Gene GeneBank accession number Forward primer Reverse primer Product size (bp)
COX2 NM_017232.3 AAGACAGATCAGAAGCGAGGAC AGGATACACCTCTCCACCGAT 159
GAPDH NM_008084 TGCCCCCATGTTTGTGATG GGCATGGACTGTGGTCATGA 160
IL-1β NM_031512.2 GCTTCCTTGTGCAAGTGTCTG GTCGAGATGCTGCTGTGAGA 200
IL-6 NM_012589.2 TCTCTCCGCAAGAGACTTCCA AGTCTCCTCTCCGGACTTGTG 121
NF-kB NM_199267.2 ACAGATACCACTAAGACGCACCC AATGGCTTGCTCCAGGTCTCG 237
NGF NM_001277055.1 AAGCTCACCTCAGTGTCTGG TGTACGGTTCTGCCTGTACG 148
NOX1 NM_053683.1 ATAGCTACTGCCCACCCCAAGT TTGAGTACCGCCGACAGCA 97
NOX2 NM_023965.1 TGTGTCGGAATCTCCTCTCCT TGTGAATGGCCGTGTGAAGT 135
NOX4 NM_053524.1 GCACAGTCCTGGCTTACCTT GCAGCTACATGCACACCTGA 136
TNF-α NM_012675.3 ATGGGCTCCCTCTCATCAGT GCTTGGTGGTTTGCTACGAC 106
untreated Elizabethan–
Elizabethan+ 0
10 20 30
EBD extravasation (µg/g)
***
**
ns Perfusion for Evans Blue Dye extraction
A
r1d 2wks cast immobilization
r2h r2d r3d
B
Elizabethan collar (just after cast removal to 3d after cast removal)
B
D
B r1d r2d r3d
0 1 2 3 4
Time after cast removal Hair loss (cm2)
r1d
B r2d r3d
Elizabethan– Elizabethan+ untreated
C
E
Evans Blue Dye
0 10 20 30 S1
L6 L5 L4 L3 L2 L1
Number of c-Fos i.r. neurons
Spinal cord levels
Elizabethan– Elizabethan+ 0
20 40 60 80 100
Number of c-Fos i.r. neurons
***
Elizabethan– Elizabethan+ 0
20 40 60
Number of c-Fos i.r. neurons
***
Elizabethan– Elizabethan+ 0
10 20 30 40
Number of c-Fos i.r. neurons
ns
Elizabethan– Elizabethan+
Elizabethan– Elizabethan+ 0 100 200 300 400 500
pERK / NK1R (µm2) ns
F G
I-II III-IV C
D E
Elizabethan– Elizabethan+
Elizabethan– Elizabethan+
A
B
F G
untreated vehicle 2 times 0
5 10 15
Acetone-evoked withdrawal score
Hind paw
***
###
B r3d r1w r2w r3w r4w r5w B r3d r1w r2w r3w r4w r5w 0
5 10 15 20
Time after cast removal
Light-evoked withdrawal score
Hind paw
vehicle
2 times
vehicle 2 times 0 20 40 60 80
AUC ###
A
Mechanical pain like behaviors (B-E) Tactile allodynia like behaviors (F)
⛄
Cold allodynia-like behevior (G) 2wks cast immobilization
r2h r1d r3d r1w r2w r3w r4w r5w r6w r7w r8w r9w r10w
saline + saline (vehicle) lidocaine + saline (1 time block) lidocaine + lidocaine (2 times block) B
B r3d r1w r2w r3w r4w r5w r6w r7w r8w r9wr10w 0
20 40 60 80 100
Time after cast removal
Withdrawal freaquency (%)
Calf skin
vehicle 1 time 2 times 0
500 1000 1500
AUC ###
B r3d r1w r2w r3w r4w r5w r6w r7w r8w r9wr10w 50
100 150 200
Time after cast removal
Withdrawal threhsold (g)
Calf muscle
vehicle 1 time 2 times 1200
1400 1600 1800 2000 2200
AUC
## ###
B r3d r1w r2w r3w r4w r5w r6w r7w r8w r9wr10w 0
20 40 60 80 100
Time after cast removal
Withdrawal freaquency (%)
Hind paw
vehicle 1 time 2 times 0
200 400 600 800
AUC ##
B r3d r1w r2w r3w r4w r5w r6w r7w r8w r9wr10w 0
20 40 60 80 100
Time after cast removal
Withdrawal freaquency (%)
Hind paw
vehicle 1 time 2 times 600
800 1000 1200
AUC
##
B C
D E
B r3d r1w r3w r5w B r3d r1w r3w r5w 0
5 10 15 20 25
Time after cast removal
Withdrawal threhsold (g)
Calf skin
B r3d r1w r3w r5w B r3d r1w r3w r5w 0
50 100 150 200
Time after cast removal
Withdrawal threhsold (g)
Calf muscle
B r3d r1w r3w r5w B r3d r1w r3w r5w 0
5 10 15 20
Time after cast removal
Withdrawal threhsold (g)
Hind paw
B r3d r1w r3w r5w B r3d r1w r3w r5w 0
10 20 30 40
Time after cast removal
Withdrawal threhsold (g)
Tail Elizabethan–
Elizabethan+ 0 20 40 60
AUC
ns
Mechanical pain-like behaviors (B-E)
A
B C
D E
r3w r5w
r2d 2wks cast immobilization
r1d r3d r1w
B
Elizabethan collar (just after cast removal to 3d after cast removal)
⛄
Cold pain-like behevior (F, G)
Elizabethan– Elizabethan+ 0 100 200 300 400 500
AUC
ns
Elizabethan– Elizabethan+ 0 10 20 30 40
AUC
ns
Elizabethan– Elizabethan+ 0 20 40 60 80 100
AUC
ns
Elizabethan– Elizabethan+ 0 5 10 15
Acetone-evoked withdrawal score
Hind paw
ns
Elizabethan– Elizabethan+ 0 5 10 15
Acetone-evoked withdrawal score
Tail
ns
F G
Push-pull gauge
