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The effects of pelvic neurectomy on collagen in the cervix of the pseudopregnant rat.

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THE ANATOMICAL RECORD 210575-581 (1984)
The Effects of Pelvic Neurectomy on Collagen in the Cervix of the
Pseudopregnant Rat
HUBERT W. BURDEN, MARILYN LEONARD, CARLTON P. SMITH,
THOMAS M. LOUIS AND IRVIN E. LAWRENCE
Department of Anatomy, School of Medicine. East Carolina University,
Greenuille, NC 27834
ABSTRACT
Bilateral section of the pelvic parasympathetic nerves (pelvicneurectomy) on day 5 of pseudopregnancy had no effect on the wet weight of
ovaries, uteri, andlor cervices, but a t day 9 serum progesterone was reduced to
approximately half that of sham-operated animals. Collagen in the cervix was
visualized with picrosirius red staining under polarization microscopy. Pelvic
neurectomy decreased the birefringence of Type 1collagen in the cervix to less
than half that of sham operated animals. The decreased birefringence, an
index of the organization of collagen fibers, is believed to be attributable to
reduced progesterone levels. Alternatively, the pelvic nerve may directly influence mucopolysaccharides or collagenolytic enzymes in the cervix.
Bilateral section of the pelvic parasym- ganization in the cervix. The pseudopregpathetic nerves (pelvic neurectomy [PN]) in nant rat was utilized since this is a wellrats is compatible with pregnancy, but par- defined endocrine model, devoid of the comturition is blocked (Carlson and DeFeo, 1965; plex hormonal interactions characteristic of
Spies et al., 1971; Louis et al., 1978). The the preparturient and parturient periods.
cause of this blocked parturition is unknown.
MATERIALS AND METHODS
We have previously studied selected endoAnimals
crine parameters in PN rats during the parNulliparous Sprague-Dawley rats (Charles
turient period (Louis et al., 1978; Burden et
al., 1982). Although there are subtle differ- River derived, Animal Resources Center,
ences in utero-ovarian vein levels of proges- East Carolina University) were housed in a
terone and estradiol, a consistent major room with controlled lighting (lights on
1”C),
quantitative difference is the failure of pros- 05.00-19.00 h) and temperature (25
taglandin F to rise a t the expected time of and provided food and water ad libitum. The
delivery in PN rats with blocked parturition estrous cycle was monitored daily by vaginal
lavages and only those rats showing two con(Louis et al., 1978).
Dramatic structural and functional secutive 4-day cycles were used in experichanges occur in the rat cervix prior to par- ments. On the morning of estrus (day 1)
turition (Williams et al., 1982). These pseudopregnancy was induced by tapping the
changes, collectively referred to as “cervical region of the cervix and/or the posterior forripening” are controlled, at least partially, nix rapidly with a polished glass rod 2-3 mm
by prostaglandins (Hillier and Wallis, 1982; in diameter (DeFeo, 1963). On day 5 , sham
Theobald et al., 1982; Rath et al., 1982; Nor- surgery or bilateral PN was performed under
strom et al., 1983; Fuchs et al., 1983; Uldbjerg ether anesthesia using the anatomical apet al., 1983)and neurotransmitters (Hollings- proach described by Carlson and DeFeo
worth, 1974; Hollingsworth and Isherwood, (1965). In the sham group, the pelvic nerves
were exposed and touched. On day 9, all ani1979).
The rat cervix has a well-developed adre- mals were decapitated, trunk blood collected,
nergic and cholinergic innervation (Adham and serum analyzed for progesterone by raand Schenk, 1969).Most of these nerves reach
the cervix via the pelvic splanchnic nerves
(Hulsebosch and Coggeshall, 1982). The purpose of the present study was to evaluate the
Received April 20,1984; accepted July 2, 1984
effects of pelvic neurectomy on collagen or-
+
0 1984 ALAN R. LISS, INC.
576
H.W. BURDEN ET AL
dioimmunoassay. Both ovaries, uterus, and
cervix were removed, trimmed, and weighed.
The cervix was defined as the less vascular
tissue with parallel lumina between the
uterine horns and the vagina (Hollingsworth
and Isherwood, 1978). The cervix was fixed
in Carnoy's fluid.
Microscopy and Morphometry
Each cervix was embedded in paraffin with
the external 0s down. Serial cross sections
were cut at 5 pm, starting a t the external os,
stained with the picrosirius method (Junqueira et al., 19791, and observed under polarization microscopy. This method is specific
for oriented polymerized collagen molecules
(collagen fibers) in the sense that only these
structures present a bright red birefringence
(Junqueira et al., 1979). Depolymerized collagen, not oriented as fibers, lacks orientation and even though it stains red, such
collagen is not birefringent. The birefringence was quantitated by counting the bright
areas (pixels) with the grain counting program utilized with a Bioquant I1 image analysis system (R and M Biometrics, Nashville,
TN). The slides were coded, hence the identity of individual cervices was unknown to
the operator (ML) of the image analysis system. Birefringence was quantitated utilizing
a 10x objective fitted to a Zeiss Photomicroscope I1 at three levels in the cervix: (1)at
the point where the lateral vaginal fornix
disappeared (reference level), (2) 100 sections
cranially from the reference level, and (3) 200
sections cranially from the reference point.
Five nonoverlapping fields per section at each
level were measured (Fig. 1).The total area
of the five fields measured a t each level was
approximately 500 pm2. Any measuring
fields which extended off a tissue section
could be ignored, since these areas contained
no bright grains.
Progesterone Radioirnmunoassay
The progesterone radioimmunoassay used
rabbit antiprogesterone-lla-BSA obtained
from Miles Laboratories, Elkhart, IN. The
only significant cross-reactivities of the antiserum were with lla-hydroxyprogesterone
and 11/3-hydroxyprogesterone(44 and 8%,respectively). The antiserum also cross-reacts
with 5a-pregnen-3,20-dione (3%) and with
17a-hydroxyprogesterone (2%).All other possible interfering steroids cross-react less than
0.4%.
E
!!
Fig. 1. Diagram illustrating the structure of the rat
cervix. E, endometrium; M, inner circular and outer
longitudinal layers of myometrium; L, lumen of uterine
cornu. Numbers 1-5 between the two horizontal lines
indicate the five nonoverlapping fields where collagen
Type I birefringence was recorded at each of three levels
in the cervix (see Materials and Methods section). The
total area of the five fields measured at each level was
approximately 500 pm2.
Assay consisted of extraction of plasma
samples (100 pl) with petroleum ether and
reconstitution of evaporated solvent phase
with 0.1 M phosphate-buffered saline (PBS
0.1% Knox gelatin, pH 7.2). Samples received
sufficient antibody to bind approximately
35% of available progesterone and were equilibrated 20 min before addition of approximately 12,000 cpm tritiated progesterone.
After incubation overnight at 4"C, bound and
free fractions were separated using dextrancoated charcoal at 4°C.
Accuracy and precision were tested by including several known quantities of progesterone and standard plasma tubes with the
assay. Two water blanks were also included
to reveal positive or negative interference on
the assay. Coefficients of variation for intraassay variation averaged 8.5%. The average blank value was 5 pg.
Statistics
Birefringence values from collagen fibers
in the cervix and serum progesterone values
were compared using the Student's t-test.
Differences were considered significant if p
was less than 0.05.
RESULTS
Effects of Pelvic Neurectomy on Urinary
Bladder
Bilateral pelvic neurectomy caused distension of the urinary bladder, apparently because of interruption of the micturition
577
PELVIC NEURECTOMY AND THE RAT CERVIX
TABLE 1. Organ weights in sham and pelvic neurectomized rats on
day 9 of pseudopregnancy'
Treatment
Combined
ovarian
weight
(mg/100 gm
body wt)
Uterine
weight
(mg/100 gm
body wt)
Cervix
weight
(mgi100 gm
body wt)
Sham (3)
Pelvic Neurectomy (8)
26.8 f 1.4
27.0 f 1.0
131.5
9.7
131.3 f 13.2
28.8 f 1.7
27.6 & 2.0
'Numbers in parentheses are number of animals.
TABLE 2. Serum progesterone values (mean f SEMI in
sham and pelvic neurectomized rats on day 9 of
pseudopregnancy
'
Treatment
Sham operated (8)
Pelvic neurectomy (9)
Progesterone
(ng/ml)
40.2
21.6
+ 5.0
+ 4.0*
'Numbers in parentheses are number of animals.
*Significantly less (p < 0.01)than sham-operated animals.
reflex. The presence of a n enlarged urinary
bladder was taken as evidence of complete
bilateral pelvic neurectomy. The animals
were able to void by overflow and by contraction of abdominal muscles. The animals did
not appear to be in distress and they were in
good health during the 4 days subsequent to
surgery.
TABLE 3. Birefringence of collagen fibers in the cervix
of pseudopregnant rats'
Treatment
Sham (3)
PN (3)
Birefringence
(total No. bright
pixels, mean & SEMI
5,131.11 & 628.39
2.064.58 + 316.49*
'Numbers in parentheses are number of cervices measured
*Significantly (p < 0.001) less than sham value.
Pelvic neurectomy did not appear to alter
the microanatomy of the cervix stained with
the picrosirius technique and visualized under transmitted light microscopy (Figs. 2,4).
However, when viewed with polarization microscopy, the total birefringence of collagen
fibers in the cervix was reduced to less than
half that of sham animals (Table 3). There
was reduction in birefringence in both the
Ovarian Weights and Progesterone
circular and longitudinal layers of the myoPelvic neurectomy had no effect on the wet metrium (Figs. 33) and in the endometrium
weight of the ovaries, uteri, or cervices (Table (Figs. 6,7).
1).Cutting the pelvic splanchnic nerves sigDISCUSSION
nificantly (p < 0.05) reduced serum progesterone values (Table 2).
The present study shows that bilateral pelvic neurectomy in pseudopregnant rats does
Gross Anatomy
not affect ovarian, uterine, or cervical
In the pseudopregnant rat, the cervix is weights but luteal function was depressed, as
compact, paler than the uterine horns, and determined by serum levels of progesterone.
of firmer consistency. The transition between The luteolytic effects of the uterus have been
the cranial cervix and uterine cornu is grad- documented by many workers (see review by
ual. In contrast, the junction between caudal Anderson, 1977)) but the role of uterine
cervix and the vagina is well defined.
nerves in uterine-mediated luteolysis is less
certain. Carlson and DeFeo (1965) showed
Microscopy and Morphometry
that pelvic neurectomy abolished the exThe histology of the cervix is similar to the tended luteal maintenance characteristic of
uterus. The myometrium consists of a n outer rats bearing deciduoma. In contrast, Wilson
longitudinal layer of smooth muscle and a n et al. (1970) showed that increased luteal
inner circular layer (Figs. 1,2). Deep to the maintenance in rats with deciduoma was not
circular layer is the endometrium with a n altered by pelvic neurectomy. We have reepithelial lining around each cervical canal. ported previously (Burden et al., 1980) that
The two canals are separated by connective unilateral PN did not alter ipsilateral uteroovarian vein values of progesterone, 20a-01,
tissue.
578
H.W. BURDEN ET AL.
PELVIC NEURECTOMY AND THE RAT CERVIX
Figs. 2-5. Photomicrographs of rat cervix stained with
picrosirius red. All photomicrographs were taken at the
same level (200 sections cranial to reference section, see
Methods).
579
Fig. 5. Same area as Figure 4, polarization microscopy. Note reduced birefringence of collagen fibers as
compared to Figure 3. X 120.
Fig. 2. Sham-operated animals, transmitted light microscopy. L, longitudinal layer of myometrium; C, circular layer of myometrium; E, endometrium. X 120.
Figs. 6-7. Photomicrographs of endometrium of rat
cervix stained with picrosirius red. Both photomicrographs were taken with polarized light a t the same level
(200 sections cranial to reference section, see Methods).
Fig. 3. Same area as Figure 2, polarization microscopy. Note numerous white (birefringent)collagen fibers.
x 120.
Fig. 6. Sham-operated animal. E, endometrium; L,
lumen of uterine cornu. Note numerous white (birefringent) collagen fibers. X 120.
Fig. 4. Pelvic neurectomized animal, transmitted light
microscopy. L, longitudinal layer of myometrium; C, circular layer of myometrium; E, endometrium. X 120.
Fig. 7. Pelvic neurectomized animal. E, endometrium; L, lumen of uterine cornu. Note reduced number
of white (birefringent) collagen fibers. x 120.
580
H.W. BURDEN ET AL.
or prostaglandin F during the preparturient
period. The possibility of a direct effect of PN
on ovarian function can be ruled out since
the pelvic splanchnic nerves do not contribute to the ovarian innervation in the rat
(Burden and Lawrence, 1978).
Collagen molecules are orderly disposed in
a parallel orientation, and a normal birefringence is one of the classic characteristics of
collagenous structures. The enhancement of
birefringence promoted by the picrosirius polarization method is specific for collagen and
has proved to be more useful for studying
collagen distribution than the routinely used
trichrome techniques (see review by Junqueira and Montes, 1983). Collagen Type I,
the classical collagen fiber (Bornstein, 1980),
appears as thick, strongly birefringent yellow or red fibers in the cervix. While pelvic
neurectomy did not affect the wet weight of
the cervix, the collagen fibers were separated
and irregularly fragmented and the amount
of collagen fibers per area was dramatically
reduced.
The pelvic nerve is a major route of sensory, adrenergic, cholinergic (Hulsebosch and
Coggeshall, 1982), and peptidergic (de Groat
et al., 1983) components to pelvic viscera.
The mechanism whereby these nerves maintain collagen fibers in the cervix is not
known. Collagen fibers are composed of
bound fibrils. These fibrils are formed from,
and are in thermodynamic equilibrium with,
a pool of less aggregated collagen protein
dispersed in the vacuoles of the ground substance (Gersh and Catchpole, 1960). The collagen when aggregated into fibrils and fibers
derives its strength from the high molecular
glycoaminoglycans or mucopolysaccharides
which act as cement or binding substances
(F'itzpatrick, 1977). Apparently, pelvic neurectomy alters the thermodynamic equilibrium between aggregated and less
aggregated collagen protein or alters the mucopolysaccharide binding substances between fibers (Golichowski et al., 1980).
Alternatively, cutting the nerve may activate cervical collagenases. Circulating progesterone was reduced in PN rats and it is
known that decreased levels of steroids activate collagenolytic enzymes in the cervix
(Wallis and Hillier, 1981). Whatever the
mechanisms operable, it appears that PN
causes distinct changes in the quality of collagen in the cervix. Possible mechanisms mediating these changes are currently being
studied in our laboratory.
ACKNOWLEDGMENTS
The authors gratefully acknowledge the
secretarial help of Alma Haddock. This work
was supported by grant HD 06899 from the
National Institute of Child Health and Human Development, United States Public
Health Service.
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