Materials and methods NaNO 0, wt vol as a vasodilator followed by 1000
T .J. McDonald et al. Brain Research 885 2000 70 –78
71
to sympathetic motor neurons of the thoracic spinal cord normoxic NORMOX n53 treatment groups. At 120 days
[9,14,21]. For example there is experimental evidence of gestational age dGA; term¯150 dGA a subset of the
which indicates that one way in which the PVN influences fetuses n53 underwent stereotaxic neurosurgical place-
blood pressure is by modifying sympathetic nervous ment of microinjections of the retrograde axon tracer
system outflow that is initiated by activation of the FluoroGold FAu; 200 or 300 nl of 5 FAu in distilled
baroreceptors [29]. The anatomy of reciprocal innervation H O, Fluorochrome Inc., Englewood, CO 80155 either
2
of the PVN and the brainstem has been extensively and bilaterally n51 or unilaterally n52; left side into the
elegantly reviewed by L.W. Swanson [33]. PVN in-house atlas coordinates: AP 18.0, V 12.0, H 1.5
Increases in the protein product Fos of the immediate under halothane general anesthesia using methods de-
early gene c-fos have been used extensively as markers of scribed previously in detail [26]. All animals receiving
neuronal activation in neuroendocrine systems for review FAu injections were allowed at least 2 weeks of recovery
see [18]. In previous studies, we used fetal hypoxemia as time for retrograde transport of FAu to occur. At least 5
a well characterized and repeatable stimulus for ACTH days prior to the hypoxemic challenge, six of the seven
secretion and as a stimulus for the production of Fos in ewes carrying HYPOXEMIC fetuses were surgically in-
CRH-immunopositive neurons of the PVN of fetal sheep strumented with one jugular and one carotid polyvinyl
[17]. In adult rats, hypoxia stimulates Fos production in catheter 18-gauge and one tracheal polyvinyl catheter
CA neurons of the ventrolateral A1 C1 and dorsal o.d.53.5 mm, i.d.52.5 mm while under halothane gener-
medulla A2 C2; [8,19] and CRH, vasopressin AVP and al anesthesia, as previously published [12]. No fetuses
oxytocin OT neurons of the PVN and supraoptic nucleus. were instrumented to keep fetal stress to a minimum.
Lesions of A1 C1 CA neurons significantly reduce Fos expression in hypothalamic CRH, AVP and OT neurons
2.2. Hypoxemic challenge [32]. However, to our knowledge, no investigations in
fetuses of any species have determined which brainstem On the day of hypoxemic challenge, fetuses ranged in
nuclei projections to the fetal PVN are developed and the age from 124–144 HYPOX and 125–141 NORMOX
extent to which the cells of these brainstem nuclei exhibit dGA. Hypoxemia was induced according to the method of
GR and are coactivated with hypothalamic neurons during Gleed et al. [12]. Briefly, at time50, N infusion to the
2
hypoxemia. maternal tracheal catheter was begun at a rate of 12 l min.
Since the fetus often displays physiological responses In order to provide variability in the degree of hypoxemia
that differ markedly from adults e.g., unlike adults, fetuses in individual fetuses after the first 10 min, N flow was
2
greatly decrease or cease their in utero breathing move- adjusted to achieve differing maternal PO values over the
2
ments during hypoxemia [4], it is impossible to extrapo- 60 min hypoxemic period. One HYPOXEMIC group ewe
late from the adult condition or to determine when a was not catheterized. In this ewe, N flow was maintained
2
system begins to function in utero other than by examining at 8 l min for the last 50 min of the hypoxemic period. We
the fetus itself. We have chosen to perform our studies in have shown previously that this method produces de-
fetal sheep at the approximate time of gestation that: 1 creases in fetal PO of approximately 10.0–14.0 Torr and
2
fetal sheep adrenals are responsive to ACTH stimulation reproducibly elicits fetal ACTH secretion [24]. At 60 min,
after a mid gestation refractory period for review see N
flow was terminated and ewes and fetuses were
2
[6,24]and 2 negative feedback begins to function in the immediately euthanized by exsanguination under deep
fetal sheep hypothalamo–pituitary–adrenal axis [36]. The halothane anesthesia.
first aim of the present study was to determine if fetal brainstem CA neurons project to the PVN, express GR and
2.3. Tissue collection and immunocytochemistry show Fos activation during a hypoxemic challenge. The
second aim was to see if any brainstem CA neuronal Perfusion and fixation of the fetal brain were performed
activation observed correlates with Fos activation of CRH via the carotid artery with a 14-gauge indwelling catheter
neurons of the fetal PVN during graded hypoxemic pointed toward the brain using techniques previously
challenges, which we know from previous studies reliably described in detail [26]. Both jugular veins were cut and
increase ACTH secretion in peripheral fetal sheep plasma. the anterior vena cava, aorta and pulmonary artery were
clamped at the heart. The fetal brain was perfused with 500 ml of normal saline containing heparin 10 IU ml and
2. Materials and methods NaNO 2.0, wt vol as a vasodilator followed by 1000
2
ml of 0.1 M phosphate-buffered acrolein–paraformal- 2.1. Care and use of animals
dehyde Sigma, St Louis, MO; 2.5 vol vol; 4 wt vol. The time from the end of hypoxemia until the start of
The study
utilized timed
pregnant Rambouillet3
fixative infusion was 20–30 min in all animals. Following Columbia ewes n510 and their fetuses n510 that were
fixation the skull vault was opened and the brain and randomly assigned to hypoxemic HYPOX n57 or
pituitary were removed. Brains were dehydrated in sucrose
72 T
30, wt vol and sectioned as a 1 in 12 series at 30 mm 2.4. Data analysis
on a sliding microtome Fisher Scientific, Springfield, N.J. 07081 while frozen with crushed dry ice. Sections were
The average number of neurons counted in each brain- stored in cryoprotectant [35] solution at 2208C to preserve
stem CAergic area e.g., A1 C1, A2 C2 in studies of the immunogenicity until the time of staining.
percent of brainstem neurons retrogradely labeled with Double label immunocytochemical staining for Fos and
FAu after FAu injection to the PVN was: A1 C1 — 307, either CRH or tyrosine hydroxylase TH, FAu and CRH
A2 C2 — 275, A5 — 231 and A6 — 1615. The average or TH, and GR and TH was accomplished using methods
number of neurons counted in studies of the correlation of previously reported [16]. Once the staining procedures
Fos activation of PVN CRH-immunoreactive neurons vs. were well established on practice tissues, one well of
Fos activation of brainstem CAergic area neurons over a sections i.e., 1 12th of all sections cut of brainstem
range of hypoxemia were PVN CRH — 347, A1 C1 — and or of hypothalamus from each animal were immuno-
651, A2 C2 — 597 and A6 — 539. stained for each pair of antigens listed in the previous
Counting of coincidence of Fos and TH in brainstem sentence. Staining of the first antigen in double reactions
A1 C1, A2 C2 and locus coeruleus A6 neuronal areas was accomplished with the avidin–biotin complex ABC
and coincidence of Fos and CRH in PVN was done under method Elite Kits, Vector Labs, Burlingame, CA 94010
light microscopy by an observer unaware of treatment. and immunoperoxidase detection with nickel–diaminoben-
Counts were expressed as a single independent value i.e., zidine Ni–DAB as the chromogen. The presence of
percentage of total CA or CRH neurons expressing Fos per antigen was detected as a blue black product, which could
brainstem neuronal area e.g., A1, A2 or PVN, per animal. be eliminated by substitution of normal rabbit serum for
The percentages of Fos and TH coincidence for each area the primary antibody or preabsorption of the primary
of CAergic brainstem neurons vs. the percent of Fos and antibody with 10 mg of the antigen per ml of antibody.
CRH coincidence in neurons of the PVN were plotted Fig. Incubations with primary antisera: CRH polyclonal, gift
7 and subjected to the Pearson Product Moment Correla- of Dr A. J. Silverman 1:40,000; FAu Chemicon, poly-
tion test with P-value set at 0.05. All data are expressed as clonal 1:100,000; TH Chemicon, monoclonal 1:250,000;
mean6S.E.M. Fos Oncogene Sciences, AB-2 or Dr T. Curran’s alu-Fos
All procedures were approved by the Cornell University antibody 1:50,000 and GR gift of Dr D. DeFranco,
Animal Care and Use Committee. All facilities were BUGR2 1:70,000 were performed for 48 h at 48C. After
approved by the American Association for the Accredita- development of the first antibody the tissue was rinsed and
tion of Laboratory Animal Care. the second primary antiserum was applied. Afterward the
tissue was again processed for ABC immunoperoxidase staining, but the chromogen used was DAB alone. The
results obtained then had either black nuclei GR or Fos