Low COMT and Stress Potentiate Functional Pain and Depressive Behavior, Especially in Female Mice
Xin Zhang
1, Katie Kanter
1, Jiegen Chen
1, Seungtae Kim
1,2, Yaoming Wang
1, Clementine Adeyemi
1, Sandra C. O’Buckley
1, Andrea G. Nackley
1*
Supplemental FiguresÂ
   Â
Baseline
A
2.0 1.5 1.0 0.5
Mechanical Allodynia B Mechanical Hyperalgesia
10 8 6 4 2
C Thermal Hyperalgesia
20 15 10 5
0.0 Vehicle+
Sham OR486+
Sham Vehicle+
Stress OR486+
Stress 0
Vehicle+
Sham OR486+
Sham Vehicle+
Stress OR486+
Stress 0
Vehicle+
Sham OR486+
Sham Vehicle+
Stress OR486+
Stress
Post-stress
D
2.0 1.5 1.0 0.5
Mechanical Allodynia E Mechanical Hyperalgesia
10 8 6 4 2
F Thermal Hyperalgesia
20 15 10 5
0.0 Vehicle+
Sham OR486+
Sham Vehicle+
Stress OR486+
Stress 0
Vehicle+
Sham OR486+
Sham Vehicle+
Stress OR486+
Stress 0
Vehicle+
Sham OR486+
Sham Vehicle+
Stress OR486+
Stress
Male Female
Figure 1s. Pain behavior profiles in males and females at baseline and following stress. At baseline, males and females in all groups exhibited a similar degree of (A) mechanical allodynia, (B) mechanical hyperalgesia, and (C) thermal hyperalgesia. Following stress, males and females in the Vehicle+Sham, Vehicle+Stress, and OR486+Sham groups exhibit a similar degree of (D) mechanical allodynia, (E) mechanical hyperalgesia, and (F) thermal heat hyperalgesia. However, females in the OR486+Stress group exhibit greater thermal heat hyperalgesia compared to males. Data are shown as Mean ± SD, N=6- 9 per group, ***P < 0.01.
***
50% Paw Withdrawal Threshold (g) 50% Paw Withdrawal Threshold (g) Paw Withdrawals (#) Paw Withdrawals (#) Paw Withdrawal Latency (s) Paw Withdrawal Latency (s)
Figure 2s. Swim stress does not alter the spinal BDNF expression in male or female mice. (A) Immunohistochemical staining shows expression of spinal BDNF on day 14 in males and females receiving vehicle in the absence or presence of swim stress. (B) Quantitative analysis of immunofluorescence intensity demonstrates that swim stress alone does not alter spinal BDNF expression. Data are shown as Mean ± SD, N=6-8 per group. Scale bar = 100 𝜇m.
Figure 3s. Swim stress does not alter the spinal GR expression. (A) Immunohistochemical staining shows expression of spinal GR on day 14 in males and females receiving vehicle in the absence or presence of swim stress. (B) Quantitative analysis of immunofluorescence intensity demonstrates that swim stress alone does not alter spinal BDNF expression. Data are shown as Mean ± SD, N=8 per group.
Scale bar = 100 𝜇m.
Figure 4s. COMT inhibition leads to decreased expression in hippocampal BDNF, in a sex- independent manner. (A) Immunohistochemical staining shows expression of hippocampal BDNF on day 14 from mice receiving the COMT inhibitor OR486 or vehicle in the absence of swim stress. (B) Quantitative analysis of immunofluorescence intensity demonstrates that male and female mice in the OR486+Sham group exhibit comparable decreases in hippocampal BDNF expression compared to those in the Vehicle+Sham group. Data are shown as Mean ± SD, N=6-8 per group, **P < 0.01, vs Vehicle+Sham group. Scale bar = 100 𝜇m.
Figure 5s. COMT inhibition does not alter hippocampal GR expression in male or female mice. (A) Immunohistochemical staining shows expression of hippocampal GR on day 14 from mice receiving the COMT inhibitor OR486 or vehicle in the absence of swim stress. (B) Quantitative analysis of immunofluorescence intensity demonstrates that in the absence of swim stress, OR486 delivery alone does not alter hippocampal GR expression in males or females. Data are shown as Mean ± SD, N=6-8 per group. Scale bar = 100 𝜇m.
GR/GAPDH (vsVehicle+Sham group)
I J
Spinal Cord Hippocampus
Male Female Male Female
A B C D
Vehicle+ OR486+ OR486+ Vehicle+ OR486+ OR486+ Vehicle+ Vehicle+ OR486+ Vehicle+ Vehicle+ OR486+
Sham Sham Stress Sham Sham Stress sham Stress Stress Sham Stress Stress
GR
BDNF GAPDH
E 3 2
1
0 Vehicle+
Sham OR486+
Sham OR486+
Stress
F2.0
1.5 1.0 0.5
0.0 Vehicle+
Sham OR486+
Sham OR486+
Stress
G2.5
2.0 1.5 1.0 0.5
0.0 Vehicle+
Sham Vehicle+
swim OR486+
Stress
H
3
2
1
0 Vehicle+
Sham Vehicle+
Stress OR486+
Stress
2.5 2.0 1.5 1.0 0.5
## K L
4 1.5
3
1.0 2
1 0.5
1.5
1.0
0.5
0.0 0 0.0 0.0
Figure 6s. Western blot analysis of BDNF and GR expression in spinal cord and hippocampus.
Quantitative analysis of (A-D) western blot bands demonstrates that OR486 delivery for 14 days increases GR expression in (E, F) spinal cord and swim stress increases GR expression in (G, H) hippocampus in both male and female mice. No interactions between OR486 and stress on GR expression are found. OR486 delivery increases BDNF expressions in (I, J) spinal cord and swim stress decreases BDNF expressions in (K, L) hippocampus in both male and female mice. OR486 and stress, together, potentiate changes in BDNF expression only in female mice. Data are shown as Mean ± SD, N=5-8 per group. *P < 0.05, **P < 0.01, vs Vehicle+Sham group.
**
** **
##
**
BDNF/GAPDH (vsVehicle+Sham group) GR/GAPDH BDNF/GAPDH(vs Vehicle+Sham group) (vs Veh+Sham group) BDNF/GAPDHGR/GAPDH (vsVeh+Sham group) (vs Veh+Sham group) BDNF/GAPDHGR/GAPDH(vs Veh+Sham group) (vs Veh+Sham group) **
Vehicle+ OR486+ OR486+ Vehicle+ OR486+ OR486+ Vehicle+ Vehicle+ OR486+ Vehicle+ Vehicle+ OR486+
Sham Sham Stress Sham Sham Stress Sham Stress Stress Sham Stress Stress
