Objective
Estrogen receptors are present within the fetal brain, suggesting that estrogens may exert an influence on cerebral development. Loss of placentally derived estrogen in preterm birth may impair development.
Study Design
Baboons were delivered at 125 days of gestation (term approximately 185 days), randomly allocated to receive estradiol (n = 10) or placebo (n = 8), and ventilated for 14 days. Brains were assessed for developmental and neuropathological parameters.
Results
Body and brain weights were not different between groups, but the brain/body weight ratio was increased ( P < .05) in estradiol-treated animals. There were no differences ( P > .05) between groups in any neuropathological measure in either the forebrain or cerebellum. There were no intraventricular hemorrhages; 1 estradiol animal displayed ectactic vessels in the subarachnoid space.
Conclusion
Brief postnatal estradiol administration to primates does not pose an increased risk of injury or impaired brain development.
During human pregnancy, the placenta is the primary site of estrogen secretion, utilizing precursors that arise from both maternal and fetal compartments. Fetal plasma estradiol (E 2 ) levels increase progressively during late gestation, rising further with the onset of parturition and falling in the early postnatal period because of the loss of the placentally derived hormone.
Estrogen receptors are present in the primate lung and throughout the primate and human brain during fetal development, suggesting that their activation by E 2 might play a role in the normal development of these organs, possibly as trophic factors. Indeed, it has been reported that in the brain, estrogen promotes axonal and dendritic growth and synapse formation and acts as a proliferative agent during critical stages of cerebral cortical development.
In addition, E 2 protects the neonatal brain from hypoxia-ischemia. Preterm infants commonly have low levels of estrogen and progesterone because of the lack of placental supply; it is possible that this has an impact adversely on pulmonary and neural development and function.
In a primate model of bronchopulmonary dysplasia (BPD) postnatal E 2 treatment has beneficial effects on cardiovascular and pulmonary function and lowers the requirements for ventilatory support. Whether postnatal E 2 treatment affects the immature brain is unknown. Thus, the aim of the present study was to evaluate the effects of postnatal E 2 administration on brain growth and the pattern of cerebral injury in prematurely delivered baboons cared for in a neonatal intensive care unit.
Materials and Methods
Animal studies were performed at the Southwest Foundation for Biomedical Research in San Antonio, TX. Animal husbandry, handling, and procedures conformed to American Association for Accreditation of Laboratory Animal Care guidelines.
Delivery and ventilatory management
Pregnant baboon dams ( Papio papio ) with timed gestations were treated with antenatal steroids before elective delivery at 125 ± 2 days of gestation (dg; term 185 days). At birth, animals were weighed, sedated, intubated, and treated with 4 mL/kg surfactant (Survanta; courtesy Ross Laboratories, Columbus, OH); ventilatory support was provided for 14 days. Animals were randomly assigned to either placebo (n = 8) or E 2 (n = 10) groups. Complete surgical procedures, animal care, and ventilator management have been described previously.
Administration of E 2
Animals assigned to the E 2 group received a 0.5 mg, 21 day extended-release pellet, placed subcutaneously in the left axilla at 1 hour of life; placebo animals received a control pellet. A second control or E 2 pellet was placed in the right axilla on day 7.
The rationale for the dosing regimen has been described previously. Briefly, the dose was chosen to achieve E 2 levels that were in the upper range of the concentrations observed in the latter third trimester in fetal baboons. Serum E 2 levels, determined by radioimmunoassay, were measured in additional fetal baboons at 125 dg, 140 dg, 160 dg, and 180 dg to determine normal fetal levels of E 2 . Levels of E 2 were determined in placebo- and E 2 -treated animals at 6 hours of life and at 1, 2, 3, 7, 10, and 14 days.
Physiological data
PaO 2 ; PaCO 2 ; pH; fraction of inspired oxygen (FiO 2 ); systolic, diastolic, and mean arterial blood pressure; and heart rate were monitored continuously throughout the experimental period. Oxygenation (OI) and ventilation (VI) indices were calculated. We also examined the relationship between the baboon’s physiological instability and measurements of brain growth and injury. The interval flux of physiological variables was calculated as a surrogate measure of the physiological instability.
We first determined the maximum and minimum values of each variable during a specified time interval; the interval flux was the difference between these values. For each animal we then performed the following: (1) identified the maximum flux; and (2) calculated the mean of the interval fluxes over the entire experimental time period. A greater degree of flux, particularly in FiO 2 , is associated with increased neuropathology.
Histological analysis
Brains were weighed and immersed in 4% paraformaldehyde in 0.1 M phosphate buffer, and 11 blocks from the right forebrain (at 5 mm intervals) and a midsagittal block from the cerebellar vermis of each brain were processed to paraffin. Ten (8 μm) sections were collected from the rostral surface of each block of the forebrain and in the sagittal plane for cerebellar sections. A section from each block was stained with hematoxylin and eosin (H&E) and assessed for gross morphologic changes, including hemorrhages, lesions or infarcts, neuronal death, axonal injury, and gliosis. Masson’s trichrome was used to assess for collagen deposition and Perl’s stain to visualize hemosiderin deposition, indicative of a bleed having occurred at least 48 hours prior to postmortem. Sections were scored for hemorrhages, infarcts, and cystic lesions (0, absent; 1, present).
Immunohistochemistry for rabbit anti-cow-glial fibrillary acid protein (GFAP; 1:500, code 20334; Sigma, St. Louis, MO) was used to identify astrocytes; rabbit antiionized calcium-binding adapter molecule 1 (Iba1, 1:1500, code 019-19741; Wako, Richmond, VA) was used to identify microglia/macrophages; mouse antihuman Ki67 clone MIB-1 (1:100; DakoCytomation, Glostrup, Denmark) was used to identify proliferating cells; mouse antichicken myelin basic protein (MBP, 1:100; Chemicon, Temecula, CA) was used to assess the extent of myelination; rabbit anti-von Willebrand factor (1:800; Abcam, Cambridge, UK) was used to identify blood vessels; rabbit anticaspase-3 (1:500; Cell Signaling Technology, Beverly, MA) was used to identify cells undergoing cell death (apoptosis and necrosis); and rabbit antigoat p27 cell cycle inhibitory marker (1:1000; Millipore, Billerica, MA) was used to identify postmitotic cells, as described previously.
All analyses were performed on all brains in the study and measurements made on coded slides blinded to the observer.
Quantitative analysis
Forebrain: for each animal, all measurements were made on a section from each block, unless otherwise stated, using an image analysis system (Image Pro version 4.1; Media Cybernetics, Silver Spring, MD). All values were calculated as mean of means for each group; measurements of cell numbers were expressed as cells per square millimeter.
Volumetric measurements: 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, San Diego, CA); volumes of the white matter (WM), neocortex, deep gray matter (basal ganglia, thalamus, and hippocampus), and ventricles were then estimated using the Cavalieri principle.
Surface folding index (SFI): the SFI, which gives an estimation of the expansion of the surface area relative to volume, was determined.
Areal density of astrocytes: GFAP-immunoreactive (IR) cells were counted (×660) in randomly selected areas (0.02 mm 2 ) of the deep and subcortical WM, neocortex (3 sites in blocks from frontal/temporal, parietal/temporal, and occipital lobes in layers 5 and 6), and hippocampus (stratum radiatum in the CA1 region).
Areal density of oligodendrocytes: MBP-IR oligodendrocytes were counted (×300) in 2 randomly selected areas (0.42 mm 2 ) in both the deep and subcortical WM from the parietal/temporal lobe.
Areal density of microglia/macrophages: in Iba1-IR sections, cells were counted in randomly selected areas (×660; sample area 0.02 mm 2 ) of both the deep and subcortical WM. In the neocortex (layers 2-6), a section from each lobe was selected and 3 regions (dorsal, lateral, ventral) sampled in each section (×660).
Percentage of white matter occupied by blood vessels: point counting was performed in von Willebrand factor-IR sections to determine the density of blood vessel profiles in deep and subcortical WM and neocortex (×660) as an indicator of vasodilation or vasculogenesis.
Ki67-IR cells: in the neocortex, Ki67-IR cells were counted (×660) in the dorsal and ventral regions of the subventricular zone (sample area 0.02 mm 2 ).
Activated caspase-3-IR cells: cells were counted (×660) in deep WM in the 5 fields (0.02 mm 2 ) with the highest concentration of positively stained cells.
GFAP-IR radial glial fibers: sections from each lobe were scored for the presence of GFAP-IR fibers on a scale of 0-3 (0, none; 1, occasional; 2, moderate; 3, considerable).
Quantitative analysis
Cerebellum: sections were scored for hemorrhages and infarcts as for the forebrain.
The width of the external granule cell layer (EGL) and the molecular layer were assessed using the image analysis system as previously described.
Ki67-IR cells: in 10 75 μm lengths of EGL (×600), the number of Ki67-IR cells was expressed as the proportion of total cells in the region. p27-IR staining was examined to determine the expression pattern in relation to Ki-67-IR. In the deep cerebellar WM, 5 regions were randomly sampled in 2 sections per animal (×600) and mean density of Ki67-IR cells determined.
Percentage of WM occupied by blood vessels: point counting was performed in 2 regions of the deep WM (×660) from 1 von Willebrand-IR section from each animal.
Statistical analysis
Linear regression analysis was carried out on data from the combined groups to determine whether there was a correlation between the following: (1) physiological variables (maximum and mean fluxes for pH, PaO 2 , PaCO 2 , FiO 2 , OI, VI, and blood pressure and cardiac output) and quantitative variables (volumetric measurements, oligodendrocyte, astrocyte, and microglial densities); and (2) volumetric measurements and oligodendrocyte, astrocyte, and microglial densities. Differences between parameters in E 2 -treated and placebo groups were tested using Student t tests; for all analyses a probability of P < .05 was considered to be significant.
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