp and cgrp exhibited statistically significant different levels . 
 although none of the endogenous opioids showed statistically significant differences , they all expressed increasing trends in the expg . 
 conclusions . 
 sp and cgrp were identified in dental pulp after seven days of controlled orthodontic intrusion movement , even in the absence of pain .