Animals and housing conditions
All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Hallym University (Permit number: Hallym-2018-84) and performed in accordance with their guidelines as well as ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments) (https://www.nc3rs.org.uk/arrive-guidelines). Nine-week-old pregnant BALB/cJ female mice, weighting 25.4 (mean) ± 2.4 g, were purchased from DBL Ltd. (Eumseong, Korea). All experimental mice were housed in individual cages at the Hallym University Laboratory Animal Resources Center under specific pathogen-free (SPF) conditions with a controlled consistent temperature (23 ± 2 °C) and lighting environment (12 h/12 h light/dark cycle). Mice were fed with the standard irradiated chow diet (Purina, Seongnam, Korea) for rodent ad libitum and drinking water. At the end of the study, the experimental mice were sacrificed by CO2 inhalation. A gradual fill rate of 20% chamber volume per minute displacement was used for CO2 euthanasia. All efforts were made to minimize the number and suffering of any animals used in these experiments.
Experimental protocols
Twenty pregnant BALB/c mice were randomly divided into two experimental groups, and were treated intraperitoneally with either 600 mg/kg valproic acid sodium salt (VPA, Sigma, MO, USA) dissolved in saline (n=12) or saline alone (n=8) on embryonic day 12.5 (E12.5). VPA- or saline (vehicle)-injected mice were housed in individual cages, and pups from VPA- or vehicle-treated dams were maintained until up to postnatal days 21 (P21). While 41 pups from VPA-treated dams and 59 pups from vehicle-treated dams were employed in this study, both brain and skin tissues for downstream assays were obtained from pups of VPA- or vehicle-treated dams on P1 (n=9 or n=13, respectively), P4 (n=11 or n=12, respectively), P12 (n=9 or n=15, respectively) and P21 (n=12 or n=19, respectively). The same experiment was repeated using 22 pregnant mice. 39 pups from VPA-treated dams and 47 pups from vehicle-treated dams were used in the 2nd batch of experiments, and brain/skin tissues were obtained from VPA- or vehicle-exposed pups on P1 (n=9 or n=11, respectively), P4 (n=8 or n=9, respectively), P12 (n=9 or n=10, respectively) and P21 (n=13 or n=17, respectively).
Epidermal functional studies
Prior to performing epidermal functional studies, mice (pups) were anesthetized with 2% isoflurane in a combination of nitrous oxide and oxygen (7:3, v/v) via an isoflurane vaporizer (VetEquip, Livermore, CA). Basal epidermal permeability barrier function was assessed by measuring transepidermal water loss (TEWL) using TM300 connected to MPA5 (C&K, Cologne, Germany) between 10:00 am and 12:00 pm during the light phase of the circadian cycle, as described previously [55]. In addition, epidermal permeability barrier function was further assessed by toluidine blue staining, as reported earlier [64]. Briefly, four-day old pups from both VPA- and vehicle-treated dams were euthanized and fixed in methanol at room temperature. After washing five times with PBS, mice were incubated with 0.1% toluidine blue solution dissolved in saline, followed by washing with PBS, then mice were examined and photographed for the penetration of the blue dye into the skin.
Behavioral study
Spatial learning and memory performance were assessed using the Morris water maze task, as described previously [65, 66] (Suppl. Fig. 1). Briefly, a 9 cm diameter platform was placed in the southeast quadrant of the 1.2 m diameter circular pool filled with a room-temperature water. After the completion of a training session which consisted of three trials, a visible trail, hidden-platform trial, and probe trial, for 4 days, the mice were given three trials for another 3 days to test their ability to locate a visual or hidden platform, or to evaluate the number of times/duration that treated mice crossed the hidden platform. Each trial was recorded with a ceiling-mounted video camera (Ganz YCH-02, Cary, NC, USA), and analyzed using automated tracking software (Ethovision XT 6, Noldus, Wageningen, Netherlands). The Morris water maze task was conducted between 09:00 am and 16:00 pm during the light phase of the circadian cycle. An hour before the behavioral test, all experimental mice were transported from the housing room to behavioral testing rooms, and they were left to acclimatize to their new surroundings, as well as recover from any stress caused by the transportation. After completing the behavioral study, mice (pups) were anesthetized with 2% isoflurane in a combination of nitrous oxide and oxygen (7:3, v/v) via an isoflurane vaporizer (VetEquip, Livermore, CA) for the downstream experiments.
All procedures were subjected to approval by the Ethical Committee on Animal Experiments, Hallym University, Korea (permit number: Hallym-2018-84) and performed accordingly.
Histological analyses
Prior to tissue preparation for histological analyses, mice (pups) were anesthetized with 2% isoflurane in a combination of nitrous oxide and oxygen (7:3, v/v) via an isoflurane vaporizer (VetEquip, Livermore, CA). The change in the overall morphology in skin and brain was assessed by hematoxylin and eosin staining, as described previously [67]. Distribution of tumor necrosis factor (TNF)α, interferon (IFN)γ, and interleukin (IL)-13 was determined using anti-TNFα, anti-IFNγ, or anti-IL-13 (Invitrogen, Carlsbad, CA), respectively, as described earlier [68]. The secondary antibody was Alexa Fluor® 488 goat anti-rabbit IgG (Invitrogen, Carlsbad, CA). Tissues were counterstained with the nuclear marker 4’,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories) for nuclear visualization. Slides were examined with a Carl Zeiss Axio fluorescence microscope.
Electron Microscopy
Skin or brain biopsies from both VPA- and vehicle-treated mice were taken for electron microscopy after anesthetization with 2% isoflurane in a combination of nitrous oxide and oxygen (7:3, v/v) via an isoflurane vaporizer (VetEquip, Livermore, CA). Briefly, tissues were fixed in modified Karnovsky's fixative overnight, and post-fixed in either 0.2% ruthenium tetroxide or 1% aqueous osmium tetroxide, containing 1.5% potassium ferrocyanide. After fixation, all tissues were dehydrated in a graded ethanol series, and embedded in an Epon-epoxy mixture. Ultrathin sections were examined, with or without further contrasting with lead citrate, in a Zeiss 10A electron microscope (Carl Zeiss, Thornwood, NJ), operated at 60 kV.
ELISA for cytokine quantification
Blood, skin or brain biopsies from both VPA- and vehicle-treated mice were collected for cytokine quantifications after anesthetization with 2% isoflurane in a combination of nitrous oxide and oxygen (7:3, v/v) via an isoflurane vaporizer (VetEquip, Livermore, CA, USA). Levels of proinflammatory cytokines, e.g, IL-4, IL-5, IL-13, IL-17A, thymic stromal lymphopoietin (TSLP), IFNγ, TNFα, and IgE were quantitated using appropriated ELISA kits obtained from Thermo Fisher Scientific (Walthan, MA, USA) or Komabiotech (Seoul, South Korea) in accordance with the manufacturer’s instructions.
Quantification of sphingolipids by liquid chromatography and tandem-mass spectrometry (LC-MS/MS)
Skin or brain biopsies from both VPA- and vehicle-treated mice were taken for sphingolipid quantifications after anesthetization with 2% isoflurane in a combination of nitrous oxide and oxygen (7:3, v/v) via an isoflurane vaporizer (VetEquip, Livermore, CA, USA). The levels of ceramide (Cer) and sphingomyelin (SM) were quantified using the LC-ESI-MS/MS (API 3200 QTRAP mass, AB/SCIEX) by selective ion monitoring mode, as described previously [68-70]. The MS/MS transitions of ceramides depending on their acyl chain length were 510→264 for C14-ceramide, 538→264 for C16-ceramide, 552→264 for C17-ceramide, 566→264 for C18-ceramide, 594→264 for C20-ceramide, 648→264 for C24:1-ceramide, and 650→264 for C24-ceramide, respectively. In addition, the sphingomyelin MS/MS transitions were 718→184 for C17 SM (d18:1/17:0) as an internal standard, 704→184 for C16 SM, 732→184 for C18 SM, 760→184 for C20 SM, 788→184 for C22 SM, 814→184 for C24:1 SM and 816→184 for C24 SM, respectively. Data were acquired using Analyst 1.5.1 software (Applied Biosystems, Foster City, CA). The results are expressed as pmol/mg protein.
Statistical analyses
Data were expressed as the mean ± standard deviation (SD). Significance between groups was
determined with unpaired Student t test. The P values were set at *P < 0.05.