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)

2

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

3

Increasing Interest in the Scientific Community

4

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.

6

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

7

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.

8

Procedure: Perfusion, Sectioning, Histology

9

Immunohistochemistry: Optimization

10

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

11

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

12

Cursor coordinates

Axiovision 4.8 Interactive Measurement

Representative Image: Control

13

Representative Image: Morphine

14

Representative Image: Pregabalin

15

Columnar Representation of Fos-like Immunoreactivity Expression

16

Effects of SC (subcutaneous) morphine and PO (per oral) pregabalin on formalin-induced c-fos expression

Relationship between Nociceptive Behavior and c-Fos Expression

17

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.

18

Acknowledgements

Yolanda Kirksey

Rhoneil Pena

Joseph McLaughlin

Irene Choi

Kathleen Gogas

Grace Kim

19

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.

20