using c-fos-like immunoreactivity as a neural marker for nociceptor activity
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Megan Atkins Preclinical Pharmacology: Nektar Therapeutics
20August2015
Using c-fos-like Immunoreactivity as a
Neural Marker for Nociceptor Activity
What is c-Fos?
Tissue-Specific Gene Expression Pattern of the Fos Gene (Genomics Institute of the Novartis Research Foundation,
GNF)
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NFAT(green), Jun(blue) and Fos(red),
bound to DNA
c-Fos is rapidly and transiently expressed in neurons in response to stimulation
c-Fos as an Indirect Marker of Functional Activity
• Studies show that various types of noxious stimulation (thermal, mechanical, chemical) induce c-Fos expression in the brain and spinal cord.
• “Induction of c-Fos-like protein in spinal cord neurons following sensory stimulation”, Hunt et al. 1987.
– Noxious stimulation (mustard oil or radiant heat) to the hind paw resulted in a massive increase in the expression of Fos in neurons in the dorsal horn of the lumbar spinal cord.
• Nociception-induced c-Fos expression in spinal neurons is suppressed by
administration of analgesic drugs
– Acclimatizing to environment conducted to reduce Fos expression due to handling, restraint, or novel environment stressors
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Increasing Interest in the Scientific Community
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The number of publications each year from 1995 to 2015 identified by a literature search on the ScienceDirect database using the keyword “fos” combined with either “pain”, “nociception”, “nociceptive”, or “noxious”.
Implementing a Translational Tool Advantages of using c-fos expression as a tool for evaluating neural basis of nociception:
– Easy to identify neuronal populations that
respond to noxious stimulation – c-fos expression can be analyzed quantitatively
(counting of neurons immunoreactively labeled for Fos)
– Provides reliable basis for comparison of
various manipulations on nociceptive processing • Basal c-fos expression is low in most
neurons – Does not require the use of anesthesia during
noxious stimulation • nociceptive processing under normal
conditions – Immuno-labeling of Fos protein with other
immunocytochemical & tract-tracing procedures (ex. retrograde labeling) • identify c-fos-expressing neuronal
projections 5
Photomicrograph of in-vivo basal c-Fos expression in the rat spinal cord. Insets are
magnified.
Figure 4A, "Sympathetic-correlated C-Fos Expression in the Neonatal Rat Spinal Cord in Vitro." Open-i. API, 2009. Web. 17 Aug. 2015.
Project Overview
Development of immunohistochemical detection of Fos protein after noxious stimuli and alterations in c-Fos expression with the addition of pain-relieving drugs
morphine and pregabalin.
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Materials & Methods: AFP Study
• Unbiased, automated measurement of response to formalin insult
• Metal bands placed on left hind paw & rats acclimated to the test chamber (approx.15 min)
• Test article administered (PO, IP, SC, or IV)
• 50 uL formalin solution administered into dorsal surface of paw
• Rat placed in test chamber- number of paw movements recorded for up to 60 minutes post-formalin injection
• Rat euthanized 1hr after study conclusion – Previous literature shows c-fos
expression peak ~2hr post-formalin injection
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Results: AFP Study Data
–First (acute) phase: 0-9 minutes; results from direct chemical activation (i.e. formalin) of myelinated and unmyelinated nociceptive afferent fibers- acute mechanical pain. –Short quiescent period –Second (tonic) phase: 10-60 minutes; characterized by persistent shaking or licking of the injected paw due to
inflammatory pain and central sensitization.
These biphasic behavioral responses are paralleled by a biphasic discharge of DH nociresponsive neurons.
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Procedure: Perfusion, Sectioning, Histology
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Immunohistochemistry: Optimization
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Calbiochem Rabbit anti-cfos 1:500–1h RT—2d @ 4
degrees Celsius
Abcam Rabbit anti-cfos 1:100–2h RT—2d @
4 degrees Celsius
Abcam Rabbit anti-cfos 1:500–2h RT—2d @ 4
degrees Celsius
Abcam Rabbit anti-cfos 1:500–4h RT—2d @
4 degrees Celsius
Abcam Rabbit anti-cfos 1:500–4h RT—3d @
4 degrees Celsius
Abcam 1:100, 1h RT 2 days
Abcam 1:500, 4h RT 2 days
Image Analysis: c-Fos Qualification
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Light microscopic images of c-fos expression in the lumbar spinal cord dorsal horn ~1h following intraplantar formalin (5%, 50 uL) injection.
Image Analysis: c-Fos Quantification
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Cursor coordinates
Axiovision 4.8 Interactive Measurement
Representative Image: Control
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Representative Image: Morphine
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Representative Image: Pregabalin
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Columnar Representation of Fos-like Immunoreactivity Expression
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Effects of SC (subcutaneous) morphine and PO (per oral) pregabalin on formalin-induced c-fos expression
Relationship between Nociceptive Behavior and c-Fos Expression
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Effects of SC (subcutaneous) morphine and
PO (per oral) pregabalin on formalin-
induced nociceptive behavior
Effects of SC (subcutaneous) morphine and
PO (per oral) pregabalin on formalin-induced
c-fos expression
Summary & Conclusions
Immunohistochemical techniques were optimized for c-Fos expression and
can be successfully applied to future studies.
There is no relevant literature on the effects of pregabalin on c-Fos expression
in the AFP model. There is literature demonstrating c-Fos expression
reduction with pregabalin administration in neuropathic pain models. This is a
novel finding.
Future studies can be conducted to profile various classes of analgesics to
determine the signature of each on noxious stimulus-evoked FLI.
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Acknowledgements
Yolanda Kirksey
Rhoneil Pena
Joseph McLaughlin
Irene Choi
Kathleen Gogas
Grace Kim
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References
• "Sympathetic-correlated C-Fos Expression in the Neonatal Rat Spinal Cord in Vitro." Open-i. API, 2009. Web. 17 Aug. 2015.
• Krukoff, Teresa L. "C-fos Expression as a Marker of Functional Activity in the Brain." Neuromethods 33 (1999): 213-30. Print.
• Hunt, S. P.; Pini, A.; Evan, G. Induction of c-fos-like protein in spinal cord neurons following sensory stimulation. Nature 328:632–634; 1987.
• Hahm, T. S., H. J. Ahn, S. Ryu, M. S. Gwak, S. J. Choi, J. K. Kim, and J. M. Yu. "Combined Carbamazepine and Pregabalin Therapy in a Rat Model of Neuropathic Pain." British Journal of Anaesthesia 109.6 (2012): 968-74. Web. 18 Aug. 2015.
• Harris, Justin A. "Using C-fos as a Neural Marker of Pain." Brain Research Bulletin 45 (1997): n. pag. Elsevier. Web. 17 Aug. 2015.
• Gogas, K. R. "The Antinociceptive Action of Supraspinal Opioids Results from an Increase in Descending Inhibitory Control." Neuroscience 42.3 (1991): n. pag. Print.
• Jasmin, L. "Differential Effects of Morphine on Noxious Stimulus-evoked Fos-like Immunoreactivity in Subpopulations in Spinoparabrachial Neurons." The Journal of Neuroscience (1994): 7252-260. Print.
• Presley, R. W.; Mene´trey, D.; Levine, J. D.; Basbaum, A. I. Systemic morphine suppresses noxious stimulus-evoked Fos protein-like immunoreactivity in the rat spinal cord. J. Neurosci. 10:323–335; 1990.
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