Animals
Timed pregnant CD dams were obtained from Charles River (Wilmington, MA) at 14 d of gestation. Rats were housed in solid plastic hanging cages under constant conditions (temperature, 22 °C; humidity, 62 %) with a 12-h dark–light cycle and were allowed to consume food and water ad libitum. On d2, rat pups were randomized to litters of 10 pups per dam (Normal growth, N) or 16 pups per dam (Restricted growth, R). On d11, R pups were re-randomized into litters creating catch-up (R-6, 6 pups/dam), normal (R-10, 10 pups/dam) or reduced growth (R-16, 16 pups/dam) groups. N pups remained in litters of 10 pups/dam (N-10). Equal numbers of males and females were included in all litters (Fig. 9). Pups were weaned at d21 to a standard, non-purified rodent diet (LabDiet 5001, Purina, Hayward, CA) fed ad libitum. Weights were monitored until d60. The University of California Institutional Animal Care and Use Committee approved all animal procedures.
The period from d2-10 in rats is typically taken to represent the period between the early third trimester and term in humans, and therefore represents early ex utero life in preterm infants. The period from d11-21 in the rat is broadly representative of the first 2 years of life in humans, and therefore reflects the period where catch-up growth is common in human preterm infants [18].
Body composition
A subset of animals had body composition assessed at d60 by carcass analysis. Frozen carcasses were cut and freeze-dried for 24 h to determine water content, calculated from change in weight before and after freeze-drying. Fat content was measured from the change in weight after diethyl ether (Fisher Scientific, Pittsburgh, PA) extraction for 7 d using a Soxhlet apparatus, followed by acetone (Fisher Scientific, Pittsburg, PA) extraction for an additional 7 d. Total ash content was determined following muffle furnace incineration for 72 h at 540 °C and desiccation for 24 h. Protein was calculated as the difference between post-fat extraction weight and ash content. Water, protein, fat and ash content of each animal were expressed as a percentage of total body weight.
Biochemical analysis
Blood samples were collected at time of sacrifice on d22 and d60. Specimens were centrifuged at 1000 × g for 15 min at 4 °C, and serum samples stored at −80 °C until analysis. Serum insulin and serum leptin were measured using ELISA kits (Millipore, Billerica, MA). Serum and hepatic triglycerides were measured with Triglyceride Reagent (Fisher Scientific, Pittsburg, PA) and read at 540 nm at 37 °C.
Insulin and glucose tolerance tests
An intraperitoneal insulin tolerance test (ITT) was performed on d50 after 4 h of food deprivation. Insulin (0.5 U/kg body weight [36]) was injected intraperitoneally and blood glucose levels were measured in tail vein blood using a glucometer (Easy Plus, Home Aid Diagnostics, Deerfield Beach, FL) at 0, 15, 30, 45, 60, 90, and 120 min after insulin injection. The area under the blood glucose curve (AUC) was calculated using a rhomboid rule. The primary comparison between groups was the total AUC for the entire study (120 min); secondary comparisons were for the AUC between 0 min and 30 min, and between 30 min and 120 min. Larger values for AUC denote poorer insulin sensitivity. In addition, the change in blood glucose from baseline (0 min) was examined. The area under baseline (AUB) was calculated for the entire period, and for the first 30 min and last 90 min separately.
After a 3-days recovery period, an intraperitoneal glucose tolerance test (GTT) was performed after 12 h of food deprivation. Rats were injected intraperitoneally with 2 g/kg of glucose solution (Sigma, St. Louis, MO) and blood glucose was measured at 0, 15, 30, 45, 60, 90, 120, 150, and 180 min after glucose injection [37]. As before, the blood glucose concentrations were used to calculate the area under the blood glucose versus time curve (AUC) for the entire study (0 min to 180 min), as well as for the first 30 min and the last 150 min. Changes in blood glucose from the 0 min baseline were also calculated and the area over the baseline (0 min) value calculated using a rhomboid rule for the time periods 0–180 min, 0–30 min, and 30–180 min.
T-Maze
Memory and learning were examined by spontaneous alternation in a T-maze on d35. In the T-maze test, rats were tested on their capability to alternate between two directions of an enclosed apparatus in the form of a T placed horizontally, as previously described [38]. Upon successful alternation of direction, animals were given a score of 1. This was repeated ten times, with the maximum score being 9.
Brain Histology and Immunohistochemistry
For brain histology studies, rats (d60) were deeply anaesthetized with pentobarbital (100 mg/kg) and fixed by transcardial perfusion with 4 % paraformaldehyde. Total brains were removed and placed in 4 % paraformaldehyde solution overnight at 4 °C. Samples were next placed in serial dilutions until fixed in 100 % ethanol and embedded in paraffin. Coronal sections were cut into 8–10 μm sections and immunohistochemically stained with goat polyclonal anti-MBP antibody (sc-13914, Santa Cruz Biotechnology, Santa Cruz, CA) at 1:100 dilution in blocking buffer and donkey anti-goat secondary antibody (sc-2020, Santa Cruz, Biotechnology, Santa Cruz, CA) at 1:500 in 1 % BSA. The staining was developed with DAB substrate (Vector Laboratories, Burlingame, pt]?>CA) and sections were counterstained with toluidine (0.1 %) blue. Images were acquired under microscope at 40X magnification (DP Olympus BX51). Areas of MBP fibers were assessed as MPB-positive per high power field and quantified using ImageJ software (NIH, Bethesda, MD).
Data analysis
Glucose homeostasis
Blood glucose data for the glucose tolerance test are expressed as area under the curve (AUC), calculated using a rhomboid rule. AUC was calculated for the entire study period (AUC0–180), for the first 30 min (0–30 min, AUC0–30) of the study and for the last 150 min (30–180 min, AUC30–180) of the study. Changes in blood glucose from the time-0 baseline are expressed as the area over the time-0 baseline (AOB) for the same time intervals.
Blood glucose data for the insulin tolerance test were converted to AUC, and are expressed for the entire study period (AUC0–120), for the first 30 min (AUC0–30), and for 30–120 min (AUC30–120). Changes in blood glucose data for the insulin tolerance test are expressed as the area under the baseline (AUB) for the same time intervals.
The primary outcome measure for the glucose tolerance test and for the insulin tolerance tests was the area under the curve (AUC) for the entire study period (AUC0–120).
Secondary outcomes for the glucose tolerance test and for the insulin tolerance test was the area under the curve (AUC) for the first 30 min, and for the rest of the study, and the changes in glucose from baseline.
Statistical analysis
Weight data were analyzed by repeated-measures ANOVA with age, sex, and group as independent variables.
The effect of group on other continuously distributed outcomes was assessed by ANOVA with sex as a covariant. If main effects ANOVA showed a significant effect of “group”, post-hoc testing to assess differences between the groups was carried out when needed using Tukey’s HSD. All statistical analyses were performed using JMP Pro 11.0 (SAS Institute, Cary, NC) and statistical significance was accepted at P < 0.05.
Data are expressed as means ± SEM.