behavior paradigms for pain - psychogenics...vehicle (sni) gabapentin (sni;100 mg/kg) * * baseline 4...
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1Confidential
Behavior Paradigms for Pain
2Confidential
Pain Models at PGI
page 2
Acute InflammatoryVisceral Writhing
NeuropathicPost
Operative
Tail FlickHot Plate
FormalinCarrageenan
CFAATC
Acetic Acid Phenylquinone
ChemotherapyNerve Ligation:
Sciatic nerveSpinal nerveSpared nerve
Brennan incisional
model
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ACUTE PAIN
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Tail Flick and Hot Plate
• Tail Flick• Each animal is manually restrained so that an infrared heat source is
directed at a specific point on the tail
• When the pain threshold is reached, the animal flicks its tail away from the heat source and the latency is recorded
• The test period is limited to 10 sec to avoid tissue damage
• An average of 3 trials is used to determine latency to pain
• Hot Plate• Each animal is placed inside an observation chamber on a hot place set to
a specific temperature
• When the pain threshold is reached, the animal respond by shaking, licking or biting their hind paws or trying to escape
• The test period is limited to 10-60 sec (dependent on species and temperature) to avoid tissue damage
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Effects of morphine on tail flick in mice
Morphine dose dependently increases the latency to tail flick in CD1 mice.
Late
nc
y t
o t
ail
fli
ck
(s
ec
)
0
2
4
6
8
10
12
*
* *
Morphine (mg/kg; s.c.)
Vehicle 0.625 1.25 2.5 5
Time after injection
Baseline 30 min 1 hour 5 hours
Late
ncy t
o t
ai fl
ick (
sec)
0
2
4
6
8
10
12
Saline
Morphine 5 mg/kg
**
Time course for the effects of morphine in the tail flick test
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Effects of morphine in the hot plate test in miceL
ate
nc
y t
o r
ep
on
se (
se
c)
0
5
10
15
20
25
30
*
* *
Morphine (mg/kg; s.c.)
Vehicle 1.25 2.5 5 10
Morphine dose dependently increases the latency to paw lift from a hot plate in ICR mice.
Baseline Test
Late
ncy t
o r
esp
on
se (
sec)
0
4
8
12
16
20
24Vehicle - Saline
Vehicle - Morphine (5 mg/kg)
Naloxone (3 mg/kg) - Morphine (5 mg/kg)
*
The analgesic effects of morphine can be reversed by naloxone.
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Effects of morphine in the hot plate test in ratsL
ate
ncy t
o r
esp
on
se (
sec)
0
20
40
60
80
Vehicle
Morphine (3 mg/kg)
Morphine (10 mg/kg)
***
***
Morphine dose dependently increases the latency to paw lift from a hot plate in SD rats.
Time (min)
0 5 10 15 20 25 30
Late
nc
y t
o r
esp
on
se
(s
ec
)
0
10
20
30
40
50
60
70
Vehicle
Morphine 5 mg/kg
*
**
Time course for the analgesic effects of morphine on pain response in the hot plate test in SD rats.
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Inflammatory Pain
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Chemical-induced inflammation
• Formalin, carrageenan or complete Freud’s Adjuvant (CFA) injected in the plantar surface of one hind paw.
• Two phases are seen during formalin-induced inflammation• Phase 1: acute pain due to burst of activity from pain fibers
• Phase 2: represents responses inflammatory hyperalgesia
• End point measurement include• Assessment of mechanical allodynia using Von Frey Filaments
• Time spent and number of behaviors (licking and biting the injected paw)
• Pain Score as shown in the table below
0: Normal weight bearing on injected paw1: Limping or resting the injected paw lightly on the floor2: Lifting injected paw3: Flinching and/or shaking injected paw4: Licking, biting or grooming injected paw
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Formalin-induced inflammation in rats and mice
Time (min)
0 10 20 30 40 50 60
Tim
e s
pen
t li
ckin
g p
aw
(sec)
0
20
40
60
80
100
120
Saline - Formalin
Acetaminophen - Formalin
Acetaminophen attenuates formalin-induced inflammatory pain response in SD rats during early Phase and late Phase
Morphine attenuates formalin-induced inflammatory pain response in C57 mice during early Phase and late Phase
Me
an
Beh
avio
ral
Sc
ore
0
1
2
3
4
5
Saline
Morphine 0.3 mg/kg
Morphine 1 mg/kg
Morphine 3 mg/kg
Acute Phase(min 1-10)
Late Phase(min 11-40)
#
* *
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CFA-induced inflammation in SD rats
Time post treatment
Baseline 1 hour 2 hours
Pa
w W
Ith
dra
wa
l T
hre
sh
old
(g
)
0
10
20
30
40Vehicle - Vehicle
CFA - Vehicle
CFA - Naproxen (10 mg/kg)
* * **
Paw withdrawal threshold (PWT) was assessed using Von Frey filaments 24 hours after CFA injection. CFA decreases PWT indicative of pain response. Naproxen this pain response by increasing PWT
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Carrageenan-induced inflammation in mice
Pa
w W
ith
dra
wa
l T
hre
sh
old
(g
)
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
Vehicle
Naproxen 30 mg/kg
Baseline 60 min
Time after naproxen administration
90 min
*
*
Oral administration of naproxen attenuates carrageenan 25ul of 1%) pain response as seen in the increased PWT response
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Visceral Pain - Writhing
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Chemical-induced writhing
• An intraperitoneal injection of an acetic acid solution or phenylquinoneinduce a transient state of somatic (visceral) pain. This manifests as constriction of abdomen, turning of trunk and extension of hind legs.
• The number of constrictions is counted for 15 minutes
Nu
mb
er
of
wri
thes
0
10
20
30
40
50
60
*
*
(-)-U50488 (mg/kg; i.p.)
Vehicle 0.3 1 3
Nu
mb
er
of
wri
thes
0
5
10
15
20
**
Morphine (mg/kg; s.c.)
Vehicle 1.25 2.5 5
Morphine attenuates phenylquinoneinduced writhing in mice
U50488 (kappa opioid agonist) attenuates acetic acid induced writhing in mice
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Neuropathic Pain
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Neuropathic Pain Models
• Nerve ligation models• Spinal (L5) nerve ligation (SNL)• Chronic constriction injury of the sciatic nerve (CCI)• Spared Nerve Injury (SNI)
• Chemotherapy-induced neuropathic pain• Paclitaxel• Bortezomib• Oxaliplatin• Vincristine
• Endpoint Measures• Evoked pain response (Von Frey)• Cold sensitivity
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Nerve Ligation Models
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Analgesic effects of gabapentin in SNL rats
Paw
Wit
hd
raw
al T
hre
sh
old
(g
)
0
10
20
30
40
Sham - Vehicle
SNL - Vehicle
SNL - Gabapentin (100 mg/kg)
**
Baseline 30 min 2 hour
Time after administration
Two week post injury, SNL rats show increased pain response compared to Sham rats as seen in the decreased PWT response when measured by Von Frey filaments. Gabapentin dose dependently attenuates this pain response and increases PWT response (left). The analgesic effects of acute oral administration of gabapentin lasts for 2 hours (right).
Paw
Wit
hd
raw
al
Th
resh
old
(g
)
0
10
20
30
40
Sham - Vehicle
SNL - Vehicle
SNL - Gabapentin (100 mg/kg)
SNL - Gabapentin (150 mg/kg)
Baseline pre-dose 90 min post-dose
***
***
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Analgesic effects of antidepressants in SNL ratsP
aw
Wit
hd
raw
al T
hre
sh
old
(g
)
0
4
8
12
16
20
Vehicle
Duloxetine (10 mg/kg)
Duloxetine (30 mg/kg)
Baseline 30 min post-dose
***
Pa
w W
ith
dra
wa
l T
hre
sh
old
(g
)
0
4
8
12
16
20
Vehicle
Venlafaxine (200 mg/kg)
Baseline 120 min post-dose
***
Acute oral administration of duloxetine (left) and venlafaxine (right) decreases neuropathic pain response as seen in the increased PWT response compared to vehicle-treated rats.
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Analgesic effects of tramadol in SNL ratsP
aw
Wit
hd
raw
al T
hre
sh
old
(g
)
0
5
10
15
20Vehicle
Tramadol 50 mg/kg
Tramadol 100 mg/kg
Tramadol 150 mg/kg
Baseline
*
~
2 hrs post-administration
Tramadol dose dependently attenuates pain response in SNL rats and increases PWT response (left) following acute oral administration. The analgesic effects of tramadol lasts for 4 hours (right).
Time post administration (hour)
0 1 2 3 4
Paw
Wit
hd
raw
al T
hre
sh
old
(g
)
0
5
10
15
20
25
30
VehicleTramadol (150 mg/kg)
BSL
* *
*
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Analgesic effects of gabapentin and carbamazepine in CCI rats
Late
nc
y t
o l
ift
pa
w (
se
c)
0
20
40
60
80
100
120
Vehicle
Gabapentin (100 mg/kg)
Carbamazepine (100 mg/kg)
*
*
*
Baseline 2 hours 5 hours
Time after administration
*
Acute oral administration of either gabapentin or carbamazepine attenuates cold allodynia as measured by the increased latency to lift the injured paw from a cold plate in CCI rats. The effect lasted 5 hours
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Analgesic effects of gabapentin and morphine in CCI ratsP
aw
Wit
hd
raw
al T
hre
sh
old
(g
)
0
4
8
12
Vehicle
Gabapentin (10 mg/kg)
Gabapentin (30 mg/kg)
Baseline 60 min post-dose
*
*
Acute administration of gabapentin (left) or morphine (right) dose dependently attenuates pain response in CCI rats two weeks post injury.
Pa
w W
ith
dra
wa
l T
hre
sh
old
(g
)
0
3
6
9
12
15
Vehicle
Morphine (2.5 mg/kg)
Morphine (5 mg/kg)
Baseline 30 min post-dose
**
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Analgesic efficacy of duloxetine in CCI rats and miceP
aw
Wit
hd
raw
al
Th
res
ho
ld (
g)
0
4
8
12
Vehicle
Duloxetine (10 mg/kg)
Duloxetine (30 mg/kg)
Baseline 60 min post-dose
**
Baseline 60 min post-dose
Paw
Wit
hd
raw
al T
hre
sh
old
(g
)
0.0
0.1
0.2
0.3
0.4
Vehicle
Duloxetine (30 mg/kg)
*
Oral acute administration of duloxetine attenuates pain response in CCI rats (left) and CCI mice (right) and increases PWT response.
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Spared Nerve Injury
Paw
Wit
hd
raw
al T
hre
sh
old
(g
)
0
10
20
30
40
Vehicle (Sham)
Vehicle (SNI)
Gabapentin (SNI;100 mg/kg)
**
Baseline 4 weeks post injury 5 weeks post injury
Acute oral administration of Gabapentin attenuates neuropathic pain response in SNI rats as seen in the increased PWT response
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Chemotherapy- induced neuropathic pain (CINP)
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Paclitaxel-induced neuropathic pain in SD rats2D Graph 2
Days post Paclitaxel injection
D0 D8 D12 D16 D23 D32 D38 D44
Paw
Wit
hd
raw
al
Th
resh
old
(g
)
0
10
20
30
40
Vehicle - Vehicle
Paclitaxel - Vehicle
** *
* **
Subchronic paclitaxel injections (4 injections of 2 mg/kg QOD) induces peripheral neuropathic pain in rats. This mechanical hyperalgesia can be seen by the decrease in PWT starting from 12 until day 44
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Analgesic effects of gabapentin on CINP2D Graph 2
Time post treatment
Baseline Day 1 Week 2 Week 3 Week 4
Pa
w W
ith
dra
wa
l T
hre
sh
old
(g
)
0
10
20
30
40
50
Paclitaxel - Vehicle
Paclitaxel - Gabapentin (50 mg/kg)
Paclitaxel - Gabapentin (100 mg/kg)
* **
*
Chronic oral administration of gabapentin (100 mg/kg) attenuates neuropathic pain response in rats injected with paclitaxel as seen with the increased PWT response.
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Bortezomib-induced neuropathic pain in SD rats
Time after injection
Baseline D2 D7 D10 D14 D17 D21
Paw
Wit
hd
raw
al
Th
resh
old
(g
)
0
10
20
30
40
Vehicle
Bortezomib (0.5 mg/kg)
Bortezomib (1 mg/kg)
**
* ***
* * * *
Vehicle or Gabapentin
D0 D13 D15 D20 D25 D30
Paw
Wit
hd
raw
al T
hre
sh
old
(g
)
0
10
20
30
40
Bortezomib (0.5 mg/kg) - Vehicle
Bortezomib (0.5 mg/kg) - Gabapentin (100 mg/kg)
* * * *
Bortezomibinjection
Post injectionbaseline
Acute injection of bortezomib (red arrow) produces long lasting neuropathic pain response as seen in the decreased PWT response compared to vehicle .
Acute injection of bortezomib (0.5 mg/kg) produced mechanical hyperalgesia that lasted for 30 days post injection. Gabapentin decreased the hyperalgesia and increased PWT of the rats compared to vehicle .
29Confidential
Oxaliplatin and vincristine-induced neuropathic pain in SD rats
Intravenous injection of oxaliplatin (twice weekly) dose dependently induces peripheral neuropathic pain in rats and decreases PWT response.
Weeks of injection
Baseline W1 W2 W3 W4
Paw
Wit
hd
raw
al T
hre
sh
old
(g
)
0
5
10
15
20
25
30
35Vehicle
Oxaliplatin (1.5 mg/kg)
Oxaliplatin (3 mg/kg)
**
*
*
*
*
*
Gabapentin (100 mg/kg; po) increases PWT during week 4 of oxaliplatin treatment.
Paw
Wit
hd
raw
al T
hre
sh
old
(g
)
0
10
20
30
40
Vehicle - Oxaliplatin (3 mg/kg; iv) Gabapentin (100 mg/kg; po) - Oxaliplatin (3 mg/kg; iv)
*
30Confidential
Vincristine-induced neuropathic pain in SD rats
*
* **
Time after injection
Baseline D1 D3 D8 D11 D14 D17
Paw
Wit
hd
raw
al T
hre
sh
old
(g
)
0
5
10
15
20
25
30
35
Vehicle
Vincristine (0.175 mg/kg)
*
* **
Intravenous injection of vincristine (QOD for a total of 5 injections) induces mechanical hyperalgesia as seen in the decreased PWT response.
31Confidential
Incisional Pain
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Brennan model of incisional pain
• Postoperative pain is a common form of acute pain for which effective treatments are not yet available.• Treatment of persistent or chronic pain can be hindered by the limited efficacy and side effects
of currently available analgesics (e.g., opioids or anti-inflammatory drugs).
• Pain behaviors are greated immediately after recovery from anesthesia and can persist for several days (Peters and Eisenach, 2010; Obata, 2010).
• Operative pain in humans can be mimicked by paw incision in rats (Brennan, et al., 1995; Kang et al., 2010). Rats are assessed based on guarding scores:
0: plantar surface, toes, and heel of ipsilateral paw are firmly on mesh floor, balancing weight uniformly with contralateral paw.
1: ipsilateral heel is raised, but the animal is still bearing some weight on the plantar surfaceand toes. At this stage, the distribution of weight between the ipsilateral and contralateral paws ceases to be equal, and signs of blanching, or whitening, of the ipsilateral paw should be a sign of such a case
2: ipsilateral paw is not bearing weight (may be flat and held out to the side) or the animal isonly on its toes. Heel, and possibly plantar surface and base of the toes may be raised up, but not totally away from the mesh floor, with an unbalanced support of weight
3: ipsilateral paw is lifted up completely and being held close to the body.
33Confidential
Analgesic effects of ketoprofen and morphineT
ota
l G
uard
ing
Sco
re
0
10
20
30
40
50
Vehicle
Ketoprofen (1 mg/kg, p.o.)
Ketoprofen (2 mg/kg, p.o.)
Ketoprofen (5 mg/kg, p.o.)
Baseline
*
60 min pose-dose
**
To
tal
Gu
ard
ing
Sco
re
0
10
20
30
40
50
Vehicle
Morphine (5 mg/kg, s.c.)
Morphine (10 mg/kg, s.c.)
Baseline
*
60 min post-dose
*
Acute administration of either the NSAID ketoprofen (left) or morphine (right) 1 hour post surgery decreases guarding scores in SD rats in a dose dependent manner.
34Confidential
Analgesic effects of gabapentin and tramadolT
ota
l G
uard
ing
Sco
re
0
10
20
30
40
50
Vehicle
Tramadol (50 mg/kg, p.o.)
Tramadol (100 mg/kg, p.o.)
Tramadol (150 mg/kg, p.o.)
Baseline
*
60 min pose-dose
*
*
To
tal G
uard
ing
Sco
re
0
10
20
30
40
50
Vehicle
Gabapentin (50 mg/kg, p.o.)
Gabapentin (100 mg/kg, p.o.)
Baseline
*
60 min post-dose
*
Acute oral administration of either tramadol (left) or gabapentin (right) dose dependently decreases guarding scores when administered 1 hour post surgery.