McNair Research Journal - University of St. Thomas

Science
Establishing a Definitive Model for
Inflammatory Hypernociception in C57B1/6
Paul L. Maitland-McKinley* T , Carolina Mora Solano* and Devavani Chatterjea*
*Macalester College - Department of Biology; T University of St. Thomas McNair Scholars Program
Abstract
Surprisingly little work has been done on characterizing the possible cell types
involved in the inflammatory hypernociceptive pathway. While a wealth of
information exists pertaining to the actual mediators involved in the transduction
of inflammatory hypernociceptive signals, the cellular origins of these
mediators remain undiscovered. However, in order to begin reliable and novel
cytokine testing to determine possible cell types that are involved, it is first
necessary to develop a reliable method of producing and measuring the
inflammatory hypernociceptive response in a model. Therefore, our aim was to
explore and characterize compound 48/80 (c48/80) induced inflammatory
hypernociception in the ND4 strain (well established and used in pain models),
followed by the C57Bl/6strain (a more established immunological strain).
We found that although mechano-hypernociception results were inconclusive,
thermo-hypernociceptive responses were much more reliable over a 24-hour
period following challenge with most significant differences between treated
and untreated groups between 15-45 minutes. Furthermore, we also found
that C57Bl/6 mice respond with the optimal hypernociceptive-indicating
actions at a slightly lower temperature (50.0 C) as opposed to ND4 (53.0 C)
mice. Nevertheless, with the finding that the thermal inflammatory
hypernociceptive model is reliable, we can now move on to more novel
potential studies involving classifying the particular secretagogues involved
and the respective cell type that are releasing them.
Keywords: Rodent, Mast Cell, Inflammation, Neuroimmunology, Skin
1. Introduction.
I. Inflammatory Pain. All organ systems within the human body are
susceptible to inflammation, which subsequently leads to either the direct
or indirect stimulation of primary afferent nociceptors in the periphery.
While inflammation of the tissue does not necessarily cause chronic pain,
pain is experienced upon either mechanical or thermal stimulation of the
inflamed site (1,2). Pain is defined by the International Association for the
Study of Pain (IASP) as an unpleasant sensory and emotional experience
associated with actual or potential tissue damage, or described in terms of such
damage. For the purposes of this proposal, it is defined as heightened
response to a noxious stimulus. Nociception, on the other hand, is
defined as the system’s physical response to the detection of noxious
stimulus which serves as an alert to the incidence or to the potential threat
of infection and injury.
Nociception can be measured either via an observed increase in the
excitability of primary sensory neurons or through behavioral responses in
the case of live organism models. A definitive and conclusive observation
of hypernociception through behavioral assays, however, typically uses
either the administration of a hypernociceptive agent or the application of
a noxious stimulus to determine if said agent has the ability to produce
one of the two following actions: 1) block the detection of the administered
stimulus (analgesia or anti-nociception) or, 2) increase the observed
response to a stimulus (hypernociception) (3).
It is generally accepted in the field of pain research that hypernociception
is essential to inflammatory pain (4,5). In the past it was believed that
sensitization of the nociceptor was produced by the excitatory actions of a
“soup of inflammatory mediators” – i.e., the combined release of a vast
amount of mediators from the site of inflamed or damaged tissues (6).
The current understanding, however, is that most likely it is a sequential
release of mediators or cellular events that initiates that inflammatory
Paul L. Maitland-McKinley
Inflammatory Hypernociception
hypernociceptive pathway. Furthermore, the release of these mediators or
events, called a cytokine cascade, appears to be dependent on time from
initial stimulation (2).
II. Cascades & Mediators. While there are no true universally understood
or accepted molecular mechanisms for the sensitization of nociceptors it is
currently believed that the stimulation of G-protein coupled receptors by
inflammatory mediators may activate the enzyme adenylate cyclase which,
in turn, produces the chemical signaler cAMP. The activation of protein
kinases (PKA and PKC) is then triggered by this cAMP, which in turn
lead to the phosphorylation of ion channels in the neuronal membrane.
The result of these successive triggers is that sodium and calcium are able
to freely enter the cell while potassium, in turn, is prevented from leaving
due to the necessity to balance the growing cellular cationic charge. This is
believed to be the basic molecular mechanism of hyperalgesia, where a
previously non-troubling stimulus – be it thermal, mechanical or chemical
– becomes painful. It is this response that is perceived at the level of the
primary afferent neurons (4).
There are two distinct types of mediators present in the system during
inflammatory pain that have been outlined by the Ferreira lab: the first is
“intermediate” mediators and second are “final” mediators. Intermediate
mediators are molecules which are released during inflammation by
resident and migrating cells within the vicinity of the damaged location
or by plasma. They are responsible for the eventual release of other
mediators, including the final mediators. Final mediators, on the other
hand, have a direct-acting effect on the primary sensory (or afferent)
neurons. Examples of final mediators include endothelin, sympathetic
amines, nerve growth factor (NGF), bradykinin and prostaglandins.
“Intermediate” mediators include cytokines such as interleukins,
TNF-alpha and IFNgamma (7).
Still, while a wealth of information exists regarding the roles of mediators,
the cellular origins of these mediators remain elusive. Research into other
possible cellular origins of mediators has been previously done, but much
still remains only speculation.
III. The specific actions of TNF-a, IL-1b, IL-15, IL-18. In a model of carrageenan-
induced mechanical hypernociception in the rat hind paw produced
by the Cunha lab, it was found that neutrophils are most likely not
responsible for the production of two very important inflammatory cytokines:
TNF-alpha and IL-1beta. In fact, an increase in cytokine production
seemed to induce neutrophil migration; and these cytokines appeared
to act in a neutrophil-dependent manner to induce hypernociception, as it
was found that neutrophils mediated the production of PGE2 by IL-1beta
(8). Furthermore, in a separate study from this lab that again made use of
carrageenan-hind paw model, it was found that two of the key cytokines
most likely involved were TNF-alpha and KC (keratinocyte-derived
chemokine). Both molecules have a role in the secretion of prostaglandins
as induced by IL-1beta. It was also found that KC stimulates the release of
sympathetic amines which resulted in the direct sensitization of nociceptors.
Whereas it was found that TNF-alpha appears to act on IL-1beta via
the TNF-R1 receptor (2).
In addition, two very similar experimental investigations in mice concluded
that both IL-15 and IL-18 seem to be key cytokines in the mediation
of the inflammatory pain cascade induced through the ovalbumin
28 University of St. Thomas McNair Research Journal