Fig 2.

(3β,5β,17β)-3-hydroxyandrostane-17-carbonitrile (3β-OH), unlike ketamine, does not cause developmental neuroapoptosis at higher doses. 3β-OH (10 mg kg⁻¹, i.p.), ketamine (40 mg kg⁻¹, i.p.) and ketamine (40 mg kg⁻¹)+β-cyclodextrin were injected in P7 rat pups every 2 h for a total of six injections. Bar graphs on the left show averaged data from multiple experiments; representative images are depicted on the right panels. (A) The CA1-subiculum region exhibits a significant increase in caspase-3 activation when compared with groups treated with 3β-OH (3β-OH 10), ketamine alone (KET 40) or ketamine+β-cyclodextrin (KET 40+CYCLO) (***P<0.001 vs KET 40 and P<0.001 vs KET 40+CYCLO) suggesting that 3β-OH, unlike ketamine, does not cause significant developmental neuroapoptosis. This was confirmed by the finding that activated caspase-3 staining in 3β—OH—treated animals was not significantly upregulated compared with saline (SAL) or β-cyclodextrin (CYCLO) controls (P=0.964 vs saline and P=0.980 vs β-cyclodextrin) (n=6 pups per data point). (B) In thalamic anteroventral nucleus (TAV) there was minimal caspase-3 activation in vehicle groups, whilst there was a significant increase in caspase-3 activation in ketamine or ketamine+β-cyclodextrin groups compared with vehicle controls (***P<0.001). The level of caspase-3 activation in 3β-OH animals was comparable with vehicle controls and significantly lower than in either ketamine or ketamine+β-cyclodextrin (***P<0.001 vs KET 40 and P<0.001 vs KET 40+CYCLO) groups (n=6 pups per data point). (C) In thalamic lateral nucleus (TL) there was minimal caspase-3 activation in vehicle groups, whilst there was significant caspase-3 activation in ketamine or ketamine+β-cyclodextrin groups (**P=0.006 vs SAL and P=0.009 vs CYCLO). Note that the caspase-3 activation in the 3β-OH group is not significant compared with vehicle controls (n=6 pups per data point). (D) In the cingulate cortex, there was no difference in caspase-3 activation in the 3β-OH group compared with vehicle controls (P>0.999 vs SAL; P=0.995 vs CYCLO), but ketamine or ketamine+β-cyclodextrin groups exhibited significant increases in caspase-3 activation compared with vehicle control (***P<0.001 vs SAL and P<0.001 vs CYCLO). 3β-OH does not exhibit neurotoxic potential compared with either ketamine or ketamine+β-cyclodextrin groups (***P<0.001 vs KET 40 and P<0.001 vs KET 40+CYCLO) (n=6 pups per data point). All statistical analyses were done using one-way analysis of variance with Tukey's post hoc test. Representative photomicrographs shown in the right-side panels depict activated caspase-3 staining in ketamine+β-cyclodextrin compared with 3β-OH or a vehicle. Scale bars in the low magnification images in panels (A–C) are 400 μm, and 800 μm in panel (D). Scale bars in all high magnification insets are 100 μm.