Expression of PGP 9.5 over the paw skin in different treatment groups.

<p>(A) Illustration of PGP9.5 over the hind-paw skin in different treatment group (B) Representative of western blot analysis in different treatment groups (C) The quantitative western blot analysis in PGP 9.5 in the paw skin (D) The quantitative analysis of nerve fiber density in epidermis. N = 6; *p < 0.05; **p < 0.01; ***p < 0.01; Scale bar length = 100μm.</p

Buprenorphine decreased plasma BDNF and serotonin content and altered signaling molecule expression.

<p>Prenatal buprenorphine (0, 0.3, and 1 mg/kg/day) exposure was started from gestation day 7 and lasted for 14 days. After birth, the male and female pups were collected at postnatal day 21. Blood samples (n = 20 per group) were collected and subjected to ELISA for the measurement of BDNF (A) and serotonin (B). Brain cortical tissues were isolated. The obtained protein extracts (n = 5 per group) were subjected to Western blot analysis for the measurement of phosphorylated TrkB, TrkB, phosphorylated ERK, ERK, phosphorylated CREB, CREB, and β-tubulin (C). The obtained nuclear extracts (n = 5 per group) were subjected to EMSA for the measurement of CREB DNA binding activity (D). The protein extracts (n = 7 per group) were subjected to the enzymatic measurement of PKA activity (E). *p<0.05 and **p<0.01 vs. each vehicle group.</p

Buprenorphine induced depression-like neurobehaviors in pups.

<p>Prenatal buprenorphine (0, 0.3, and 1 mg/kg/day) exposure was started from gestation day 7 and lasted for 14 days. After birth, the male and female pups were collected at postnatal day 21 and subjected to neurobehavioral test. Forced swimming test (n = 6 per group) was conducted for a period of 5 min and the climbing time, swimming time, and immobility time were recorded (A). Tail suspension test (n = 6 per group) was performed for a period of 6 min and the climbing time and immobility time were recorded (B). **p<0.01 vs. each vehicle group.</p

Illustration of various parameters of the CatWalk XT system related to various intensities of nerve damage at different time frames.

<p>(A) Illustration of stand phase in different treatment related to different time frames, (B) Illustration of swing phase in different treatment related to different time frames, (C) Illustration of printed area in different treatment related to different time frames, (D) Illustration of maximum contact maximum intensity in different treatment related to different time frames, (E) Illustration of single stance in different treatment related to different time frames, (F) Illustration of regularity index in different treatment related to different time frames. Printed area, Maximum contact maximum intensity, Stand phase, Swing phase, Single stance, and Regular index: see text. *p < 0.05; **p < 0.01; ***p < 0.001 for sham vs. CCI group;^#p < 0.05; ^##p < 0.01; ^###p<0.001 for CCI vs CCI+AFS group.</p

Buprenorphine had negligible effects on learning/memory and locomotor activity in pups.

<p>Prenatal buprenorphine (0, 0.3, and 1 mg/kg/day) exposure was started from gestation day 7 and lasted for 14 days. After birth, the male and female pups were collected at postnatal day 21 and subjected to neurobehavioral test. The time required to reach the hidden platform (latency) was recorded in Morris water maze task (n = 6 per group). Each animal underwent three sessions (test 1–3) per day for three consecutive days (A). The spontaneous locomotion (n = 6 per group), including travel distance and moving time, was measured for a period of 20 min using open field test (B).</p

Expression of TNF-α and synaptophysin in hippocampus and somatosensory cortex after hAFMSCs treatment.

<p>(A) Depiction of synaptophysin and TNF-α expression over the hippocampus (CA3) and somatosensory cortex in different treatment groups, (B) Representative of western blot analysis of synaptophysin and TNF-α over the hippocampus in the different treatment, (C) Representative of western blot analysis of synaptophysin and TNF-α over the somatosensory cortex in the different treatment, (D) Quantitative western blot analysis of synaptophysin over the hippocampus in different treatment, (E) Quantitative western blot analysis of TNF-α over the hippocampus in different treatment, (F) Quantitative western blot analysis of synaptophysin over the somatosensory cortex in different treatment, (E) Quantitative western blot analysis of TNF-α over the somatosensory cortex in different treatment. N = 6 for western blot analysis, Scar bar length = 100μm,*p < 0.05; **p < 0.01; ***p < 0.001.</p

Imipramine attenuated buprenorphine-induced depression-like neurobehaviors in pups.

<p>Prenatal buprenorphine (0 and 1 mg/kg/day) exposure was started from gestation day 7 and lasted for 14 days. After birth, the male and female pups were collected at postnatal day 21 and subjected to neurobehavioral test. Male and female pups (n = 6 per group) were subjected to intraperitoneal imipramine (0 and 20 mg/kg) administration 1 h before forced swimming test (A) and tail suspension test (B). **p<0.01 vs. each vehicle group and ##p<0.01 vs. each buprenorphine group.</p

Expression of immune cells and associated inflammatory cytokines in dorsal spinal cord after hAFMSCs treatment.

<p>(A) Illustration of OX-42 and TNF-α expression over the spinal dorsal horn in the different treatment groups, (B) Quantitative analysis of OX-42 determined by the optic density in different treatment, (C) Quantitative analysis of TNF-α determined by optic density in the different treatment. Scar bar length = 100μm. N = 6 for analysis. *p < 0.05; **p < 0.01; ***p < 0.001.</p

Buprenorphine decreased organ mass in pups.

<p>Prenatal buprenorphine (0, 0.3, and 1 mg/kg/day) exposure was started from gestation day 7 and lasted for 14 days. After birth, the male and female pups were collected at postnatal day 21. At this time, the body mass and brain mass of dams (A) and pups (B) were weighed and the ratio of brain mass in body mass (%) was calculated. The body mass of dams was recorded prior to the start of experiments (beginning) and at the end of experiments (end). **p<0.01 vs. each vehicle group, n = 20 per group.</p

Relative density of TNF-α over the various tissue in CCI subjected to different treatment.

<p>Relative density of TNF-α over the various tissue in CCI subjected to different treatment.</p