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Endocrinology Vol. 144, No. 6 2350-2359
Copyright © 2003 by The Endocrine Society

Estradiol Exacerbates Hippocampal Damage in a Model of Preterm Infant Brain Injury

Joseph L. Nuñez and Margaret M. McCarthy

Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201

Address all correspondence and requests for reprints to: Dr. Joseph L. Nuñez, Department of Physiology, University of Maryland School of Medicine, 5-014 Bressler Research Building, 655 West Baltimore Street, Baltimore, Maryland 21201. E-mail: jnune001{at}umaryland.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have developed a model for prenatal hypoxia-ischemia in which muscimol, a selective {gamma}-aminobutyric acid A (GABAA) receptor agonist, administered to newborn rats, induces hippocampal damage. In the neonatal rat brain, activation of GABAA receptors leads to membrane depolarization and neuronal excitation. Because of our previous detection of sex differences in this model and the considerable interest in the neuroprotective effects of estradiol in the adult brain, we now investigate the effect of pretreatment with high physiological levels of estradiol in our model of prenatal hypoxia-ischemia. We used unbiased stereology to assess neuron number in the hippocampal formation of control, muscimol-treated, and estradiol- plus muscimol-treated animals. Muscimol decreased neuron number in the hippocampus, with damage exacerbated by pretreatment with estradiol. A hippocampal culture paradigm was developed to mirror the in vivo investigation. We observed elevated cytotoxicity (using the lactate dehydrogenase assay) by 48 h after treatment with estradiol plus muscimol, but decreased cytotoxicity between 2 and 24 h after treatment. To determine whether the actions of estradiol on muscimol-induced damage were via the estrogen receptor, hippocampal cultures were pretreated with ICI 182,780, a selective estrogen receptor antagonist. Treatment with ICI 182,780 blocked the potentiating effect of estradiol on the late period of cytotoxicity, but had no effect on the protective actions of estradiol during the early period of cytotoxicity. There appears to be a biphasic action of estradiol in our model of neonatal brain injury that involves early nongenomic, nonreceptor-mediated protection, followed by late deleterious receptor-mediated effects.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PREMATURELY born human infants are at high risk for hypoxia-ischemia due to preeclampsia, compression of the umbilical cord, seizures, and cerebral vascular accidents (1, 2, 3, 4, 5, 6). These catastrophic events and the damage caused by the subsequent hypoxia-ischemia can lead to neuropathological and behavioral deficits throughout the life of the individual (3, 6). Pioneering work by Vanucci and colleagues (7, 8, 9, 10) has demonstrated that, similar to neonatal human infants, the young (postnatal d 7) rat is extremely susceptible to hypoxic-ischemic brain injury. Hypoxia-ischemia induced in young rats produces anatomical and behavioral deficits analogous to those observed in asphyxiated newborn humans (4). Although a topic of much debate, the developmental stage of the postnatal d 7–10 rat is generally considered to be equivalent to that of the newborn human infant, making the newborn rat analogous to the premature human (7, 8, 9, 10). One common event after hypoxia-ischemia and other neonatal brain insults is a marked increase in extracellular levels of amino acids such as {gamma}-aminobutyric acid (GABA), glutamate, and glycine (11). The release of these amino acids remains elevated for 2 h after insult (11) and plays a primary role in the development of neonatal brain injury.

We have recently developed a model for premature infant hypoxia-ischemia in which muscimol, a selective GABAA receptor agonist, is administered to the postnatal d 0 and 1 rat (12, 13) to mimic the 3- to 4-fold increase in extracellular GABA following hypoxia-ischemia in the neonatal rat (11). After neonatal muscimol treatment, there is a 25–30% reduction in the number of neurons in the hippocampus and dentate gyrus of both male and female rats when examined on postnatal d 7 (12). Damage persists throughout development, as documented by reduced numbers of hippocampal and dentate gyrus neurons on postnatal d 21 along with deficits in water maze and open field task performance (13), two behaviors that are sensitive to functioning of the hippocampus (14, 15, 16).

In the adult brain, activation of the GABAA receptor is a major source of synaptic inhibition via hyperpolarization of the neuronal membrane due to an influx of chloride ions through the GABAA channel (17). However, in the developing brain, the transmembrane chloride gradient is such that opening of the GABAA channel produces an efflux of chloride and depolarization of the neuronal membrane (17, 18, 19). Of particular interest is that the membrane depolarization produced by GABAA receptor activation in immature neurons is sufficient to open L-type voltage-sensitive calcium channels, resulting in markedly increased intracellular calcium (19, 20). In our model of prenatal brain damage, pretreatment with diltiazem, a selective L-type calcium channel blocker, prevents muscimol-induced damage in the hippocampus and dentate gyrus (12). This is consistent with other models of hypoxia-ischemia in which excessive calcium entry through L-type calcium channels is an important mitigator of injury (21, 22, 23, 24).

The gonadal steroid estradiol confers a protective effect against neuronal damage in several models of brain injury in the adult hippocampus and cerebral cortex (25, 26, 27, 28, 29, 30, 31, 32, 33, 34). Prenatally, the brain is exposed to high levels of estradiol originating from the circulation of the mother as well as from the activity of neuronal aromatase in the fetus (35, 36, 37, 38). The latter is particularly important in males where there is an additional source of estradiol, the secretion of testicular androgens, which are aromatized to estrogens locally within neurons (38, 39). Therefore, we investigated the effect of pretreatment with high physiological levels of estradiol in our model of premature infant hypoxia-ischemia in male and female rats. The number of neurons and pyknotic cells in the hippocampal formation (CA1, CA2/3, and the dentate gyrus) of animals treated with muscimol was compared with that in estradiol- plus muscimol-treated and control animals. To investigate whether the effects of estradiol on muscimol-induced damage were independent of connectivity to or from the hippocampus, a hippocampal culture paradigm was developed. Using this system, we were able to more thoroughly document the rapid effects of estradiol on muscimol-induced cytotoxicity. Likewise, this paradigm allowed us to observe whether the effect of estradiol pretreatment on muscimol-induced cytotoxicity was via the estrogen receptor. To this end, estradiol-treated hippocampal neurons were pretreated with the estrogen receptor antagonist ICI 182,780.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental animals
Animals were Sprague Dawley albino rats from Charles River Laboratories, Inc. (Wilmington, MA). Female rats were time bred with male breeders in the animal colony at University of Maryland School of Medicine (Baltimore, MD). Pregnant females were checked every morning for the presence of pups. The day of birth was designated postnatal d 0. All animals were housed under a 12-h light, 12-h dark cycle, with food and water provided. All animal procedures were approved by the University of Maryland institutional animal care and use committee.

Experiment 1: effect of estradiol pretreatment on muscimol-induced damage in vivo.
Treatment of animals.
Rats were removed from their mothers approximately 4 h after birth and placed on a heated pad to avoid any drop in body temperature. Each litter contained at least four male and four female pups. Litters that did not meet this criterion were excluded. Male and female pups were administered 17ß-estradiol (50 µg) or sesame oil vehicle in a 0.05-cc sc injection on postnatal d 0. Numerous reports have documented that between embryonic d 18 and postnatal d 1, male rats encounter elevated levels of estradiol (37, 38, 39). In a recent report from our laboratory, newborn female rats were treated with 50 µg estradiol at 2 and 26 h after birth, with tissue collected at 30 h after birth. Although control males and females had 10–18 pg estradiol/mg protein in the hippocampus, estradiol-treated females had approximately 32 pg estradiol/mg protein (Amateau, S. K., C. L. Stamps, and M. M. McCarthy, unpublished observation). Therefore, our treatment paradigm, although leading to significantly elevated levels of estradiol, does not lead to supraphysiological levels. This dose of estradiol was chosen because it induces masculinization of the female rat brain, but has no effect on body weight (40, 41). Four hours later, animals were administered either muscimol (5 µg) or saline in a 0.05-cc sc injection. This entire procedure was repeated on the next day, for a total of two estradiol injections and four muscimol injections. The injection sites were sealed with cyanoacrylate Vetbond Surgical Adhesive (3M Animal Care Products, St. Paul, MN). Pups were marked, depending upon the manipulation they underwent, by injecting India ink into their paws and were returned to the dam within 15 min. There were a total of eight groups: 1) sham males, 2) sham females, 3) males treated with muscimol, 4) females treated with muscimol, 5) males treated with estradiol, 6) females treated with estradiol, 7) males treated with estradiol and muscimol, and 8) females treated with estradiol and muscimol. There were a total of four or five animals in each of the eight groups.

Histological analysis.
Pups were euthanized on postnatal d 7 by decapitation, and their brains were fixed overnight in 4% paraformaldehyde with 2.5% acrolein, then for 24 h in 4% paraformaldehyde. Brain weights were taken before fixation. Brains were stored in 30% sucrose in paraformaldehyde for 72 h, then sectioned on a cryostat. Two sets of consecutive 60-µm sections were made through the entire hippocampus. One set was used for cresyl violet staining, and the other was used for neuronal nuclear antigen immunocytochemistry.

Neuronal nuclear antigen (NeuN) is a protein exclusively expressed in neurons. The tissue labeled with NeuN was used for quantification of neuron number. To detect NeuN immunocytochemically, free floating tissue sections were rinsed with 0.1 M PBS and cleared of endogenous phosphatase activity by exposure to sodium borohydride. After rinsing, the monoclonal mouse anti-NeuN antibody (Chemicon, Temecula, CA; 1:70,000 in 0.1 M PBS/0.4% Triton X-100) was applied, and the tissue was incubated for 48 h at 4 C. On the third day, tissue was rinsed before exposure to biotinylated goat antimouse IgG secondary antibody (Vector Laboratories, Inc., Burlingame, CA), followed by rinses and addition of Vectastain Elite ABC reagents (Vector Laboratories, Inc.). The tissue was visualized via addition of nickel-enhanced diaminobenzidine in sodium acetate. After the reaction, the tissue was rinsed, mounted onto gelatin-subbed slides, dehydrated, and coverslipped. The other set of tissues was stained with cresyl violet for quantification of hippocampal volume and pyknotic cell number. Cresyl violet densely stains the condensed clumps of nuclear chromatin that are characteristic of apoptosis.

Stereological analysis.
Using the Neurolucida program (MicrobrightField version 2.01, Colchester, VT), tracings were made from the anterior through the posterior extent of the hippocampal formation. In the tracings, a distinction was made between the CA1 and CA2/3 fields and the dentate gyrus. A total of seven or eight tracings were made per hippocampal formation, per hemisphere in each animal. The traced sections were evenly spaced 240 µm apart. The first plane was a randomly chosen section within 180 µm of the most anterior plane of the hippocampus [similar to Plate 27 in the atlas of Paxinos and Watson (42)]. The last traced section for every animal was the last plane in which the hippocampal formation was present [see Plate 44, left side, in Paxinos and Watson (42)].

Volumetric estimation.
To obtain the volume of each individual subfield (CA1 and CA2/3), the dentate gyrus, and the total volume of the hippocampus, we first had to estimate the volume shrinkage factor (43). If the correction for shrinkage is not performed, the volume of the region of interest may be over- or underestimated. One male and one female brain were sectioned fresh-frozen on a cryostat. The 60-µm sections were made through the entire hippocampal formation, and the tissue was immediately mounted onto slides, lightly stained, then coverslipped. This unprocessed tissue was compared with the fixed and immunocytochemically processed tissue. Although stereology allows for unbiased estimates of particle number, calculation of regional volume using Cavilieri estimation is based upon knowing the actual thickness of each section on the slide (h), as in the formula , where V(ref) is the volume of the region of interest, and {Sigma}nAi is the cross-sectional area of the ith section of the region of interest (for n sections). However, because errors inherent to determining h for each section may occur, we used the equation , where (SF)v is the volume shrinkage factor, t is the constant section thickness (as recorded from the cryostat), and n is the number of sections measured (43). As evident from these two equations, . We calculated (SF)v using the equation , where Vf is the volume of the region of interest in the experimental brain (perfused, stained, and coverslipped), and Vs is the volume of the region of interest in the control (fresh-frozen) brain. There were no differences in (SF)v between males and females. In the current experiment, (SF)v = 0.262. This value was used in the equation , where t = 60 µm, n = 7–8 sections, and Ai is the cross-sectional area of each region in a given section. Ai was measured using the program Neuroexplorer (MicrobrightField, version 2.01). Using the above formula, the volumes for CA1, CA2/3, and the dentate gyrus were determined.

Cell number estimation.
Using an unbiased stereological technique, the optical dissector (44), NeuN-immunoreactive (NeuN-ir) cells and pyknotic cells were counted within three distinct regions of the hippocampal formation: CA1 field, CA2/3 field, and dentate gyrus. The optical dissector technique eliminates bias in counting as a result of cell size and shape. Pyknotic cells were counted in cresyl violet-stained sections and were characterized by clumps of condensed chromatin within the nucleus. Using the Neurolucida program package (MicrobrightField version 2.01), coronal sections were imaged onto a computer screen. A counting frame (100 x 100 µm with x60 objective for NeuN-ir cell counts, and 100 x 100 µm with x40 objective for pyknotic cell counts) was used. The counting frame was moved in a raster fashion throughout CA1, CA2/3, and the dentate gyrus. A total of 8–10 counting frames were sampled per region per hemisphere in each section. Cell counts were performed through a defined 25-µm depth of the tissue section. The defined depth allowed for a 5- to 10-µm border, thus avoiding the issue of lost caps or bottoms. To obtain cell number, either NeuN-ir or pyknotic, the area of the counting frame (Aframe) was multiplied by the defined depth of the tissue section (25 µm) to obtain the volume of the counting frame (Vframe). The cell counts made within this volume (P) were divided by the volume of the counting frame (Vframe) to obtain cell density measures (Nv). To determine the total number of cells (N), the cell density (Nv) was multiplied by the total volume of the region of interest (Vref).

Experiment 2: effect of estradiol pretreatment on muscimol-induced damage in vitro
Primary cultures of hippocampal neurons.
Primary cultures of hippocampal neurons were prepared based on the method described by Banker and Goslin (45). Briefly, a timed pregnant Sprague Dawley female was killed, and the embryonic d 18 fetuses were removed and placed in a petri dish containing HBSS+ [88 ml sterile H2O, 10 ml 10x Hanks’ Balanced Salt Solution (Ca2+ and Mg2+-free), 1 ml 1.0 M HEPES buffer (pH 7.3), 1 ml antibiotic/antimycotic, and 100x liquid]. Hippocampi were dissected into a centrifuge tube containing HBSS+. After all hippocampi were collected, HBSS+ was added to the tube to a volume of 4.5 ml, with 0.5 ml trypsin (2.5%). Cells were incubated for 15 min in a 37 C water bath. The supernatant was removed and washed three times for 5 min each time with HBSS+. Cells were dissociated by pipetting up and down using a Pasteur pipette, and cell number and viability were determined by trypan blue exclusion. Cells were plated on 18-mm poly-L-lysine-coated coverslips at a density of approximately 300,000 cells/coverslip and placed in 60-mm dishes containing 4 ml plating medium [86 ml MEM, 10 ml horse serum, 3 ml glucose (filter sterilized, 20%), 1 ml 100 mM pyruvic acid]. Cells were allowed to adhere for 4 h in a 37 C, 5% CO2 incubator.

The neuron cultures were removed from the plating dishes and placed in glial feeders neuron side down, prepared according to the method of Banker and Goslin (45). The 60-mm glial dishes contained 4 ml serum-free, glutamate-free neuronal maintenance medium [86 ml MEM, 10 ml ovalbumin, 1% in MEM, 10 ml N2 solution (97.5 ml MEM, 1 ml putrescine solution 16.1 mg 1 ml H2O, 1 ml selenium dioxide, 330 µg 100 ml H2O, 0.5 ml insulin solution (10 mg/ml), 100 mg transferrin, (human), 3 ml glucose (filter sterilized, 20%), 1 ml pyruvic acid, 100 mM, and 1 ml antibiotic/antimycotic, 100x liquid)].

Treatment of cultures.
Cultures were treated on days in vitro (DIV) 3 and 4 with 17ß-estradiol dissolved in dimethylsulfoxide (DMSO) or vehicle (DMSO). To determine a physiologically relevant dosage of estradiol to administer in cell culture, culture dishes were treated with 1 nM 17ß-estradiol in DMSO (2 µl/2000 µl culture medium). Medium was collected in triplicate from the culture dishes at 5 min, 1 h, 2 h, 4 h, 8 h, 24 h, and 48 h after estradiol administration. As a control, medium was collected from a culture dish that was not treated with estradiol. The culture medium underwent RIA for estradiol at the Center for Cellular and Molecular Studies in Reproduction, University of Virginia (Charlottesville, VA). From the data (Fig. 1Go), we determined that 2 µl 1 nM estradiol administered every other day were sufficient to maintain culture medium estradiol levels at about 200 pg/ml, within normal physiological levels for the neonatal brain (37). Estradiol levels in control (untreated) cultures were less than 1.50 pg/ml.



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  		Figure 1. Levels of estradiol as assessed by RIA in hippocampal culture medium from 5 min to 48 h after treatment. Hippocampal culture medium was treated with 1 nM 17ß-estradiol. Note that from 1–48 h after treatment, estradiol levels were maintained between 187–232 pg/ml.

 
Estradiol, at a final concentration of 1 nM, was added to 2.0 ml culture medium. One third of the culture medium was replaced with fresh astrocyte-conditioned medium every 2 d. On DIV 5, cultures were treated with 1 µM muscimol or sterile saline. The treatment was repeated 4 h later. The entire process was repeated on DIV 6. There were a total of four treatment groups: 1) control, 2) muscimol alone, 3) estradiol alone, and 4) estradiol plus muscimol.

Cytotoxicity assay.
Using the lactate dehydrogenase assay, a measure of neurotoxicity, samples were collected 2, 24, 48, and 72 h after the last muscimol treatment. The lactate dehydrogenase (LDH) assay is an assay of cellular injury in response to a cytotoxin. Briefly, 200-µl aliquots of culture supernatant and controls (background control, glia cultured with conditioned medium only; low control, HEPES-treated cultures; high control, cultures treated with 2 µl Triton X-100) were obtained and stored at 4 C until assay. LDH release was assayed using an ELISA reader and the Cytotoxicity Detection Kit (Roche, Indianapolis, IN). The LDH value obtained from the background control was subtracted from all measures. To obtain an accurate assessment of cytotoxicity, the following formula was used:

One of the caveats in using the LDH assay is that in all treatment paradigms, there is a gradual decline in LDH levels after the start of sample collection (not noticeable until 3–4 d after the start of collection). Although this may represent a decrease in toxicity, it may also be a reflection of the loss of cultured neurons, with fewer cells being able to release LDH. Therefore, although LDH release is an important indicator of cell death, it may actually underestimate the total amount of cell loss. There were a total of three samples in each group for one individual LDH assay run, with a total of three LDH assay runs, to give a final n of nine per group.

Experiment 3: effect of estradiol receptor antagonism
To determine whether the action of estradiol on muscimol-induced damage was via the estrogen receptor, a separate set of cultures was investigated. Using the same manner of hippocampal culture tissue collection as that mentioned above, cultures were treated twice daily on DIV 3 and 4 with the selective estrogen receptor antagonist ICI 182,780 at a final concentration of 1 µM or with sterile water. This concentration of ICI 182,780 was chosen because it is 100 times higher than the concentration of estradiol administered to the cultured hipopocampal neurons. Also, this concentration is similar to those used in previous reports (46, 47). On DIV 3, 30 min after the first administration of ICI 182,780, cultures were treated with 1 nM 17ß-estradiol or DMSO. On DIV 5 and 6, cultures were treated twice daily with muscimol or sterile saline. There were a total of eight treatment groups: 1) control, 2) estradiol alone, 3) muscimol alone, 4) ICI 182,780 alone, 5) muscimol plus ICI 182,780, 6) estradiol plus ICI 182,780, 7) estradiol plus muscimol, and 8) ICI 182,780, estradiol, plus muscimol.

Using the LDH assay (described above), samples were collected 2, 24, 48, 72, and 96 h after the last muscimol treatment. There were a total of three samples in each group for one individual LDH assay run, with a total of three LDH assay runs, to give a final number of nine per group.

Statistical analysis
One-way ANOVA (treatment) were run on measures of brain weight, body weight, NeuN-ir cell number, and pyknotic cell number. Males and females were analyzed separately. NeuN-ir cell counts were made individually from CA1 and CA2/3, with the data recorded and analyzed as the addition of the two areas for clarity. This is referred to as the hippocampus and is separated from the dentate gyrus. Two-way ANOVAs (treatment, time) were performed on in vitro LDH data. Post hoc Newman-Keuls and t test comparisons were conducted, and a level of P < 0.05 was required for statistical significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Experiment 1: effect of estradiol on muscimol-induced hippocampal cell loss in vivo
To assess the possibility that estradiol and muscimol treatment had a general deleterious effect on development, body and brain masses were determined at the time of sacrifice. There was an overall effect of treatment on brain and body masses in both males (body: F3,12 = 5.08, P < 0.02; brain: F3,12 = 4.16, P < 0.03) and females (body: F3,12 = 14,00, P < 0.0003; brain: F3,12 = 5.20, P < 0.02; see Table 1Go). Post hoc tests indicated that although estradiol alone and muscimol alone had no significant effect on brain or body weight relative to controls (both males and females), the combination of estradiol and muscimol led to decreased brain and body weight measures relative to all other groups (P < 0.05 for each measure). There was a substantial decrease in overall body mass (~25% less than controls of each sex); however, the alteration in brain mass was much more subtle (~10%).


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  		Table 1. Effect of neonatal treatment on brain and body mass

 
Stereological estimates performed on postnatal d (PND) 7 indicated significant overall differences between the groups in neuron number in the hippocampus and dentate gyrus in both male (hippocampus: F3,12 = 20.66, P < 0.0001; dentate gyrus: F3,12 = 10.32, P < 0.001; Fig. 2AGo) and female (hippocampus: F3,12 = 9.52, P < 0.002; dentate gyrus: F3,12 = 21.25, P < 0.0001; Fig. 2BGo) rats. Photomicrographs of NeuN-ir neurons in the hippocampal formation area shown in Fig. 3Go (A–F). Post hoc tests indicated a significant reduction in neuron number in the overall hippocampus and dentate gyrus of estradiol- plus muscimol-treated pups (both males and females) compared with all other groups (P < 0.01 for each measure). In the male hippocampus, there was a significant effect of muscimol alone on neuron number relative to control males (P < 0.05) and relative to estradiol alone-treated males (P < 0.05). Estradiol plus muscimol treatment of males led to a 28% decrease in the number of neurons in the hippocampus (P < 0.01) and a 48% decrease in that in the dentate gyrus relative to control males (P < 0.01). In estradiol- plus muscimol-treated female pups there was a 37% reduction in hippocampal neuron number compared with control females (P < 0.01) and a 49% decrease that in the dentate gyrus. Although not significant, there was a modest decrease in female hippocampal neuron number after neonatal muscimol treatment. We have previously observed a significant decrease in hippocampal neuron number in females after neonatal muscimol treatment (12), but to a lesser extent than that observed in males. The reduction in neuron number in estradiol- plus muscimol-treated animals (between 28–49%) was greater than the 10% decrease in overall brain mass.



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  		Figure 2. Neonatal estradiol plus muscimol treatment in both males (A) and females (B) induces a loss of hippocampal and dentate gyrus neurons. Stereological estimates of neuron number were obtained from four or five animals in each group. Animals were treated on PND 0 and 1, then examined on PND 7. Tissue was labeled with NeuN. Data are the mean ± SEM. Columns that share the same letter are significantly different from one another (by ANOVA, P < 0.05).

 


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  		Figure 3. Representative photomicrographs illustrating NeuN-ir neurons in the CA1 region of the hippocampus in sham males (A), males treated with muscimol (B), males treated with estradiol and muscimol (C), sham females (D), females treated with muscimol (E), and females treated with estradiol and muscimol (F). Both sham males and females have more neurons than their muscimol- and estradiol- plus muscimol-treated counterparts. Scale bar, 100 µm.

 
Complementary to the changes in neuron number, there were significant differences in the number of pyknotic cells observed in the hippocampus and dentate gyrus on PND 7 in both male (hippocampus: F3,12 = 34.12, P < 0.0001; dentate gyrus: F3,12 = 5.38, P < 0.01) and female (hippocampus: F3,12 = 42.78, P < 0.0001; dentate gyrus: F3,12 = 4.63, P < 0.02) rats (Fig. 4Go, A and B). In the hippocampus, estradiol- plus muscimol-treated males had significantly more pyknotic cells than all other groups (P < 0.01), whereas in the dentate gyrus, estradiol- plus muscimol-treated males had significantly more pyknotic cells than control males (P < 0.05), but numbers equivalent to those in muscimol alone-treated males. In the female hippocampus and dentate gyrus, neonatal estradiol plus muscimol treatment led to an increased number of pyknotic cells on PND 7 relative to all other groups (P < 0.01). Although muscimol-treated females had more pyknotic cells than controls in the hippocampus, there was no effect of treatment on pyknotic cell number in the dentate gyrus. Estradiol alone led to a small, but significant, increase in the number of pyknotic cells in the hippocampus relative to that in control females. This was not matched by a change in neuron number and is of unknown significance. For photomicrographs of pyknotic cells in the dentate gyrus of male and female rats, see Fig. 5Go (A–F). These data indicate that in the hippocampus and dentate gyrus of males and females, muscimol treatment combined with estradiol on PND 0 and 1 leads to the persistence of elevated levels of cell death for a minimum of 6 d after exposure.



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  		Figure 4. Neonatal estradiol plus muscimol treatment in both males (A) and females (B) leads to an increase in pyknotic cell number in the male rat hippocampus and dentate gyrus. Tissue was labeled with cresyl violet for pyknotic cell analysis. Stereological estimates of pyknotic cell number were obtained from four or five animals in each group. Animals were treated on PND 0 and 1, then examined on PND 7. Data are the mean ± SEM. Columns that share the same letter are significantly different from one another (by ANOVA, P < 0.05).

 


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  		Figure 5. Representative photomicrographs illustrating pyknotic cells in the CA1 region of the hippocampus in control males (A), males treated with muscimol (B), males treated with estradiol plus muscimol (C), control females (E), females treated with muscimol (F), and females treated with estradiol plus muscimol. Both males and females treated with estradiol plus muscimol have more pyknotic cells than all other groups. Arrows denote the locations of pyknotic cells. Higher magnification photomicrographs of pyknotic cells in the CA1 region of the hippocampus are also illustrated (D and H). Scale bar, 10 µm.

 
Experiment 2: effects of estradiol on muscimol-induced hippocampal cell loss in vitro
There was an overall effect of treatment (F2,54 =4.11, P < 0.02) and time (F3,54 =3.59, P < 0.02) and a treatment by time interaction (F6,54 =3.00, P < 0.01) on LDH release (Fig. 6Go). Post hoc t tests indicated that at all time points investigated, muscimol alone-treated cultures displayed greater release of LDH than controls (P < 0.01 for each measure), consistent with previous reports from our laboratory (12). Even as early as 2 h after muscimol treatment, cytotoxicity was elevated in the muscimol-treated cultures relative to controls. The elevated levels of LDH release persisted for at least 3 d posttreatment, with a peak about 48 h after treatment. Of interest was that the estradiol- plus muscimol-treated cultures were similar to controls (vehicle-treated cultures) at 2 and 24 h posttreatment, but had a dramatic increase in cytotoxicity at 48 h that persisted until at least 72 h posttreatment. At both 48 and 72 h posttreatment, estradiol- plus muscimol-treated cultures had significantly higher levels of cytotoxicity than all other groups (P < 0.01). Estradiol alone-treated cultures were similar to controls at all time points.



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  		Figure 6. Pretreatment with estradiol is protective against the early phase, but exacerbates the delayed phase of cell death. Cytotoxicity was assessed by the LDH assay in cultured neonatal hippocampal neurons. There were a total of three samples in each group for one individual LDH assay run, with a total of three LDH assay runs, to give a final number of nine per group. a, Significant difference from control (baseline measure) at the same time point; b, significant differences from estradiol- plus muscimol-treated cultures at the same time point (by ANOVA, P < 0.01).

 
Experiment 3: effect of estrogen receptor antagonism on the deleterious effects of estradiol on muscimol-induced cell loss
We again documented a significant overall effect of treatment (F4,56 =19.96, P < 0.0001) and time (F6,56 =30.97, P < 0.0001) and a time by treatment interaction (F24,56 =3.70, P < 0.0001) on LDH release (Fig. 7Go, A and B). As in Experiment 2 and previous work (12), muscimol alone treatment led to elevated LDH release by 2 h, with persistence at all time points, relative to controls (P < 0.05 for each measure). Peak LDH levels were observed at 48 h after muscimol treatment, with a gradual decline until 96 h. As expected, the cultures treated with ICI 182,780 plus muscimol were similar to the muscimol alone-treated cultures. As in Experiment 2, LDH release at 2 and 24 h in the estradiol- plus muscimol-treated cultures was equivalent to that in controls. However, there was a rapid rise in LDH release by 48 h and a peak at 72 h, with a modest level persisting at 96 h after estradiol plus muscimol treatment. At 48–96 h, LDH release in the estradiol- plus muscimol-treated cultures was significantly greater than that in controls, muscimol alone-treated cultures, and ICI 182,780-, estradiol-, and muscimol-treated cultures. Of major interest was that ICI 182,780-, estradiol-, and muscimol-treated cultures had LDH release at 2 and 24 h after treatment equivalent to controls, with a modest, but significant, rise at 48 h that persisted until 72 h. This brief peak was followed by a return to baseline levels by 96 h, the last time point investigated. Thus, the ICI 182,780-, estradiol-, and muscimol-treated cultures appeared to follow two distinct patterns. Although they displayed LDH release equivalent to estradiol- and muscimol-treated cultures at 2 and 24 h posttreatment, they displayed LDH release equivalent to muscimol alone-treated cultures at 48–96 h. It appears that the blockade of the estrogen receptor with ICI 182,780 is able to attenuate the late rise in cytotoxicity observed in the estradiol- plus muscimol-treated cultures, but does not alter the apparent protective effect of estradiol that occurs from 2–24 h. Across all time points examined, ICI 182,780 alone-, estradiol alone-, and estradiol- plus ICI 182,780-treated cultures were similar to control cultures, indicating that neither ICI 182,780 nor estradiol was toxic or protective independently of muscimol-induced damage in vitro.



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  		Figure 7. Pretreatment with the estrogen receptor antagonist ICI 182,780 blocks estradiol-mediated exacerbation of muscimol-induced damage in cultured neonatal hippocampal neurons as assessed by the LDH assay (A and B). There were a total of three samples in each group for one individual LDH assay run, with a total of three LDH assay runs, to give a final number of nine per group. Data are the mean ± SEM. a, Significant difference from control (baseline measure) at the same time point; b, significant differences from estradiol- plus muscimol-treated cultures at the same time point (by ANOVA, P < 0.01).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present experiment demonstrates that estradiol, when administered to newborn male and female rats, exacerbates hippocampal neuron loss in a model of preterm infant hypoxia-ischemia. In our model of neonatal brain injury, muscimol, a selective GABAA receptor agonist, when administered over postnatal d 0 and 1 induces a 20–30% reduction in the number of neurons in the hippocampus and dentate gyrus. However, after pretreatment with physiological levels of estradiol, there was a 30–50% reduction in hippocampal and dentate gyrus neuron number.

The hippocampus is sexually dimorphic, with males having a greater volume and more neurons in all subregions (CA1, CA2/3, and the dentate gyrus) than females (12, 13, 34, 48). This region is also a well known target for the actions of estradiol in development (49). Both estrogen receptors {alpha} and ß are expressed at high density in the developing rat hippocampus along with elevated levels of estrogen receptor binding (50, 51, 52, 53, 54). In a previous report we observed a sex difference in response to neonatal muscimol-induced injury in the hippocampus, with males being more sensitive to the same neonatal muscimol treatment than females (12). The sex difference in sensitivity to injury was hippocampal region specific, with males experiencing a greater loss of neurons in CA1 and the dentate gyrus than females. Not only was the loss of neurons more substantial in males by postnatal d 7, but the number of pyknotic cells was also higher in males than in females, suggesting the persistence of cell death in males (12). More importantly, juvenile males performed worse than females on a spatial learning test after neonatal muscimol exposure (13). A similar pattern of findings was observed in the present study. Although muscimol alone led to a significant decrease in hippocampal neuron number in males, the same treatment failed to significantly decrease hippocampal neuron number in females. These findings are important given the large number of developmental injuries in both humans and rats in which males are more sensitive than females to the same insult (3, 55). Males experience higher endogenous tissue levels of estradiol subsequent to the production of androgens by the testis during a perinatal sensitive period (37, 38). The peripherally derived testosterone is converted to estradiol within neurons by the enzyme aromatase (35, 36). We hypothesize that the higher endogenous tissue levels of estradiol during early development in males are an important component of the increased sensitivity. However, we have not directly tested this possibility due to the limitations of the estrogen receptor antagonist ICI 182,780, which does not readily cross the blood-brain barrier (46, 47). Administering ICI 182,780 in vivo would therefore require intracerebroventricular injections, which in itself would induce neuronal damage.

Results obtained in vitro paralleled those seen in vivo and allowed for preliminary examination of the mechanism of estrogen action. One of the benefits of in vitro investigation is the extended time course over which cytotoxicity can be assessed. Using the lactate dehydrogenase assay as a measure of toxicity, cultured hippocampal neurons pretreated with estradiol did not display elevated cell loss until 48 h after muscimol treatment. This is intriguing given that hippocampal neurons treated with muscimol alone displayed appreciable levels of cell loss by 2 h. Also, although cytotoxicity declined in the muscimol alone-treated cultures by 72 h after treatment, cell loss remained elevated at this time in the estradiol-pretreated hippocampal neurons and persisted until 96 h. The differences in the timing of cell loss is important given that the early phase of cell death (between 0 and 24 h) after insult is most likely necrotic in nature (56, 57, 58). Although this observation is not novel, what is of interest is that estradiol pretreatment attenuates the early necrotic phase of cell loss, in accordance with previous studies in the adult (27, 28, 29, 59, 60). Furthermore, it has been established that the later phase of cell loss (between 48 and 96 h) after insult is of an apoptotic nature (56, 57, 58). Our data put forward the premise that estradiol exacerbates the later phase of cell loss. Estradiol enhances this late period of damage by apparent action through the estrogen receptor. Pretreatment with the selective estrogen receptor antagonist ICI 182,780 on neurons subsequently treated with estradiol and muscimol showed that blockade of the estrogen receptor attenuates this late phase of cytotoxicity. The attenuation appears to be complete, given that ICI 182,780-, estradiol-, and muscimol-treated cultures displayed LDH release in the late period (48–96 h) equivalent to muscimol alone-treated cultures. Thus, estradiol appears to affect muscimol-induced cell loss by two distinct mechanisms that are specific to the two forms of cell death, apoptosis vs. necrosis. Estradiol has been implicated in both forms of death in the adult hippocampus and cerebral cortex, exerting neuroprotective effects in both cases (27, 28, 29, 59, 60, 61). In contrast, in the neonate, estradiol appears to have a mixed effect, being neuroprotective against the rapid (necrotic) phase of cell death, while exacerbating the later (apoptotic) phase of cell death when induced by excitatory GABA action.

The production of free radicals subsequent to injury is a mediator of necrotic cell death (56, 58). Estradiol and several estrogenic compounds are potent scavengers of free radicals and at high doses provide protection from free radical-induced damage (25, 27, 28, 29, 59, 60, 62). Prevention of free radical-induced cell death by estrogen is independent of the estrogen receptor and requires no gene transcription or protein synthesis, thus imposing no temporal constraints on its action (25, 27, 28, 59, 62). Our observation of a protective effect of estradiol against short-term muscimol-induced cell death in neonatal hippocampal neurons (that was unaffected by the estrogen receptor antagonist, ICI 182,780) is consistent with this mechanism of action.

The second phase of injury induced cell death, apoptosis, is considered to be a continuum of necrosis (57, 58, 63), but involves the activation of caspases (64, 65, 66). Preliminary data from our laboratory indicates that Bax, caspase-9, and cytochrome-c protein levels are elevated by 24 h after the last muscimol treatment (Nuñez, J. L., unpublished observations). The increase in the levels of these three proteins integral in the cell death pathway after neonatal muscimol treatment gives credence to the involvement of apoptotic mechanisms in our model of prenatal brain injury. In adult models of injury and in our recent study on kainic acid-induced damage in neonatal female rats, estradiol protects against apoptotic cell death. The precise mechanism of protection remains unclear, but it appears to require the estrogen receptor (33, 67, 68, 69) and is likely to involve a mixture of actions that include MAPKs (29, 30, 59, 60) and transcriptional regulation of cell death-associated proteins such as Bax and Bcl-2 (29, 70, 71). In our current model estradiol pretreatment exacerbates apoptotic cell death induced by muscimol and appears to involve the estrogen receptor. The mechanism of estrogen receptor-mediated increases in muscimol-induced damage is known at this time.

Although muscimol alone led to increased cytotoxicity in vitro and enhanced neuron loss in vivo, estradiol alone failed to alter cytotoxicity in vitro or hippocampal neuron number when given exogenously to male and female rats. Thus, we propose that estradiol enhances muscimol-induced damage by acting through two potential mechanisms. One is by directly increasing the levels of GABA, thereby leading to sustained activation of the GABAA receptor and prolonged membrane depolarization. Both males and females injected neonatally with testosterone have higher hippocampal levels of GABA and GAD, the rate-limiting enzyme for GABA synthesis, than females (72, 73). Alternatively, estradiol may promote an increase in intracellular calcium levels. Work in neonatal hypothalamic cultures has demonstrated that estradiol increases the magnitude and duration of calcium influx through the L-type voltage-sensitive calcium channel after muscimol application (74). We have established that damage in our model is subsequent to calcium influx via L-type voltage-sensitive calcium channels (12), and excessive calcium influx is an integral mitigator of hypoxia-ischemia induced damage (21, 22, 23, 24).

In summary, we have documented that estradiol pretreatment of neonatal male and female rats exacerbates damage subsequent to muscimol exposure, a model of prenatal hypoxia-ischemia. We propose that estradiol, which is elevated during early development in males, is an important component determining why males are more sensitive then females to many forms of neonatal insult. In vitro, we documented that selective antagonism of the estrogen receptor led to a blockade of the damaging effects of estradiol pretreatment. Placing the present findings in the context of the existing literature on estradiol and brain damage is complicated by the lack of a clear consensus on both the mechanism of damage and the type of effect. The majority of rodent models have focused on damage to the cerebral cortex after middle cerebral artery occlusion and have generally found estradiol to be neuroprotective (26, 29, 61, 70), although disagreement about the timing, receptor involvement, and dose abound (25, 27, 28, 32, 33, 59, 60, 62, 68, 69, 71). For the hippocampus, which is particularly vulnerable to global ischemia and seizure-related damage (3, 6, 30, 75), estradiol pretreatment has been reported as both protective (30, 31, 75) and damaging (76). An equally confusing picture is emerging for the developing brain. We have previously reported that pretreatment with estradiol protects against kainic acid-induced damage to the neonatal rat hippocampus (77), and Ikonomidou et al. report that estrogen protects against damage caused by antiseizure medications given to neonates (78). The current results, however, show a clearly deleterious effect of estradiol to damage induced by excitatory GABA. Thus, it appears that the effects of estradiol are complex, being regionally and situationally specific. Further research will help to elucidate general principles and establish the best therapeutic approaches.


   Acknowledgments
 
We thank Jesse J. Alt for his expert technical assistance with the culture of hippocampal neurons.


   Footnotes
 
This work was supported by NIH Grant R01-MH-52716 (to M.M.M.) and a Society for Neuroscience minority postdoctoral fellowship (to J.L.N.).

Abbreviations: DIV, Days in vitro; DMSO, dimethylsulfoxide; GABA, {gamma}-aminobutyric acid; -ir, immunoreactive; LDH, lactate dehydrogenase; NeuN, neuronal nuclear antigen; PND, postnatal day.

Received August 12, 2002.

Accepted for publication February 4, 2003.


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