Article Figures & Data
Figures
- Figure 1.
Microglial subtypes. Representative images of the three classifications of microglia based on morphology are shown. “Nonramified”: round/amoeboid activated microglia; “intermediate”: reactive microglia with numerous thick processes; “ramified”: microglia with thin long processes. Scale bars, 2 μm in all images.
- Figure 2.
Sex, but not morphine treatment or time, affects microglia activation in the vlPAG. a, No significant differences in microglia cell number were noted as a function of sex, time (collapsed), or treatment (handled: n = 5 males, 6 females; saline: n = 15 males, 17 females; morphine: n = 22 males, 17 females). b, Females had significantly more nonramified/amoeboid (NR) and intermediate (I) microglia than males regardless of treatment (t(80)=3.90, p < 0.001). As shown in the scatterplot at right, the percentages of NR and I microglia were highly variable within each sex (line indicates median). c, Females had significantly more NR and I and fewer ramified (R) microglia in the vlPAG than males.
- Figure 3.
Percentage of activated microglia correlates with morphine effective dose. Bivariate correlations of morphine ED50 for the percentage of total nonramified and intermediate microglia (a) and by morphological subtype within the PAG (b) are shown. Degree of relatedness for morphine ED50 and microglia morphology are also shown for the MeA (c), rostral ventral medulla (d), SC (e), and SN (f). n = 15 males, 8 females.
- Figure 4.
Treatment with TLR4 agonist LPS increases microglia activation in females. a, LPS induces greater proportions of nonramified/amoeboid (NR) and intermediate (I) microglia in the PAG of females than males. b, LPS-treated females had a higher percentage of I microglia in the vlPAG than saline-treated females and show decreased percentage of ramified (R) microglia compared with all other groups. c, Representative images of microglia in the vlPAG of an LPS-treated male and female. Note that females have a greater density of nonramified and intermediate microglia than males. Scale bar, 5 μm. d, No sex differences in febrile response were noted after LPS administration. e, Percentage of nonramified and intermediate microglia for males and females after saline or LPS administration. LPS-induced increases in activated microglia were region and sex dependent. Endotoxin LPS, n = 7 males, 6 females; saline, n = 7 males, 6 females.
- Figure 5.
LPS-induced changes in cytokine and TLR4 transcription in the vlPAG of males and females. Peripheral administration of LPS increased the transcription of TNF (a) and IL-1β (b) in the vlPAG. c, No change in IL-6 was noted. d, Transcription of the anti-inflammatory cytokine IL-10 significantly decreased in females after LPS. e, No differences in TLR4 transcription were noted for sex or treatment. Endotoxin LPS, n = 5 males, 5 females; saline, n = 3 males, 3 females.
- Figure 6.
Pharmacological blockade or activation of vlPAG microglia has a sex-dependent impact on morphine antinociception. a, LPS-induced activation of vlPAG microglia significantly attenuates the antinociceptive effects of morphine in both males and females, as indicated by a significant rightward shift in the morphine dose–response curve. Saline + Saline (n = 5 males, 5 females), LPS + Saline (n = 5 males, 5 females), Saline + Morphine (n = 8 males, 11 females), and LPS + Morphine (n = 7 males, 8 females). b, In contrast, inhibition of TLR4 via intra-vlPAG (+)-naloxone results in a significant leftward shift in morphine dose–response curves in females such that (+)-Naloxone + Morphine-treated females were not significantly different from Saline + Morphine-treated males. Naloxone + Saline (n = 5 males, 5 females); Naloxone + Morphine (n = 7 males, 9 females).
Tables
GAPDH Forward GAG GTG ACC GCA TCT TCT TG Reverse CCG ACC TTC ACC ATC TTG TC IL-1β Forward CCC TGA AGG ATG TGA TCA TTG Reverse GGC AAA GGG TTT CTC CAC TT IL-6 Forward AAG ACC CAA GCA CCT TCT TT Reverse AGA CAG CAC GAG GCA TTT TT IL-10 Forward TGT ACC TTA TCT ACT CCC AGG TTC TCT Reverse GTG TGG GTG AGG AGC ACG TA TNF Forward TGT ACC TTA TCT ACT CCC AGG TTC TCT Reverse GTG TGG GTG AGG AGC ACG TA TLR4 Forward TCC CTG CAT AGA GGT ACT TC Reverse CAC ACC TGG ATA AAT CCA GC Sequences are listed for GAPDH (control gene) and the proinflammatory cytokines IL-1β, IL-6, IL-10, and TNF, as well as the immune receptor TLR4.
