Effect of combined repeated treatment with PROG and TMZ on the viability of U87MG and U118MG cell lines.

<p>Cells were grown in 24-well plate and repeatedly treated with PROG and TMZ at different concentration for 3 and 6 days. For repeated exposure, culture medium was replaced daily and the drugs were added to the medium every day. On day 4 and 7, cell viability test was performed using MTT reduction assay. PROG and TMZ stocks were prepared in absolute DMSO and further diluted in culture medium. The final concentration of DMSO was kept at <5μl/ml. Data are expressed as means ± SD of three separate replication experiments (n = 3 each). Statistically significant difference: *<i>P</i><0.05 compared with control group; ^#<i>P</i><0.05 compared to T100 alone group. P5 = PROG (5 μM); P80 = PROG (80 μM); T100 = TMZ (100 μM).</p

Progesterone Treatment Shows Benefit in Female Rats in a Pediatric Model of Controlled Cortical Impact Injury

<div><p>Purpose</p><p>We recently showed that progesterone treatment can reduce lesion size and behavioral deficits after moderate-to-severe bilateral injury to the medial prefrontal cortex in immature male rats. Whether there are important sex differences in response to injury and progesterone treatment in very young subjects has not been given sufficient attention. Here we investigated progesterone’s effects in the same model of brain injury but with pre-pubescent females.</p><p>Methods</p><p>Twenty-eight-day-old female Sprague-Dawley rats received sham (n = 14) or controlled cortical impact (CCI) (n = 21) injury, were given progesterone (8 mg/kg body weight) or vehicle injections on post-injury days (PID) 1–7, and underwent behavioral testing from PID 9–27. Brains were evaluated for lesion size at PID 28.</p><p>Results</p><p>Lesion size in vehicle-treated female rats with CCI injury was smaller than that previously reported for similarly treated age-matched male rats. Treatment with progesterone reduced the effect of CCI on extent of damage and behavioral deficits.</p><p>Conclusion</p><p>Pre-pubescent female rats with midline CCI injury to the frontal cortex have reduced morphological and functional deficits following progesterone treatment. While gender differences in susceptibility to this injury were observed, progesterone treatment produced beneficial effects in young rats of both sexes following CCI.</p></div

Combined treatment effect of PROG and TMZ on U87MG cell migration in a wound healing assay.

<p>Cells were grown in multi-well plates and pre-treated with <b>(A)</b> PROG alone and <b>(B)</b> in combination with TMZ at different concentrations for 2 h. A scratch/wound was formed with a 200-μl tip and the cells were incubated with PROG, TMZ or their combinations for the next 24 h. Photographs (4x) were taken at 0 h and 24 h post-wound formation. In the vehicle group, a large number of cells migrated from both sides to heal the wound at 24 h compared to 0 hr. PROG and TMZ individually decreased U87MG cell migration 24 h after wound formation compared to vehicle. The combination of the highest concentrations of both the drugs inhibited cell migration better than either drug alone. Representative photomicrographs from three separate replication experiments (n = 3 each).</p

Individual treatment effect of PROG on U87MG cell migration in a wound healing assay.

<p>Cells were grown in multi-well plates and pre-treated with PROG alone at different concentrations for 2 h. A scratch/wound was formed with a 200-μl tip and the cells were incubated with PROG for the next 24 h. Photographs (4x) were taken at 0 h and 24 h post-wound formation. In the vehicle group, a large number of cells migrated from both sides to heal the wound at 24 h compared to 0 hr. PROG decreased U87MG cell migration 24 h after wound formation compared to vehicle. Representative photomicrographs from three separate replication experiments (n = 3 each).</p

Individual and combined treatment effect of PROG and TMZ on the viability of primary human dermal fibroblasts (HDF).

<p>Cells were grown in 24-well plate and repeatedly treated with PROG and TMZ at different concentration for 3 and 6 days. For repeated exposure, culture medium was replaced daily and the drugs were added to the medium every day. On day 4 and 7, cell viability test was performed using MTT reduction assay. PROG and TMZ stocks were prepared in absolute DMSO and further diluted in culture medium. The final concentration of DMSO was kept at <5μl/ml. Data are expressed as means ± SD of three separate replication experiments (n = 3 each). Statistically significant difference: ^#<i>P</i><0.05 compared to control group; *<i>P</i><0.05 compared to T100 alone. P5 = PROG (5 μM); P10 = PROG (10 μM); P40 = PROG (40 μM); P80 = PROG (80 μM); T100 = TMZ (100 μM).</p

Tabulated comparative deficits between male and female controlled cortical impact injury.

<p>Bold numbers with asterisk (<b>*</b>) indicate differences between treatment groups within the same gender, <i>p</i> < .05. Darker-shaded cells indicate where male rats either found the hidden MWM platform faster or made fewer visits to the closed arm of the EPM than similarly treated female rat pups (between-gender analysis), <i>p</i> < .05. Lighter-shaded cells indicate where female rat pups either weighed less, had smaller lesions, spent less time in the open arm of the EPM, tended to travel longer distances (Open Field), remained on the rotarod longer, or found the MWM platform faster than similarly treated male counterparts (b/w gender analysis), <i>p</i> < .05; PID 0 = day of injury but prior to surgery; PID -1 = day(s) before surgery; MWM: Morris Water Maze; CCI: controlled cortical impact; Mean +/- SEM; n = 6–11. Male data (columns 3, 5, 7, and 9) were previously published [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146419#pone.0146419.ref026" target="_blank">26</a>].</p

Dose-response effect of progesterone on learning and memory task as assessed by latency to locate the MWM platform.

<p>During Run 1 (<b>a</b>) on the acquisition phase there was a significant effect of CCI injury and all three doses of progesterone proved beneficial by decreasing the mean latency latency to find a hidden platform in the MWM compared to the CCI+ vehicle treated group. There was no clear effect of CCI injury on Run 2 (<b>b</b>) or during the reversal phase (p>0.05). * =  mean values are significantly different from sham; n = 8–9. PROG =  progesterone.</p

Dose-response effect of progesterone on mean velocity in the MWM.

<p>During Run 1 (<b>a</b>) in the acquisition phase there was a significant effect of CCI injury. While progesterone treatment eventually improved performance, when collapsed across trials the 4–16 mg/kg CCI+PROG groups were not statistically different from CCI+ vehicle-treated rats. During Run 2 (<b>b</b>) swim speed for the CCI+ vehicle group compared to Sham Vehicle was significantly different. PROG (16 mg/kg)+CCI groups showed the most improvement on swim speed during reverse phase learning. * =  significantly different from CCI+ vehicle; # =  significantly different from CCI+8 mg/kg (<i>p</i>'s<.05). n = 8–9. PROG =  progesterone.</p

Dose-response effect on Spontaneous Activity test.

<p>Mean distance travelled (<b>a</b>), mean time spent at rest (<b>b</b>), Ambulatory responses (<b>c</b>) and stereotypic responses (<b>d</b>). Although CCI had a significant effect on spontaneous activity across test days, across the group there was no observed significant effect of Lesion/Treatment. * =  significant difference from baseline within each group. Values are mean ±SEM (n = 8–9). PROG =  progesterone.</p

Effects of progesterone (PROG) on learning and memory task as assessed by latency to locate the Morris water maze (MWM) platform.

<p>During Run 1 <b>(a)</b> on the acquisition phase there was a significant effect of controlled cortical impact injury. Eight mg/kg PROG proved beneficial by decreasing the mean latency to find a hidden platform in the MWM compared to the CCI+vehicle-treated group. There was no clear effect of PROG treatment on Run 2 <b>(b)</b> or during the reversal phase (<i>p</i> > 0.05). The Sham+PROG group was given 16 mg/kg doses of PROG. * = different from Sham+Vehicle; # = different from CCI+Vehicle (<i>p</i>’s < 0.05). ‡ = different from baseline within each group; n = 6–11.</p