ATTENUATION OF INJURY EVOKED PAIN-RELATED BEHAVIOR BY BLOCKADE OF NEUROPEPTIDE Y Y2 RECEPTOR Damir Sapunar, Katarina Vukojevic, Sandra Kostic, Livia Puljak

1
PH 180
ATTENUATION OF INJURY EVOKED PAIN-RELATED
BEHAVIOR BY BLOCKADE OF NEUROPEPTIDE Y Y2
RECEPTOR Damir Sapunar, Katarina Vukojevic,
Sandra Kostic, Livia Puljak Laboratory for Pain
Research, Department of Anatomy, Histology and
Embryology, University of Split School of Medicine
Background
Results
Neuropeptide Y (NPY) is a highly conserved
peptide from the pancreatic polypeptide family
that has marked and diverse biological effects.
Besides its effects in the regulation of neuronal
and antimicrobial activity, cardiovascular
performance, and food intake, it is also involved
in pain processing. However, its mechanism of
action in neuropathic pain is incompletely
defined and conflicting results have been
reported. Since in neuropathic pain important
early changes occur in peripheral neuron itself
and because of the clinical opportunity for
delivery of drugs selectively to peripheral
nerves and DRG, we decided to test the effect of
NPY application directly to DRG immediately
following the SNL injury. The study was designed
to determine whether NPY modulates behavioral
signs of neuropathic pain. With its role in
neuroinflammation in mind, we extended our
analysis to see if topically applied NPY can
initiate neuroinflammation within DRG and dorsal
horn (DH). In order to discriminate NPY action,
specific Y1 and Y2 antagonists were delivered
directly to DRG. Since inhibition of
neuroinflammation attenuates the development but
not existing hypersensitivity in a rat model of
neuropathy we opted for simultaneous application
of NPY antagonists and SNL.
Immunofluorescence intensity is more pronounced
in SNL/saline and SNL/NPY groups compared to
control (A). Representative examples of GFAP
immunoreactivity in ipsilateral dorsal horn of
the B) SNL/saline and C) SNL/NPY 0.01 nM groups.
Scale bar for B and C 100 ?m. Data are presented
as mean SD. Legend significant differences
from control group (n 6-15).
Effect of NPY, BIBO3304 and BIIE0246 injection to
acutely injured DRG on astrocyte activation in
dorsal horn. Quantification of GFAP
immunofluorescence was done along line overlaid
from central canal to dorsal root entry zone
(DREZ). The highest fluorescence intensity was
observed in SNL/NPY group in lamina I, II and III
(0.2 mm from DREZ) (A). Representative examples
of GFAP immunoreactivity in ipsilateral dorsal
horn of the rat spinal cord following B)
SNL/saline and C) SNL/BIIE 20 nM group. Scale
bar 0.5 mm.
SNL produced significant changes in A) vonFrey
withdrawal threshold and E) hyperalgesia
responses. DRG targeted delivery of NPY performed
simultaneously with SNL additionally exacerbated
mechanical hyperalgesia (F). However, mechanical
allodynia (decreased threshold to von Frey
fibers) (B) was enhanced but in the same manner
as in SNL/saline group. When BIBO3304, antagonist
of Y1 receptor, was injected into DRG, it
produced similar von Frey threshold reduction as
the one measured in SNL/saline and SNL/NPY group
(C). BIIE0246 application completely abolished
injury effect induced by SNL or by additive
effect of NPY (D) (in the case of mechanical
hyperalgesia). Each value represents the mean
difference between left and right paws. Data are
presented as mean SEM. Asterisk denotes
significant difference from baseline.
Figure 2.
Representative examples of GFAP immunoreactivity
in ipsilateral dorsal root ganglion of the A)
control and B) SNL/NPY 0.01 nM group. Most of the
ganglion cells in SNL/NPY group are surrounded
with activated satellite cells (A). Higher
magnification of GFAP positive satellite cells
surrounding neurons (C and D) reveals that
activation is accompanied with increase in their
thickness. The same neurons in DIC bright field
view (D). The percent of the GFAP positive
satellite cells in control and experimental group
(E). In SNL/saline, SNL/NPY and SNL/BIBO the
percentage of GFAP positive "rings" was
significantly higher compared to control group.
Scale bar 50 ?m for A and B and 20 ?m for C and
D. Data are presented as mean SD. Legend
significant differences from control group (n
3-5).
Materials and Methods
Experimental animals and surgical procedure A
total of 57 male Sprague-Dawley rats were
assigned to one of the following groups control
group (n5), SNL group accompanied with saline
injection into DRG (SNL/saline) (n5), and groups
with DRG injection of different doses of NPY
(n17), BIBO3304 (n16) and BIIE0246 (n11).
Drugs (NPY, BIBO3304 and BIIE0246) were
delivered (4 ?L) immediately following SNL using
our previously described method for DRG injection
in rats (J Neurosci Methods. 200915177(2)397).
Behavioral testing was performed on the day
preceding the SNL surgery and three times
postoperatively (4th, 8th and 15th day). Our
overall strategy involved measuring changes in
sensory test responses between ipsi- and
contralateral paws over time and in different
experimental groups according to the methodology
previously described Anesthesiology.
2004101(2)476. Immunohistochemistry Two weeks
following the surgery, the L5 ganglion and
corresponding spinal cord segment were removed,
sectioned and treated rabbit anti-GFAP polyclonal
antibody.
Conclusion
The main finding of our study is that NPY,
applied directly to acutely injured DRG, is
increasing post-injury pain-related behavior in a
modality-specific manner. When the saline was
injected into DRG after SNL expected increase in
hyperalgesia response to needle stimulation was
present on the 4th and 8th postoperative days.
Two weeks following SNL values returned to
baseline. When we substituted saline injection
with injection of different doses of NPY,
hyperalgesic responses were increased in the
SNL/NPY group compared to the SNL/saline group.
Application of different NPY doses increased
responses compared to the control group. In order
to discriminate NPY action, specific Y1 and Y2
antagonists were also delivered directly to DRG.
We found that the behavioral actions of NPY were
abolished by Y2 receptor antagonist. We assumed
that the NPY effects are result of its role in
immunity so we used immunohistochemical staining
with GFAP in order to investigate the activation
of DRG satellite cells and dorsal horn (DH)
astrocytes. Exacerbation of pain-related behavior
following NPY injection was accompanied with
astrocyte activation in ipsilateral DH and with
satellite cells activation in DRG. This
activation was reduced following Y2 receptor
antagonist application. These findings indicate
an important link between pain-related behavior
and neuroimmune role of NPY and its Y2 receptor.
SNL did not induce significant change in heat or
cold withdrawal latency (A and E). DRG targeted
delivery of NPY exacerbated cold hypersensitivity
(B) but did not change withdrawal latency to heat
stimulus (F). Application of BIIE0246 completely
abolished post injury increase in withdrawal
latency following cold stimulus (D). NPY Y 1
receptor blockade did not influence responses to
cold stimuli (C). Heat withdrawal threshold was
not influenced by Y1 or Y2 blockade (G and H).
Each value represents the mean difference between
left and right paws. Data are presented as mean
SEM. Asterisk denotes significant difference from
baseline.
Poster session date 9/2/2010, No.
10-A-3542-IASP Supported by MZOS grant no.
216-2160528-0522