Pharmacology of peripheral neuropeptide and inflammatory

mediator release
Kenneth M. Hargreaves, DDS, PhD,” James Q. Swift, DDSb
Mark T. Roszkowski, DDSC Walter Bowles, DDS, MSC Mary G. Garry, PhD,C and
Douglass L. Jackson, DMD, MSC Minneapolis, Minn.
UNIVERSITY OF MINNESOTA SCHOOL OF DENTISTRY

Resetarch conducted in the last 10 years has increased our knowledge on pain mechanisms substantially. Although
many local tissue mediators, including neuropeptides, are known to exert pro-inflammatory effects, comparatively little is known
about the actual tissue levels of these inflammatory mediators and their pharmacologic regulation. This article describes two
new methods, clinical microdialysis and superfusion of dental pulp, which provide data on the pharmacology of peripheral
neuropeptide and inflammatory mediator release. Collectively, these methods provide a biochemically based approach toward
determining the mechanisms and management of orofacial pain. (ORAL SURC ORAL MED ORAL PATHOL 1994;78:503-10)

The management of pulpal and periapical pain and
inflammation is a central component of the field of
endodontics. Pain represents a major stimulus for pa-
tients who seekemergency dental treatment, and fear
of pain is a major barrier that prevents many patients
from seekingroutine dental care. Research conducted
in the last 10 years has lead to numerous advancesin
our understanding of pain, forcing reevaluation of
many of our classical assumptions about pain mech-
anisms and pain management.
Although a complete review of these advances is
beyond the scope of this article, selective examples
will serve to highlight these conceptual changes. In
terms of new hypothesesof pain mechanisms, excita-
tory amino acids, such as glutamate and aspartate,
are now believed to be a major neurotransmitter class
involved in pain perception. As of yet, however, no
practical and clinically available analgesic blocks
these neurotranslmitters. Second, the central nervous
system (CNS) is no longer viewed as a static mono-
lith tha,t does not respond to peripheral nociceptive
input. Instead the CNS has been proposed to develop
a “central hyperalgesia” in responseto nociceptor in-
put. This central component of hyperalgesia may
contribute to acute as well as chronic pain states.
Third, peripheral nerve terminals undergo a sensiti-
zation in responseto release of certain inflammatory
Supported in part by NIDR grants R29DE09860, R03DE10096,
K16DE0027, and P30DE09737.
aDivision of Endodontics, Department of Restorative Sciences, and
Department of Pharmacology.
bDivision of Oral and Maxillofacial Surgery, Department of Sur-
gical Sciences.
CDivision amfEndodontics, Department of Restorative Sciences.
Copyright @ 1994 by Mosby-Year Book, Inc.
0030-4220/94/$3.00-i-O T/15/58090
mediators and undergo sprouting and an increase in
their content of neuropeptides in responseto chronic
inflammation.’ In terms of new concepts about pain
management, aspirin-like drugs probably produce
much of their analgesic activity through actions in the
central nervous system (CNS),2 opioids have periph-
eral analgesic activity,3, 4 and acetaminophen has pe-
ripheral analgesic activity in dental pain patients.5
These brief examples highlight some of the major re-
search advancesin the field of pain and analgesia that
have occurred in the last 10 years.
This article will focus on one new evolving area in
the field of pain control, the pharmacology of periph-
eral neuropeptide and inflammatory mediator release.
This area is of importance becausean increased un-
derstanding of the Imechanismsthat lead to activation
and sensitization of nociceptors (pain fibers) may well
lead to improved therapeutic and diagnostic methods.
PERIPHERAL NOCICEPTORS
The perception of pain is thought to signal the oc-
currence of tissue damage or the potential for dam-
age.6Noxious stimuli such as chemical, mechanical,
and thermal stimulation are detected by the terminal
endings of two major classesof nociceptive afferent
nerve fibers: A-delta fibers and C fibers. These noci-
ceptive fibers are distributed throughout periapical
bone and pulp, as well as mucosa, skin, periosteum,
muscles, and joints. Although additional research on
the classification and functions of these fibers is
ongoing, the following is a brief overview about their
contributions to peripheral pain mechanisms. (for
further information, see references 7- 10).
The lightly myelinated and relatively fast-conduct-
ing nociceptive fibers are termed A-delta nerve fibers.
These fibers that respond primarily to noxious me-
chanical stimuli m,ay also respond to chemical or
503
So$ Hargreaves et al.
Table 1.Signs of hyperalgesia and dental diagnostic
tests
Sign of Related diagnostic
hyperalgesia test or symptom
Spontaneous pain Spontaneous pain
Reduced pain Percussion test,
threshold palpation test,
throbbing pain
Increased response Increased response
to suprathreshold to pulp test
stimuli (EPT or thermal test)
thermal stimuli. A-delta fibers have been proposed to
mediate the initial for first sensation of pain, which
has a sharp, electric, or bright perceptual quality.7-‘0
Periosteum, ligaments, joints, and muscles are inner-
vated by similar finely myelinated A-delta fibers
known as group III fibers.
The second major group of nociceptive fibers are
unmyelinated slowly conducting nerve fibers. These
nerves are termed C-polymodal fibers because they
respond to several types of stimuli including thermal
and mechanical stimulation and numerous chemical
mediators. For example, C-polymodal nociceptors are
responsive to bradykinin, potassium, acetylcholine,
leukotrienes, protons (for example, low pH), and
prostaglandins. 11-13The C fibers most likely mediate
the sensation of “second pain.” Second pain occurs
after the initial “sharp pricking” pain and is generally
described to have a dull, aching, or burning perceptual
quality. 7-10Periosteum, ligaments, joints, and mus-
cles are innervated by similar unmyelinated C noci-
ceptors known as group IV fibers, which have poly-
modal response properties. Peripheral nerves in
buman beings contain approximately 3 to 5 times
more C fibers than A-delta fibers, though not all of
these fibers are necessarily nociceptors.‘4
It has been suggested that inflammatory pain
caused by pulpitis may be more representative of
C-fiber activation, or secondpain, becauseit often has
a dull aching quality. Conversely, dental hypersensi-
tivity, often characterized as a sharp sensation, more
closely represents A-delta fiber activity.
HYPERALGESIA
Pain causedby inflammation produces a perception
that is distinctly different from pain induced by tran-
sient noxious stimuli. Inflammatory pain is usually
characterized by its spontaneousoccurrence. In addi-
tion, innocuous stimuli often produce a prolonged
painful reaction. The pattern of this pain report dif-
fers qualitatively from the brief “sharp” and “bright”
sensations described after transient stimulation of
normal teeth with an electric tooth pulp tester.
ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY
October 1994
In general, inflammatory mediators have two main
effects on the peripheral nociceptive nerve ending.
Mediators activate and sensitize certain peripheral
nerve terminals and, as described here, mediators also
evoke peripheral secretion of neuropeptides such as
substance P or calcitonin gene-related peptide
(CGRP).7M10> l5 Peripheral nociceptors become sensi-
tized in responseto certain inflammatory mediators
released during tissue injury. Sensitized nociceptors
have spontaneousactivity, a decreasedthreshold, and
prolonged responses(afterdischarges) to suprathresh-
old stimuli.
These electrophysiologic changes in the response
properties of peripheral nociceptors together with
changes in the CNS result in the perceptual state of
hyperalgesia. Hyperalgesia is characterized by spon-
taneous pain, a decreasedpain threshold, and an in-
creasedmagnitude of perceived pain for a given stim-
~1~s.~As a commonplace example, a sunburn evokes
the signs of hyperalgesia becausepain is often spon-
taneous, exhibits reduced thresholds (wearing a shirt
is painful), and increased responsivenessto suprath-
reshold stimuli (it hurts even more if someoneslaps
your sunburn).
The central component of hyperalgesia may be due
to a number of changesin the CNS. Space precludes
a thorough review of CNS changes proposed to con-
tribute to the central component of hyperalgesia.
However, a brief listing of major hypothesesinclude:
altered release of neurotransmitters, increased levels
of second messengersand nitric oxide, alterations in
receptor number or binding affinity, rapid changesin
proto-ocogenes, and increased receptor field size of
second order neurons. These central changes may
persist beyond the period of activation of peripheral
nociceptors. For example, phantom limb pain may
persist evenwhen no peripheral input can be detected.
In a similar fashion, it is possible that central hyper-
algesia occurs in patients who experience prolonged
pain causedby irreversible symptomatic pulpitis. Ac-
cordingly, patients may experience postendodontic
pain even in the presence of reduced or absent peri-
pheral input.
As clinicians, we use the signs of hyperalgesia to
evaluate for the presenceof symptomatic pulpitis and
periapical inflammation (Table I). For example, the
evaluation of pain after tooth percussion is actually
the evaluation of a reduced pain threshold to me-
chanical stimuli. Investigations by Narhi have led to
the suggestionthat the throbbing pain of pulpitis may
be due to the mechanical threshold reduced to the ex-
tent that the arterial pressure wave of a heartbeat is
sufficient to activate pulpal nociceptors. In addition,
the presence of pain of long duration or excessive
magnitude after pulp vitality testing is simply another
ORAL SIJRCERY ORAL MEDICINE ORAL PATHOLOGY Hargreaves et al.
Volume 78, Number 4

Aracbridonic
Acid
/ \

IFig. 1. Schematic figure illustrates the positive-feedback relationship between inflammatory mediators that
develops during the course of inflammation. Note that several inflammatory mediators both stimulate (or
sensitize) nociceptors and evokeperipheral secretion of substanceP and CGRP.In turn, these neuropeptides
stimulate inflammatory responses,such as, plasma extravasation, and evoke releaseof additional inflamma-
tory mediators. Nerve growth factor and possibly related factors may both activate nociceptors and stimulate
sprouting of terminals and increased content of SP and CGRP. (Figure modifiedfrom Hargreaveset al.15

sign of hyperalgesia. An understanding of the signs of
hyperalgesia provides a physiologic rationale for
many of the diagnostic tests and evaluation criteria
used in clinical endodontics. Many of the clinical
testing procedures used in endodontics are directed
toward evaluating for the presenceof hyperalgesia. It
has even been suggestedthat “hyperalgesic pulpitis”
is a more accurate clinical term than “symptomatic
pulpitis” to describe the neurophysiologic status of the
pulp and periapil;al tissue.
REGULATION OF INFLAMMATORY MEDIATOR
RELEASE:MICRODIALYSIS STUDIES
In contrast to a transient pain-producing stimulus
such as electric lpulp test, needle insertion, or veni-
puncture, pain associated with inflammation is often
characterized by a prolonged period of hyperalgesia.
This is due in part to the sustained actions of periph-
eral inflammatory mediators that are thought to
interact in the development of a local positive feed-
back cycle (Fig. 1) and to plasticity changes in the
CNS (central hypeiralgesia). Tissue trauma or pulpal
or periapical inflammation participates in the local
positive feedback cycle by activating the synthesis of
prostaglandins and evoking the release of bradykinin
from its blood-borne precursor, kininogen, and of his-
tamine from mast cells. Because prostaglandins,
bradykinin, and histamine all increase either the per-
meability or dilatation of local blood vessels,they act
synergistically to increase plasma extravasation (Fig.
1). It is this accumulation of extravasated fluid into
tissue spacesthat produces the clinical sign of edema.
In addition, plasma extravasation replenishes these
short-lived local mfediatorsby providing a fresh sup-
ply of kininogen, prostaglandins (activated by brady-
kinin), histamine, and other mediators. The continued
synthesis or release of these inflammatory mediators,
the processof nociceptor sensitization, and persistent
changesin the CNS may contribute to the prolonged
duration of inflammatory pain.
506 Hargreaves et al.
Table 11.Tissue levels of selected inflammatory
mediators as measured by microdialysis probes
implanted into dental surgical sites”6-21
Approximate
peak time j concentration
Mediator (in minutes) (in nmol/L)
iPGE2 120-240
iBradykinin 150-180
isubstance P 120
iLTB4 240
Immunoreactive levels of these mediators were determined by RIA or
BLISA assay of tissue dialysates collected from oral surgery patients (n = IS
to 50).
However, many studies describing the pro-inflam-
matory effects of these factors merely document the
ability of mediators and neuropeptides, usually ad-
ministered at supraphysiologic doses, to stimulate
processes related to inflammation or nociception.
Comparatively little is known with respect to the ac-
tual tissue concentration of these chemical mediators
during inflammation and pain, or their pharmacologic
modulation, or to what extent they actually are
involved in mediating pain caused by different types
of orofacial inflammation.
To address these issues, our research team at the
University of Minnesota has developed a microdialy-
sis method that permits collection of inflammatory
mediators in awake dental pain patients who can si-
multaneously provide verbal pain reports.‘6-21 Previ-
ous attempts to measure tissue levels of mediators
generally have used methods such as needle aspira-
tions or the insertion of a “push-pull” catheter, in
which saline solution is pumped into the tissue and
then collected. These older methods have several dis-
advantages because they permit (1) concurrent col-
lection of peptidases (for example, kininases, kal-
likrein) that can alter mediator concentrations during
the collection process leading to inaccurate determi-
nation of mediator levels; (2) collection of precursors
(for example, kininogen) that can interfere with mea-
surement assays; (3) dilution of mediator concentra-
tions by an unknown factor leading to inaccurate cal-
culation of mediator levels, that is, by introduction of
saline solution into the inflamed tissue compartment);
and (4) alteration of the inflammatory process by
pumping exogenous fluid into the tissue.
Implanting microdialysis probes into inflamed tis-
sue avoids the above limitations.‘6-21 By implanting a
probe that has a semipermeable membrane with a
molecular weight cutoff of 3,000 to 20,000 daltons, it
is possible to collect a selective dialysate that contains
inflammatory mediators, most of which have a mo-
lecular weight less than 2,000 daltons. Advantages of
the microdialysis method include (1) exclusion of pep-
tidases and precursors at the tissue level; (2) quantita-
ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY
October 1994
live recoveries of inflammatory mediators, and (3)
minimal disruption of the inflamed tissue (that is, sa-
line solution is not being pumped into the tissue).
These clinical studies have used microdialysis
Peak
probes to collect immunoreactive bradykinin (iBK),
prostaglandin E2 (iPGE2), leukotriene B4 (iLTB4),
substance P (iSP), and other mediators present in in-
5-l
flamed peripheral tissue. These inflammatory media-
12-17
1 tors were selected because of their known pro-inflam-
2-4 matory actions. The oral surgery model was selected
for these studies because it is a well-recognized clin-
ical model of surgically induced pain and inflamma-
tion, which easily permits the temporary implantation
of microdialysis probes into patients who are awake
and who can simultaneously provide pain reports.
The time-course studies indicate that iPGE2, iBK,
iLTB4, and iSP are all detectable in tissue dialysates
collected from the extraction sites of awake patients
after surgical removal of impacted third molars.i6-21
The data presented in Table II illustrate typical val-
ues for the time of peak release and corresponding
peak concentrations of these mediators. Importantly,
the peak concentrations of all four of these mediators
are greater than the Kd values for binding of the me-
diators to their respective receptors. The Kd value is
the concentration of the mediator that binds to 50% of
its receptors. This comparison suggests that all of these
mediators are present at physiologically relevant con-
centrations and that they may contribute to the devel-
opment of acute dental pain and inflammation.
Corresponding pharmacologic studies have evalu-
ated the effects of nonsteroidal anti-inflammatory
drugs (NSAIDs) and systemic steroid treatment on
tissue levels of inflammatory mediators in oral surgery
patients. As compared to placebo-treated patients,
administration of either flurbiprofen, ibuprofen, or
methylprednisolone significantly reduces tissue levels
of iBK, iPGE2, and iSP.16-*r The results of a double-
blind comparison of systemic methylprednisolone
(125 mg) to placebo for altering postsurgical tissue
levels of iBK is presented in Fig. 2. Interestingly, a
single preoperative injection of methylprednisolone
produced a substantial and prolonged reduction in
tissue levels of iBK and postsurgical pain.‘” Methyl-
prednisolone also reduces tissue levels of mediators
such as iPGE2 and iSP supporting the clinical effi-
cacy of glucocorticoids.
Collectively these microdialysis studies provide a
biochemically based approach for (1) identifying in-
flammatory mediators released in peripheral tissue of
orofacial pain subjects, (2) determining their phar-
macologic regulation, and (3) evaluating their inter-
actions and contributions to the development of pain
and inflammation. We are currently extending this
method to clinical cases of surgery performed on
chronically inflamed tissue, such as, endodontic sur-
ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY
Volume 78, Number 4
z 15000
L
E
S
10000
z
z
3
9 5000
ii
.’
0
0 30 60 90 120
TIME (min after surgery)
Fig. 2. Effect of pretreatment with methylprednisolone (125 mg intravenously 2 hours before surgery) or
placebo on tissue levels of immunoreactive bradykinin in 36 oral surgery patie.nts.Tissue levels of immunor-
eactive bradykinin were collected by microdialysis probes implanted into the extraction site and measured
by RIA. (**p < 0.01.) (Figure modified from Hargreaves and Costello.16)
gery or periodontal surgery. Our preliminary find-
ings indicate that the inflammatory responsein chron-
ically inflamed t,issue is even greater than that ob-
served in the oral surgery mode1.22This difference in
tissue response may be related to the presence of
chronic inflammatory cells that are already in place
in tissues selected for periapical or periodontal sur-
geries.
REGULATION OF NEUROPEPTIDE RELEASE:
SUPERFUSION OF DENTAL PULP
In addition to the activation and sensitization of
certain nociceptors, another effect of inflammatory
mediators is to modulate the release of neuropeptides
stored in the peripheral nociceptive nerve ending (Fig.
1). The cell body of the primary afferent nerve fiber
synthesizesthe neuropeptides substanceP and CGRP
(as well asother substances)that are transported both
to the CNS and to the periphery. Theseneuropeptides
are highly concentrated in peripheral tissue such as
dental pulp and periapical tissue. Electrical or chem-
ical stimulation of peripheral nerves is a sufficient
stimulus for these fibers to release iSP and iC-
GRP.23-25
Both substance P and CGRP have pro-inflamma-
tory effects. Fig. 3 illustrates the effect of substance
P on evoking plasma extravasation. The peripheral
injection of substance P produced a dose-related
increase in plasma extravasation, as measured by
the accumulation of Evans blue dye in the injected
tissue. As illustrated in Fig. 1, plasma extravasa-
tion is a. critical process in inflammation that con-
tributes to the development of edema and the modu-
lation of inflammatory mediator release. Thus the re-
Hargreaves et al. 507
METHYLPRED
150 180 210 2;ro
lease of neuropeptides may alter the inflammatory
process.
Peripheral neuropeptides may also modulate in-
flammation by altering the release or metabolism of
inflammatory mediators. Neuropeptides, such as sub-
stance P, act with other inflammatory mediators to
stimulate the release of histamine, prostaglandin E2,
collagenase, interleukin- 1, interleukin-6, and tumor
necrosis factor.26-28Additional studies29-31indicate
that these neuropeptides and possibly additional fac-
tors of primary afferent fiber origin are critical for the
resolution of inflammation and the initiation of suc-
cessful healing. In amelegant study recently published
by Byers and Taylor,31 it was shown that denervation
of the inferior alveolar nerve substantially increased
the magnitude of pulpal necrosis after pulp exposure.
Taken together,thesestudiesindicate that the local reg-
ulation of neuropeptidereleasemay play a critical role in
the modulation of tiissueinflammation and healing.
Peripheral tissue:content of immunoreactive sub-
stance P and CGR.P is substantially altered during
inflammation, suggesting that these neuropeptides
modulate chronic inflammation after pulp expo-
sure.1,32-33Byers let al. t, 32-33have used immuno-
histochemistry (IHC) to localize the peripheral
neuropeptide response to pulp exposure. They dem-
onstrated a marked increase in staining for immuno-
reactive calcitonin gene-related peptide (iCGRP) in
pulpal areas adjacent to abscessesand in nearby pe-
riapical tissue.
Our laboratory has followed up on these studies
with the use of radioimmunoassays (RIA) for mea-
surement of both iSP and iCGRP. RIA is a quanti-
tative method that permits measurement of tissue
 ---_
508 Hargreaves et al. ORAL SURGERY ORAL MEDICIKE ORAL PATHOLOGY
Orlober I994

Substance P (pmol)
Fig. 3. Dose-related effect of substance P on producing plasma extravasation. Rats were anesthetil.cd with
pentobarbital and injected intravenously with Evans blue dye that binds to albumin and serves as a marker
for plasma extravasation. Skin biopsies were collected 15 minutes after subcutaneous injection of substance
P. Evans blue was extracted with formamide (3 days) and then measured in a spectrophotometer (620 nm).
(M. G. Garry and KM. Hargreavcs, unpublished observations).

levels of substances rather than the anatomic location
information provided by immunohistochemistry. Our
initial studies demonstrated that pulpal exposure with
a no. 7002 bur resulted in significant decreases in tis-
sue levels of iSP and iCGRP.34 This was consistent
with the finding of Byers et al., assuming that our in-
jury was similar to their moderate-to-severe level of
injury. We have recently reevaluated this hypothesis
by preparing small occlusal exposures using a no. 33%
bur under visual magnification. This smaller type of
in.jury produces about a two-fold increase in pulpal
levels of iSP and iCGRP, with peak levels observed 7
to 14 days after exposure. 35 Interestingly, concurrent
changes were observed in trigeminal ganglion levels of
iSP and iCGRP with a trend toward periapical
changes at latter points in time.35 Collectively these
studies indicate that certain primary afferent fibers
not only detect and signal the occurrence of tissue
damage, they also respond to the development of tis-
sue inflammation and healing.
In contrast to the relative wealth of studies that de-
tail the propharmacology of peripheral ncuropcptides
and their response to pulpal inflammation, there is a
relative lack of studies on the mechanisms regulating
peripheral neuropeptide secretion. As aforemen-
tioned, it is likely that this is an important area of rc-
search because these peptides can modulate inflam-
mation and healing and undergo dynamic responses in
tissue content in response to inflammation. Accord-
ingly drugs that alter secretion of neuropeptides may
have clinically important anti-inllammatory, analge-
sic, or healing properties.
To determine the pharmacologic regulation of pe-
ripheral neuropeptide secretion, we have developed an
in vitro method of superfusing dental pt~lp.~~,2s,36-39
In addition to the clinical significance to endodontics,
dental pulp is an ideal tissue for conducting these
studies because it contains relatively high concentra-
tions of neuropeptides, and the predominant sensation
after stimulation of human pulp is pain. These
anatomic and psychophysical characteristics suggest
that superfusion of this tissue would permit the eval-
uation of a relatively rich supply of nociceptors. Pulp
tissue was removed from freshly extracted bovine
mandibular incisors (obtained from a nearby slaugh-
terhouse), sectioned into 200 pm cubes, and loaded
into superfusion chambers. As seen in Fig. 4, oxygcn-
ated Kreb’s buffer is perfused through the tissue and
collected by a fraction collector. The tissue release of
iCGRP, as measured by RIA, is altered by adding
chemicals and drugs to the Krcb’s buffer at various
points in time.
Our initial validation studies demonstrated that
this method appears to measure physiologic secretion
of peptides because (1) depolariting concentrations
of potassium (for example, 50 mm) evoke release of
iCGRP, (2) capsaicin, an agent that stimulates cer-
tain nociceptors, evokes release of iCGRP, (3) the re-
ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY Hargreaves et al. 509
Volume 78, Number 4

-rc Krebs Bdfer:

- Stimdant

Radioimmunoassay

Fig. 4. Experimental setup for superfusing dental pulp to determine the pharmacologic regulation of
peripheral neuropeptide secretion. Bovine dental pulp (200 Mmcubes) are suplerfusedwith oxygenated Kreb’s
buffer containing either test drug or vehicle. The superfusate is collected by a fraction collector and assayed
for immunoreactive CGRP by RIA.

lease of iCGRP is calcium dependent (calcium is re- Table III. Effects of selected drugs on altering
quired for most types of secretion), and (4) the release of iCGRP from dental pulp24925,36-39
iCGRP that is released co-elutes with authentic Effect on Examined
CGRP by high-performance liquid chromatogra- Mediator release doses
2425
phy.
The results of our current pharmacology studies are Potassium Increased 2.5-50 mM
Capsaicin Increased I-30 FM
summarized in Table III. Mediators that are released BK/PGE2/histamine Increased l-100 /AM
peripherally during inflammation, such as PGE2, serotonin
bradykinin, hislamine and serotonin, or that are Lidocaine Decreased 2%
newly synthesized during inflammation, such as nerve Epinephrine Decreased 1:100,000
growth factor, produce dose-related stimulation of Norepinephrine Increased and 0.0001-100 KM
decreased
iCGRP from pulpal terminals.37,39Thus the bovine
Clonidine Decreased 100 nM
superfusion method permits examination of the ac- Salbutamol Decreased 100 nm
tions, receptors, and second messenger systems in- Nerve growth factor Increased 0.01-10 nM
volved in activating peripheral terminals of primary Corticotropin releasing Decreased 100 nM
afferent fibers. factor
In addition to mediators that stimulate release, a Immunoreactive levels of iCGRP were determined by RIA of in vitro super-
number of drugs inhibit release. For example, fusates of bovine dental pulp (n = 5 to 50).

lidocaine blocks the evoked-release of iCGRP from

dental pulp. 25Interestingly, adrenergic agents such as
epinephrine, norlepinephrine (at certain doses),cloni-
dine (an az-receptor agonist), and salbutamol (a
P-receptor agonist) have potent actions for inhibiting
the capsaicin-evoked release of iCGRP.36 This raises
the possibility that the enhanced efficacy observed
with the vasoconstrictor-containing local anesthetic
drugs may be due not only to vasoconstriction but also
to an inhibitory action of adrenergic agonists on cer-
tain primary afferent fibers. If subsequent research
supports this hypothesis, then it is possible that
adrenergic agents other than epinephrine or levonor-
defrin may provide superior local anesthetic adjuncts
for control of peripheral hyperalgesia.
CONCLUSIONS
Research conducted in the last 10 years has forced
reexamination of many of the traditional concepts of
pain mechanismsand pain management. A number of
substanceshave beenproposedto act as inflammatory
mediators, in large part on the basis of their pro-in-
flammatory pharmacology. However, clinical trials
evaluating the leviels and regulation of these tissue
mediators have been hampered by the lack of an ef-
ficient sampling method. The development of mi-
crodialysis probes and their implantation into surgi-
cal wounds of patients who can provide verbal pain
report permits biochemically based studies on tissue
inflammation that can be conducted in the clinical
setting. Similarly, comparatively few studies have
evaluated regulatory mechanisms of primary afferent
terminal activity. The in vitro superfusion method of
dental pulp permits isolated examination of the
actions and interactions of inflammatory mediators
and prototype and novel drugs on altering the func-
510 Hargreaves et al.
tional activity of certain primary afferent fibers. To-
gether, these methods may provide greater insight
mto mechanisms of endodontic pain and its efficient
control.
We thank the following for their excellent technical sup-
port in these projects: Heidi Geier, Jennelle Durnett-Rich-
ardson, Pat Kane, and Julie Heller, and for their superb
dental student research projects: Mark Engelstad, Charles
McCann, Karen Reese,and Susan Buck of the University
of Minnesota.
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18. Roszkowski MT, Swift JQ, Hargreaves KM. Prostaglandin E2
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19. Roszkowski MT, Swift JQ, Hargreaves KM. Corticosteroids
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20. Roszkowski M, Swift JQ, Hargreaves KM. Local tissuerelease
ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY
October 1994
of immunoreactive substance P, bradykinin, PGE2, and LTB4
in a model of surgically-induced inflammation. Abstract Sot
Neurosci 1993;19:521.
21. Roszkowski M, Swift JQ, Hargreaves KM. Effects of ibu-
profen on iPGE2, iLTB4, ibradykinin, and isubstance P in
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34. Grutzner E, Garry M, Hargreaves KM. Effect of injury on
pulpal levels of immunoreactive substance P and CGRP. J En-
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35. Buck S, Reese K, Cane P, Hargreaves KM. Pulpal exposure
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36. Engelstad M, Garry M, Jackson D, Geier H, Hargreaves KM.
Adrenergic inhibition of iCGRP release from capsaicin-sensi-
tive fibers in dental pulp. Abstract Sot Neurosci 1992;18:690.
37. Jackson D, Garry M, Engelstad M, Geier H, Hargreaves KM.
Evaluation of iCGRP secretion from dental pulp in response to
inflammatory mediators. Abstract Sot Neurosci 1992;18:689.
38. Jackson D, Aanonsen L, Durnett-Richardson J, Geier H,
Hargreaves KM. An evaluation of the effects of excitatory
amino acids in bovine dental pulp. Abstract Sot Neurosci
1993;19:966.
39. Bowles W, Jackson D, Kane P, Hargreaves KM. Effect of
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Reprint requests.
Kenneth M. Hargreaves, DDS
Div. of Endodontics
University of Minnesota School of Dentistry
8-166 Moos Tower
Minneapolis, MN 55455