IUGR induction, experimental groups, and therapy administration
All procedures were performed following all the applicable regulations and guidelines of the Animal Experimental Ethics Committee (CEEA) of the University of Barcelona and were approved with the license number 03/17. Also, animal work has been conducted fulfilling ARRIVE’s guidelines and reported accordingly59.
At 25 days of gestation, a total of 19 pregnant rabbits were subjected to the IUGR induction protocol. The selection of the sample size was made taking into account the mortality related to the IUGR model following previous experience in the group7,14,29,60. This sample size would allow us to include the required number of animals for each analysis of the study following previous evidence (see each paragraph for detailed information). The IUGR induction protocol was performed as previously described29. Briefly, IUGR was induced by ligating 40–50% of the uteroplacental vessels of gestational sacs, whereas non-ligated gestational sacs provided normally-grown controls. At the time of IUGR induction, the pregnant rabbits were randomly assigned to 3 groups: no treatment (n = 9), treatment with DHA (n = 5) and treatment with Lf (n = 5). In the mothers that had been randomized to receive DHA or Lf treatment at the time of the IUGR induction all the gestational sacs in both horns were ligated, whereas in the untreated mothers sacs from one horn were ligated while sacs from the contralateral horn were not ligated, providing normally grown controls. At 30 days of gestation, cesarean section was performed obtaining living and stillborn animals and their placentas. Control and untreated IUGR pups were obtained from untreated mothers, whereas IUGR-DHA and IUGR-Lf pups were obtained from mothers treated with DHA or Lf, respectively.
DHA or Lf were orally administered to the pregnant rabbits once per day from day 25 until the day of the cesarean section. The therapy was administered using a syringe to cautiously release the solution into the mouth. The specific dose of DHA (37 mg/kg/day) and Lf (166 mg/kg/day) was calculated taking into account previous evidence19,61. Doses were adjusted with the formula for interspecies translation based on the body surface area (BSA)62, obtained as follows:
and on the Km value, obtained by:
Finally, the formula used for interspecies translation was as follows63:
The DHA was obtained from Rendon Europe Laboratories and was presented in 3 g 100% pure powder from Microalga oil Schizochytrium sp. The Lf administrated was bovine Lf containing a low concentration of iron (9.2 mg of iron per 100 g of protein) and was obtained from Farmalabor.
Due to differences in the mortality rate obtained in the experimental groups, the final sample differed between groups: 15 normally-grown controls, 15 untreated IUGR, 18 IUGR-DHA, and 19 IUGR-Lf. At postnatal day 1, general motor skills, tone, reflexes, and olfactory sensitivity were evaluated in all offspring following the previous methodology described by Derrick et al.55. For each animal, the testing was videotaped and variables were scored on a scale of 0 to 3 (0 = worst and 3 = best), except for tone that was scored 0 to 4 according to the Ashworth scale64 by 2 blinded observers (MI, LP). A detailed explanation of how each variable was assessed can be reviewed in the supplementary material (Supplementary methods and Supplementary Table S2).
After the cesarean section, placentas were obtained, carefully washed in saline solution, weighed, fixed for 24 hours in 10% formalin and embedded in paraffin.
After functional evaluation, newborns were weighed and sacrificed by decapitation after the administration of ketamine (35 mg/kg) and xylazine (5mg/kg) intramuscularly. All brains were carefully dissected and fixed according to the analysis performed. Four brains from each group were randomly selected for the Golgi-Cox staining protocol and were processed according to it (detailed below). The other brains (n = 11 control, 11 untreated IUGR, 14 IUGR-DHA, 15 IUGR-DHA) were fixed for 24 hours in 10% formalin, dehydrated for 48 hours with sucrose 30% and finally frozen at −80 ºC.
Five placentas from each group were randomly selected for histological evaluation based on findings from previous work in pregnant rabbits7. All the placentas were evaluated except one from the IUGR-DHA group, in which the analysis could not be performed owing to technical problems with the processing. Two consecutive slices (4 µm) of each placenta from paraffin blocks were stained following a hematoxylin and eosin standard protocol. The pathologist was blinded to the experimental groups. The analyses were performed on two different regions of the placenta: the decidua basalis (maternal part) and the labyrinth zone (fetal part). Ischemia, necrosis, fibrin, and calcifications were assessed in the decidua, while ischemia, vascular collapse, congestion, and calcifications were evaluated in the labyrinth zone. Ischemia and fibrin were expressed as a percentage, necrosis was assessed by the presence of karyorrhexis and karyolysis, and the remaining variables were evaluated with a semiquantitative system that graded each lesion from 0 to 5. The grading criteria followed were: 0) Unremarkable; 1) Minimal; 2) Mild; 3) Moderate; 4) Marked; 5) Severe.
Eight formalin-fixed brains per group were selected for the O4-immunoreactive oligodendrocyte evaluation. The sample size for oligodendrocyte evaluation was calculated taking into consideration previous studies with the same animal model7,60. Three coronal sections of 40 μm at the level of the genu CC were obtained and processed as previously described7. Briefly, slices were incubated overnight with anti-oligodendrocyte marker O4 (1:50, Chemicon) followed by 1% Hoechst 33258 (1:1000, Thermofischer) incubation for cellular nucleus visualization. Finally, slices were incubated with the specific secondary antibody conjugated to 488 Alexa Fluor (1:400, MoBitec). Immunoreactive sites were observed under the confocal microscope and images were taken as previously described7. The number of the total cell nuclei (stained with Hoechst) and the number of cells with fluorescent staining around the nucleus (O4-immunoreactive oligodendrocytes) were counted using the software ImageJ. O4-immunoreactive oligodendrocyte density (immune-reactive cells/mm2) was then calculated. The evaluation for each experimental group was blinded for the evaluator (LP). The anti-O4 antibody was selected as a marker for the latter stages of oligodendrocyte maturation (O4-OL), which include pre-OLs, pre-myelinating OL, and myelinating OL65. Since pre-OL cells are the most predominant oligodendrocytes in rabbits at the day of evaluation, we assumed that the O4 marker used mainly stained the pre-OL cells56.
Neuronal arborization (Golgi-Cox staining)
Four brains per group were included in the neuronal arborization analyses, similarly to previous studies7,8. Neuronal arborization evaluation was performed as previously described7. Briefly, vibratome was used to obtain 100 µm serial and coronal sections that were processed for Golgi-Cox impregnation with the FD Rapid Golgi Stain kit (FD Neurotechnologies Inc.). Five pyramidal neurons that fulfilled the inclusion criteria from the frontal cortex per brain hemisphere were selected randomly and one image per neuron was obtained. The inclusion criteria were: pyramidal neurons within layer II and III and complete filling of the dendritic tree with well-defined endings. The evaluation for each experimental group was blinded for the evaluators (LP, MM). The parameters evaluated were: i) area of the soma; ii) total basal dendritic length; iii) basal dendritic complexity, including number of dendrites and number of intersections for each Sholl ring. The study design is summarized in Figure 4.
The software packages STATA14.0 and GraphPad 5 were used for statistical analyses and graphical representation. For quantitative variables, normality was assessed by the Shapiro-Wilk test and homoscedasticity was determined by Levene’s Test, except for variables with more than 30 observations (Golgi-Cox variables) for which normal distribution was already assumed. For ordinal variables, non-normal distribution was assumed. The descriptives of the variables were expressed as mean and standard deviation (SD) for normal distributions, whereas median and interquartile range (IQR) were used for non-normal distributions and ordinal variables. Appropriate statistical tests were used according to the variable: ANOVA with Dunnett’s multiple comparison test was used for continuous variables, Kruskal-Wallis with Dunn’s multiple comparison test for ordinal variables and chi-squared for binary variables. Statistical significance was declared at P < 0.05 in all variables evaluated.