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Decidua-associated changes in guinea pig uterine blood flow and volumeRelation to uterine norepinephrine concentrations.

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THE ANATOMICAL RECORD 211:410-413 (1985)
Decidua-Associated Changes in Guinea Pig Uterine
Blood Flow and Volume: Relation to Uterine
Norepinephrine Concentrations
DAVID R. GARRIS AND M.S. DAR
Department of Anatomy (0.R. G.) and Department of Pharmacology (M.S.D.),East Carolina
University, School of Medicine, Greenville, NC 27834
ABSTRACT
Changes in uterine blood flow (UBF) and uterine blood volume
(UBV) during decidual tissue (DT) formation in the guinea pig were assessed at 2,6,
and 10 days postuterine trauma (PT).Uterine weight increased steadily between
days 2 and 10 PT in DT-bearing animals as compared with the constant uterine
weights observed in controls. Both UBF and UBV levels were elevated between days
2 and 10 PT in DT-bearing animals as compared with controls with the vascular
changes being histologically related to the progressive stromal differentiation. Uterine norepnephrine (NE) levels were significatly (P < 0.05-0.01) depressed in the
hyperemic, DT-bearing uteri as compared with controls. These results indicate that
the uterine hyperemia associated with DT formation in the guinea pig involves a
sustained increase in uterine vascular volume as well as vascular perfusion rate
under conditions of depressed uterine NE levels. These events are intimately associated with the support of stromal differentiation in the guinea pig.
The process of stromal differentiation into decidual
tissue (DT) in the guinea pig is associated with uterine
hyperemia (Loeb, 1908; Orsini and Donovan, 1971; Garris, 1984a). An increased uterine blood flow (UBF) persists throughout the period of maximal DT development
(Garris, 1982, 1984a) in the guinea pig and has been
associated with a n increased vascular bed expansion
similar to that observed during nidation (Garris and
Whitehead, 1981; Garris et al., 1983a). Associated with
the development of uterine hyperemia in normal animals is a depression of tissue norepinephrine (NE) levels
(Garris et al., 1983b). These observations suggest that
the decline in noradrenergic innervation of the uterine
vascular plexus may account for the hyperemic responses that have previously been associated with nidation (Garris and Whitehead, 1981), elevated serum
estradiol levels (Garris and Whitehead, 1981; Garris et
al., 1983b, 1984; Garris 1984a,b), stromal differentiation
(Garris 1984a), and early placental formation (Garris,
1984c; Garris et al., 1983a).The depression in noradrenergic vascular tone, in turn, was associated with a n
increased sensitivity to cholinergic modulation of uterine vasodilation (Bell, 1968, 1970; Garris et al., 1984).
Similar vascular changes have been noted in the pseudopregnant rat, in which uterine hyperemia has been
associated with the process of DT formation and regression (Garris et al., 1983~).However, the relationship
between the DT-associated increase in UBF and any
accompanying changes that occur in uterine blood volume (UBV) and uterine NE levels during stromal differentiation remains to be determined. The present study
was undertaken to determine the changes that occur in
UBF, UBV, and uterine NE levels during DT formation
in the guinea pig.
0 1985 ALAN R. LISS, INC
MATERIALS AND METHODS
Adult female virgin guinea pigs (650-85Ogm) were
used in this study. All animals were maintained under
controlled environmental conditions with a n established
photoperiod of 12 hr light/day (lights on: 0600 hr). Food
(Wayne Guinea Pig Chow, Granville Milling Co., Creedmoor, NC) and water were available ad libitum. Daily
vaginal examination was used to denote reproductive
cyclicity, with vaginal patency in association with cornified cells in a lavage used to denote day 0 of the
estrous cycle.
The induction of DT was performed on day 5 of the
estrous cycle via a midventral laparotomy used for uterine exposure using methoxyflurane (Pittman-Moore,
Washington Crossing, NJ) anesthesia (Garris, 1984a).
Each uterine horne was incised along the antimesometrial border with a scissor cut. Sham-operated animals,
in which the uterus was exposed but not manipulated,
served as controls. Each animal was allowed to recover
and was subsequently assigned to a n experimental group
for UBF and UBV determinations on either day 2, 6, or
10 posttrauma (PT).
Uterine BF measurements were performed using a n
electromagnetic blood flow monitor (Carolina Medical
Electronics, King, NC) and appropriately sized (1.0-2.0
mm diameter), precalibrated flow probes corrected for
hematocrit and body temperature (39"C), as previously
described (Garris et al., 1983a; Garris, 1984a). In brief,
guinea pigs were anesthetized with Innovar Vet (0.3 mll
kg; Pittman-Moore) and supplemented with methoxyflurane (Pittman-Moore) as needed. The uterus was exposed via a midventral laparotomy and a 0.5-cm segment
Received April 24, 1984; accepted November 19, 1984.
411
UTERINE BLOOD FLOW AND DECIDUA GROWTH
of the uterine artery was isolated by blunt microdissection with extreme care taken not to contact directly any
of the uterine vasculature. A 10 to 15 min recording of
from each
UBF was
and the probes and
monitor were subsequently checked for precalibration
accuracy. Failure of the monitor to recalibrate to within
0.1 mumin of the established baseline was used as a
criterion for discarding a measurement from analysis.
All probes used in this study were found to measure
stable UBF rates, as indicated by common group mean
flow rates as well as repeatable, individual BF recordings. All usable values were expressed as milliliters per
minute. Each uterus was subsequently removed,
cleaned, and weighed to the nearest 0.1 mg.
Uterine BV ws determined as previously described
(Garris, 1984~).In brief, each animal was lightly anesthetized with methoxyflurane and injected with 10 pCi
of 1251-BSA, (New England Nuclear Co., Boston, MA)
suspended in 0.2 ml of physiological saline containing
10-15 mg of albumin per milliliter by intracardiac puncture. Each animal was allowed to regain consciousness
and resume normal activity for 15-20 min in order to
allow for systemic equilbration of the UBV tracer. At 20
min postinjection, each animal was sacrificed by cervical
dislocation and immediately immersed in liquid nitrogen long enough to stop blood flow but not freeze the
internal organs (approximately 25-30 sec). The thoracic
cavity was rapidly opened and a 0.2 ml sample of blood
was collected from the left ventricle to serve as a n index
of radioactivity per milliliter of systemic blood. Subsequently, the uterus was exposed, weighed, sliced into
smaller fragments, and placed in gamma counter tubes.
Total radioactivity was calculated for both uterine and
DT tissues and compared to the radioactivity level in
the sample of systemic blood. Final UBV values were
expressed as microliters of blood per gram of tissue.
The concentration of norepinephrine (NE) in fresh
uterine and decidual tissue was determined by high
performance liquid chromatography (Garris et al., 1984)
using dih droxybenzylamine as a n internal standard
and a C1'reverse phase p-Bondapak column (Waters
Associates, Milford, MA). The column was attached to
a n LC-4 electrochemical detector with a glassy carbon
electrode and a n AG+/AgCl reference electrode (BioanThe mobile phase
alytical Systems, West Lafayett, IN).
was 0.05 M sodium acetate b a e r (pH 5.0) to which 0.05
mM sodium octyl sulfonate and 1 mM tetra sodium
EDTA were added. Separation was accomplished a t ambient temperature (22-24°C) at a flow rate of 1.5 ml/min
which generated a back pressure of 1,000-1,500 psi with
a detector potential of +0.7 V and a sensitivity range of
1-2 M (Garris et al., 1984). The injected volume of
each sample ranged from 20-30 pl.
Samples of both uterine and DT tissue were collected
from each group of guinea pigs for histological analysis
of stromal and DT changes. Tissues were prepared for
microscopic analysis following paraffin sectioning and
staining with hematoxylin and eosin using conventional
techniques.
All UBF, UBV, NE, and uterine weight values were
expressed as group means (k SEM) with P < 0.05 accepted as denoting statistical differences between groups
using the Student's t-test and analysis of variance exams, where appropriate. Correlation coefficients were
determined by regression analysis.
RESULTS
Uterine weight increased between days and
(Fig. 1) in DT-bearing guinea pigs as
with
controls. In contrast, both UBF and UBV were at maximal levels by day m and remained elevated through
(Fig. 1) in DT-bearing animals as
day
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Fig. I. Changes in uterine weight, UBF and UBV, and uterine NE
levels in control (C) and decidual tissue (DT)-bearing guinea pigs at 2,
6, and 10 days post-uterine trauma. All values are expressed as group
means (* SEM) for the indicated number of animals with intergroup
differences between corresponding C and DT groups denoted by *P <
0.05; ** P < 0.01;***P < 0.001.
412
D.R. GARRIS AND M.S. DAR
Fig. 2. Photomicrographs ( ~ 5 0of
) control (A) and DT-bearing (€9 uteri from guinea pigs in
the day 10 PT group. The stroma of the traumatized uterus exhibits extensive cellular differentiation into DT as compared with control tissue. The conspicuous endometrial glands present
in control tissue are notably absent in the differentiated and proliferated DT. Arrows point to
expanded capillary vascular beds in the DT samples (B), which accounted for the elevated UBV
measurements in these guinea pigs.
with control levels. Increased UBF and UBV mesurements were associated with DT development, whereas
corresponding tissue NE levels decreased relative to control values (Fig. 1)between days 2 and 10 F'T. Day 10
NE levels in DT tissue continued to remain below those
of control tissue while UBF and UBV levels were elevated. Between days 2 and 10 PT,uterine weight and
UBF and UBV levels in DT-bearing guinea pigs remained significantly elevated over corresponding control parameters. In contrast, uterine NE concentrations
were significantly depressed relative to control values
throughout the study. A significant, inverse correlation
coefficient (r = - 0.5891; df = 12; P < 0.05) was noted
between changes in DT weight and NE concentrations
as well as between UBF and tissue NE in DT-bearing
uteri (r = - 0.5282: df = 12; P 6 0.05).
Histological analysis of the uterine tissues indicated
that the stroma of traumatized uteri had undergone
extensive differentiation into decidual tissue (Fig. 21,
which accounted for the increase in uterine weight in
DT-bearing guinea pigs. Typical of guinea pig DT was
the noted absence of endometrial glands and the expanded vascular plexus which accounted for the observed elevations in UBV in traumatized uteri. The
days 2-10 structural changes were gradual, with maximal tissue differentiation being completed by day 10
posttrauma (Fig. 2).
DISCUSSION
The results of the present study confirm previous reports indicating that uterine hyperemia is associated
with stromal differentiation in the guinea pig (Loeb,
1908; Orsini and Donovan, 1971; Garris, 1982, 1984a).
The concomitant increases in both UBF and UBV between days 2 and 10 PT indicate, for the first time, that
both the vascular perfusion rate and vascular volume of
the stromal capillary bed increase in response to DT
formation. The decline in tissue NE levels associated
with DT formation is probably a physiological response
associated with tissue differentiation which, in turn,
allows for vasodilation that was observed to be associated with stromal proliferation (Garris, 1984a). Similar
findings have been observed for the rat in which UBF
remained elevated during the period of maximal DT
growth and declined prior to DT resorption (Garris et
These findings suggest that the ability of a n
al., 1983~).
ovarian steroid-primed endometrium to respond to either
a natural or a n induced deciduogenic stimulus also depends on the capability of the endometrial vascular bed
to expand to support cellular growth, as well as demonstrate compensatory changes in vasomodulating agents
(e.g., NE). Uterine blood flow has been previously demonstrated to be responsive to the ovarian steroid milieu
as well as the type of trauma used to evoke stromal
differentiation in the guinea pig (Garris, 1984a).The use
of nondeciduogenic stimuli or improper steroid priming
of the uterus both result in the failure to induce uterine
hyperemia in this species (Garris, 1984a). The observation that UBV increases during the early phase of DT
formation supports the anatomical observations of
Lundkuist (1978) in which capillary bed expansion occurred prior to stromal differentiation in the rat. The
stable uterine weights observed in the present study
suggest that the vascular changes were not associated
with cyclic fluctuations in estrogen levels. The experimental suppression of UBF has been demonstrated to
413
UTERINE BLOOD FLOW AND DECIDUA GROWTH
result in the curtailment of intrauterine growth in pregnant rats and guinea pigs, with the decidual component
of the placental unit being most severely affected (Garris, 1983; Franklin and Brent, 1964). Thus, DT growth
demands a n increased UBF rate as well as expanded
vascular capacity, with both parameters serving to support subsequent endometrial differentiation.
The increase in UBF observed in association with DT
formation parallels the uterine hyperemia related to
both the nidatory process (Garris and Whitehead, 1981)
and early placentation (Garris et al., 1983a; 19844 in
the guinea pig. Of particular interest was the concomitant elevation in UBF and UBV observed between days
2 and 10 PT in association with DT formation. These
data indicate that both the vascular perfusion rate (i.e.,
UBF) and vascular bed capacity (i.e., UBV) increase in
a similar, temporal manner during stromal differentiation. These data contrast with the vascular patterns
observed for the guinea pig placenta (Garris, 19844 in
which changes in UBF rates did not necessarily indicate
that concomitant changes occurred in the placental vascular bed (maternal portion) volume. The present results
suggest that both perfusion rate through the uterus and
capillary bed volume increase in a similar manner to
support the cellular nutrient requirements associated
with stromal differentiation in the guinea pig.
The decline in tissue NE levels that occurred in association with DT development mimic the similar changes
that occur during placental development in this species
(Bell and Malcolm, 1978; Owman et al., 1978). This
decline in uterine NE levels also parallels the observed
depression in tissue NE concentrations observed in estrogen-induced, hyperemic uteri (Garris et al., 1983b;
1984). The decline in tissue NE concentrations has been
suggested to be associated with the cholinergic modulation of vasodilation in the guinea pig uterine vascular
plexus (Bell, 1968; Garris et al., 1984). The exact mechanisms responsible for these changes in tissue bioamine
levels remains to be determined.
In summary, the present studies indicate that in association with DT formation in the guinea pig, parallel
elevations in UBF and UBV occur in a tissue system
that exhibits depressed NE concentrations. The decline
in noradrenergic activity is suspected to be causally
associated with the occurrence of the uterine hyperemic
response. The increase in UBF and UBV are recognized
to be essential for the successful development of DT
tissue in the guinea pig (Garris, 1984a), and the changes
that occur in these uterine parameters appear to parallel the changes associated with blastocyst implantation
and early placentation in this species (Garris and White
head, 1981; Garris et al., 1983a; Garris 1984b,c).
ACKNOWLEDGMENTS
This work was supported in part by a grant from the
Rockefeller Foundation.
LITERATURE CITED
Bell, C. (1968) Dual vasomotor and vasodilator innervation of the
uterine arterial supply in the guinea pig. Cir. Res., 23t279-284.
Bell, C. (1970) Effects of circulating estrogen on function of the cholinergic dilator nerves supplying the guinea pig artery. Bri. J. Pharmacol., 39t190-196.
Bell, C., and S.J. Malcolm (1978)Observations on the loss of catecholamine fluorescence from intrauterine adrenergic nerves during
pregnancy in the guinea pig. J. Reprod. Fertil., 53t51-58.
Franklin, J.B., and R.L. Brent (1964) The effect of uterine vascular
clamping on the development of rat embryos three to fourteen days
old. J. Morphol., 115t273-290.
Garris, D.R. (1982) Effects of maximal deciduoma formation on uterine
blood flow in the rat and guinea pig: Correlation with serum
progesterone levels. IRCS Med. Sci., lOt149-150.
Garris, D.R. (1983)Regional variations in guinea pig uterine bloof flow
during pregnancy: Relationship to intrauterine growth of the fetalplacental unit. Teratology, 27t101-107.
Garris, D.R. (1984a) Decidual tissue growth and regression in the
guinea pig: Regulation by uterine blood flow and relation to circulating progesterone concentrations. Endocrinology, 114t1599-1604.
Garris, D.R. (1984b) Uterine blood flow, pH and pCOz during nidation
in the guinea pig: Ovarian regulation. Endocrinology, 114r12191224.
Garris, D.R. (1984~)
Uteroplacental vascular perfusion and blood flow
during pregnancy in the guinea pig. Biol. Neonate, 45t33-40.
Garris, D.R., L.T. Blankenship, and D.S. Whitehead (1983a) Regional
variations in uterine blood flow in the guinea pig: Relationship to
pregnancy site distribution. Gynecol. Obstet. Invest., 15t33-40.
Garris, D.R., M.S. Dar, and M.M. McConnaughey (1983b) Effects of
ovarian steroids on uterine vascular responsivity to catecholamines. Fed. Proc., 34t634.
Garris, D.R., J.H. Fair, D.S. Whitehead, and P.D. Muse (1983~)
Relationship between deciduoma formation and uterine blood flow in
the pseudopregnant rat. Gynecol. Obstet. Invest., 15t84-92.
Garris, D.R., A.J. Ingenito, M.M. McConnaughey, and M.S. Dar (1984)
Regulation of estrogen-induced uterine hyperemia and contractility in the guinea pig: Cholinergic modulation of an alpha-adrenergic response. Biol. Reprod., 302363-868.
Garris, D.R., and D.S. Whitehead (1981) Uterine blood flow and timing
of blastocyst implantation in the guinea pig. Am. J. Physiol.,
241tE142-El45.
Loeb, L. (1908) The production of deciduomata and the relation between the ovaries and the formation of decidua. JAMA, 50t18971901.
Lundkuist, 0. (1978) Morphological studies of the stromal changes
after artificial induction of the decidual reaction. Cell Tissue Res.,
194t287-296.
Orsini, M.W., and B.T. Donovan (1971) Implantation and induced decidualization of the uterus in the guinea pig, as indicated by pontamine blue. Biol. Reprod., 5270-281.
Owman, C., P. Alm, E. Rosengren, N.O. Sjoberg, and G. Thorbert
(1978) Variations in the level of uterine norepinephrine during
pregnancy in the guinea pig. Am. J. Obstet. Gynecol., 122t961-964.
~~
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