All animal studies were performed at the Southwest Foundation for Biomedical Research in San Antonio, TX. All animal husbandry, animal handling, and procedures were reviewed and approved to conform to American Association for Accreditation of Laboratory Animal Care (AAALAC) guidelines.
Delivery and Instrumentation
Pregnant baboon dams (Papio papio) with timed gestations underwent elective hysterotomy under general anesthesia. Study animals were delivered at 125±2 (dg). The dams did not receive antenatal glucocorticoids. At birth, animals were weighed, sedated, intubated and treated with 4ml/kg surfactant (Survanta, courtesy Ross Laboratories, Columbus, OH) prior to the initiation of ventilatory support.
Newborn baboons were mechanically ventilated for 14d as described previously (9
). A complete description of the details of the surgical procedures and animal care (including ventilator management, target goals for PaO2
, tidal volume, and nutritional, fluid, transfusion, antibiotic and blood pressure management) has been previously described (9
). Animals were randomized before delivery to either undergo surgical ductal ligation on day 6 of life (ligated, n=7), or receive no intervention (unligated, n=5). Animals in the unligated group did not receive anesthesia or sham surgery since our intention was to mimic the clinical care of human newborns. None of the unligated animals closed their ductus spontaneously. Fetal gestational control animals (n=7) were delivered at 140dg and euthanized immediately with sodium pentobarbitone.
, pH, FiO2
, systolic, diastolic and mean arterial blood pressure (BP), and heart rate were monitored continuously throughout the experimental period. Oxygenation Index (OI=mean airway pressure (cmH2
O) × iO2
) and Ventilation Index (VI=peak inspiratory pressure × ventilator rate × PaCO2
/1000) were also calculated. We also examined the relationship between a newborn baboon's physiological instability and measurements of brain growth and injury (see below). To do this we calculated the “interval flux” of physiological variables as a surrogate measure of the physiological instability. We first determined the maximum and minimum values of each variable during a time interval (6 hourly time periods for the first 48 hours and daily periods thereafter), the interval flux was the difference between these values during the specified time period. For each animal we then: 1) identified the maximum flux; and 2) calculated the mean of the interval fluxes over the entire experimental time period. Other findings relating to the clinical course, cardiovascular performance, and proinflammatory cytokines of the two newborn groups have been published elsewhere (9
Brains were weighed, immersed in 10% buffered formalin and sectioned into 5mm coronal blocks (10-12 blocks per animal) (6
). Blocks from the right hemisphere of each brain were processed to paraffin and 10 (8μm) sections collected from the rostral surface. A section from each block was stained with hematoxylin and eosin (H&E) and assessed for gross morphologic changes, including the presence of hemorrhages, lesions or infarcts, neuronal death, axonal injury, gliosis and perivascular cuffing. Masson's trichrome was used to assess for collagen deposition; Van Gieson's stain for elastic fibers, reticulin for reticulated fibers; Perls stain to visualize hemosiderin deposition.
Immunohistochemistry for rabbit anti cow-glial fibrillary acid protein (GFAP, 1:500, Sigma, St Louis, MO, USA) was used to identify astrocytes; rabbit anti-ionized calcium-binding adapter molecule 1 (Iba1, 1:100, Wako Chemicals, Osaka, Japan) to identify microglia/macrophages; mouse anti-human Ki67 clone MIB-1 (1:100; DakoCytomation, Glostrup, Denmark) to identify proliferating cells; and mouse anti-chicken myelin basic protein (MBP, 1:100; Chemicon, USA) to assess the extent of myelination, as previously described (6
All analyses were performed on all brains in the study. Qualitative and quantitative measurements were made on coded slides blinded to the observer.
Sections were scored for hemorrhages (present-1; absent-0) or overt injury such as infarcts, cystic white matter lesions or neuronal death. Iba1-immunoreactive (IR) sections were assessed for the presence of reactive microglial/macrophage cells in the grey and white matter.
All quantitative measurements were made for sections from each block using an image analysis system (Image Pro v4.1, Media Cybernetics, Maryland, USA). Measurements of cell numbers were expressed as cells/mm2; all values were calculated as mean of means for each group.
Cross-sectional areas of regions in the right forebrain were assessed in H&E-stained sections using a digitizing tablet (Sigma Scan Pro 4, Media Cybernetics, California, USA); volumes of the white matter, neocortex, deep grey matter (basal ganglia, thalamus and hippocampus) and ventricles were then estimated using the Cavalieri principle (10
Area of subventricular zone (SVZ)
The area of the SVZ was assessed (×30) at 3 levels in the parietal/temporal region in H&E-stained sections. The high density of cells in this region of the SVZ compared to the adjacent striatum and white matter allowed for clear delineation of the region (11
Surface folding index
The surface folding index (SFI), which gives an estimation of the expansion of the surface area relative to volume, was determined (6
Percentage of white matter occupied by blood vessels
Point counting (12
) was used to determine the density of blood vessel profiles in deep and subcortical white matter (×660) as an indicator of changes such as vasodilation or vasculogenesis. Assessment was performed in GFAP-IR sections as blood vessel profiles are clearly delineated (13
Areal density of astrocytes
GFAP-IR cells were counted (×660) in randomly selected areas (0.02mm2) in each of the deep and subcortical white matter regions; the cerebral neocortex (three sites in blocks from frontal/temporal, parietal/temporal and occipital lobes in layers 5 and 6); the hippocampus (stratum radiatum in the CA1 region).
Areal density of oligodendrocytes
MBP-IR oligodendrocytes were counted (×300) in two randomly selected areas (0.42mm2) in both the deep and subcortical white matter from the parietal/temporal lobe.
Ki67-IR cell density in the SVZ and in the subgranular zone (SGZ) in the hippocampus
To assess cell proliferation, counts were made of Ki67-IR cells in lengths of the SVZ in the anteromedial striatal neuroepithelium and subventricular zone by 2 observers. Three regions (0.02mm2) were randomly sampled 40μm from the ependymal surface (×660) in two sections for each animal (6 measurements/animal). Counts were also made in 3 randomly selected regions (0.02mm2) in the SGZ of the dentate gyrus (3 measurements/animal; ×660).
Apoptotic cell counts
Apoptotic figures (14
) were counted in stratum radiatum in CA1 region in the hippocampus and in layers 5 and 6 of the neocortex in 5 sites (0.09mm2
) in 2 sections per animal and expressed as apoptotic figures/mm2
In gestational control brains myelination as evidenced by (MBP-IR) was most advanced in the internal capsule with fibers extending into the subcortical white matter towards the subplate region; this was given a score of 3. The extent of myelination in the prematurely delivered groups was scored against this standard in the parietal/temporal region (0-no myelination; 1-a few myelinated fibers; 2-bundles of myelinated fibers; 3-similar extent of myelination to controls).
Perivascular cuffing in the subcortical white matter
The extent of perivascular cuffing was scored as: 0-not observed; 1-occasionally observed; 2-moderate degree; 3-considerable number of vessels with cuffing.
GFAP-IR radial glial fibers
Sections from the frontal/temporal, parietal/temporal and occipital regions were scored for the presence of GFAP-IR radial glial fibers on a scale of 0-3 (0-not observed; 1-occasionally observed; 2-moderate degree; 3-considerable number of fibers observed).
Growth and Development and Brain Damage Indices
Growth and development and brain damage indices were constructed as previously described (15
). We acknowledge that in constructing both of the above indices we have given equal weighting to all variables. At this stage of gestation it is difficult to predict which variables of development might be the most relevant predictors of long lasting deficits.
Linear regression analysis was carried out to determine if there was a correlation between: a) physiological variables (maximum and mean fluxes for pH, PaO2, PaCO2, FiO2, OI, VI and blood pressure and mean Qp/Qs and cardiac output) and brain growth and development or brain damage indices; b) physiological variables and quantitative variables (volumetric measurements, oligodendrocyte and astrocyte densities); c) volumetric measurements (white matter volume) and brain growth and development indices; and d) volumetric measurements (white matter volume) and quantitative variables (oligodendrocytes and astrocyte densities); a probability of p<0.05 was considered to be significant.
The statistical significance of differences between prematurely-delivered and control groups were tested using a one-way ANOVA with post-hoc analysis (Tukey's test) for histological variables. T-tests were used to compare between ligated and unligated groups for comparison of maximum and mean flux results and one-way ANOVAs were used for comparison of all other physiological variables; a probability of p<0.05 was considered to be significant. Results are expressed as mean ± SEM (weights and areas) and mean of means ± SEM (histological variables).