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Studies of the hemopoietic microenvironment. VIII andrenergic and cholinergic innervation of the murine spleen

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Studies of the Hemopoietic Microenvironment
Department of Anatomy, College of Medicine; Uniaersity of Cincinnati,
Cinci?i~iuti,Ohio 45267
Neurohistochemical techniques were used to confirm rnorphologically the distribution of adrenergic and cholinergic nerves to the splenic
microvasculature. The results form the basis of this report.
Using these methods, adrenergic innervation was observed only in the adventitia of arteries and arterioles. No cholinergic innervation was found in this
site. N o adrenergic or cholinergic innervation could be demonstrated to the
channels of the red pulp, venules or veins. These data provided morphological
evidence that in the murine spleen only splenic arteries and arterioles are innervated; and these have only an adrenergic innervation.
The murine spleen is a hemopoietic organ which unlike bone marrow, is easily
accesible for in vivo microscopic study of
the microvascular dynamics associated
with erythropoiesis. Recently, it was reported by McCuskey et al. ('72a,b) and
McCuskey and Meineke ( ' 7 3 ) that erythropoietin responsive stem cells may be the
source of a vasoactive substance or metabolite which locally regulates blood flow
through the red pulp of the erythropoietic
murine spleen. As part of an attempt to
characterize the mechanism of action of
this substance, in vivo microscopic and
nerve stimulation studies were conducted
to identify the pharmacologic receptors and
innervation in the splenic microvasculature. These studies suggested the presence
of adrenergic receptors and innervation
only in arterioles; cholinergic receptors appeared to be sparse, also restricted to arterioles, and not innervated (Reilly and
McCuskey, '74, '76a,b). As a result of
these studies, neurohistochemical techniques were used to confirm morphologically this distribution of adrenergic and
cholinergic nerves to the splenic microvasculature, and to identify the neurotransmitter in the adrenergic nerves. The results form the basis of this report.
ANAT. REC.,185: 109-118.
Falck-Hillnrp newohistochemical
Twenty-four CF, mice were anesthetized
with either urethane ( 2 . 5 mg/g) or PentobarbitalE (0.03 mg/g), and the spleens
and adrenals removed and subjected to
the fluorescence method of Falck and Hillarp (Falck and Owman, '65) to determine
the distribution of adrenergic nerves. The
sodium borohydride reduction method of
Corrodi et al. ('64) was performed on the
excised organs of four mice; and nine animals were pretreated with reserpine ( 2 . 5
mgikg) for periods of either 18 or 36
hours before sacrifice. Whole organs were
frozen in isopentane within a metal cup
suspended in liquid nitrogen which was
stored in a Dewar flask. Then they were
freeze-dried in a Virtis tissue drier, treated
with paraformaldehyde (equilibrated previously with sulfuric acid at 80.5% relative
humidity) at 80°C for 2-3 hours and embedded in paraffin. Sections 10 thick were
mounted on slides in Entellan (Scientific
Received Sept. 19, '75. Accepted Nov. 28, '75.
Supported in part by NIH AM-10507 and the S . W.
Ohio Heart Association.
2 Recipient
NIH Research Career Development
Award, AM-42370.
Products) and examined using a Zeiss
Photornicroscope equipped with an Osram
HBO-200 mercury light source, a BG-12
excitation filter, and a barrier filter which
excluded light below 500 nm. Optical
images were recorded on Kodak High
Speed Ektachrome (EHB) film pushed to
an ASA of 200. Black and white prints of
these later photographs are included to illustrate the presence and distribution of
catecholamine-specific and auto-fluorescence in the spleen. In these prints, the
apple-green, catecholamine-specific fluorescence appears bright white.
Acetylcholiiiestcrase nerirohistochemiccil technique
Eight female, CF, mice were euthanized
by cervical dislocation and portions of the
spleen and skeletal muscle excised and
subjected to the cholinesterase method of
Karnovsky and Roots ('64), as modified
by El-Badawi and Schenk ('67) to determine the distribution of cholinergic nerves.
The pieces of tissue were frozen in isopentane in a metal cup which was submerged
in liquid nitrogen, and stored in plastic
bags in a freezer at -20°C. Frozen sections were cut 10 thick using a cryostate
(-2O"C), mounted on slides, and rapidly
dried at room temperature (18-22°C) with
a current of air for three minutes. After
fixation of the tissue in 4'2, formalin; the
sections were incubated for 1, 1.5, 2 , 2.5
and 4 hours with and without the substrate
acetylthiocholine iodide (Sigma Chemical
Company). In all incubations, tetraisopropylpyrophosphoramide ( Sigma Chemical Company) was used to inhibit pseudocholinesterase (butylcholinesterase). After
incubation, the sections were examined
using a Zeiss photomicroscope equipped
with phase contrast optics, and photographed on Kodak High Speed Ektachrome
(EHB) film. Black and white prints of
these later photographs were reproduced
and included to illustrate the sites of acetylcholinesterase in the spleen. In these prints,
the enzyme-specific, reddish-brown precipitate appears as a finely granular black
Atlreneryic innervation
Using the fluorescence method to demonstrate catecholamine, adrenergic inner-
vation throughout the spleen was found to
be sparse. Only arteries, and arterioles of
the hilum, trabeculae and red and white
pulps were found to be innervated (figs.
1, 2 ) . The fluorescence produced in the
adventitia of these vessels was an applegreen color (bright white on figures).
which suggested that either norepinephrine
and.'or dopamine was the neurotransmitter. Most of these fluorescent fibers had a
yellow-beaded appearance (bright white on
figures), which was characteristic of regions of nerves where high concentrations of neurotransmitter were present. No
nerves could be demonstrated to be associated with terminal arterioles in the red
Of the remaining vasculature in the
spleen, the hilar, trabecular and splenic
red pulp veins and venules, and the channels in the red pulp were found not to
have fluorescent structures about them
(fig. 4 ) . Furthermore, avascular trabeculae
and the splenic capsule (fig. 2 ) also lacked
fluorescing nerves and, thus, adrenergic
Before and after treatment with paraformaldehyde there were areas of green
and yellow-green autofluorescence in the
spleen (bright white in figures). This background fluorescence was due to the presence of elastic tissue and was most noticeable in the internal elastic lamina and adventitia of arteries and arterioles (fig. l ) ,
in the splenic white pulp (fig. 2 ) , and in
the capsule (fig. 2 ) . Brightly fluorescent
yellow bodies (white in figures) which have
been described previously in the cat (Fillenz. '70) and dog (Dahlstrom and Zetterstrom, '65) also were present in the red
pulp of the murine spleen (fig. 3), and to
a lesser extent in the white pulp. To distinguish between catecholamine-specific
fluorescence and auto-fluorescence, the sodium borohydride technique (Corrodi, '64)
and the depletion of stored catecholamine
resulting from reserpine treatment was
utilized. Autofluorescence in all regions of
the spleen was resistant to the reduction
and depletion techniques. The fluorescence
which was reduced by the technique and
which was specific for catecholamine was
reestablished by reexposure of the reduced
tissue sections to paraformaldehyde. Reserpine pretreatment of the animals caused
a decrease in the concentration and in-
tensity of the apple-green fluorescent product due to catecholamine after 18 hours.
and complete depletion of this fluorescence
after 36 hours.
Sections of adrenal glands which were
used as controls also were positive for the
catecholamine, epinephrine. The fluorescent product was of a yellow-green color
and was localized in the chromaffin cells
of the adrenal medulla. This catecholamine
specific fluorescence was labile to both sodium borohydride reduction and reserpine
depletion while the autofluorescence in the
cells of the adrenal cortex was not.
Cholinergic innervation
Cholinergic nerve fibers within the
spleen of the mouse were not found with
the cholinesterase method of El-Badawi
and Schenk (’67) (figs. 5 , 6, 7). Megakaryocytes, erythrocytes, and sequestered
platelets in the red pulp of the spleen were
found to the enzyme positive (figs. 5 , 6. 7 )
and a positive reaction for acetylcholinesterase was seen occasionally in the
white pulp.
Skeletal muscle fibers of the external
abdominal muscles of the mouse also were
found to be enzyme deficient. Large motor
end plates which were associated with individual muscle fibers were acetylcholinesterase positive. All sites of acetylcholinesterase activity were marked by a sharply
defined granular reddish-brown precipitate
(finely granular black precipitate in
figures ) .
Tissue sections of both skeletal muscle
and spleen which were incubated without
the substrate, acetylthiocholine iodide, were
not marked by precipitation.
The recent interest in the mouse spleen
as a model for studying erythropoiesis, and
the reports of McCuskey et al. (’72a,b)
and McCuskey and Meineke ( ’ 7 3 ) that
erythropoietin responsive stem cells may
be the source of a vasoactive substance or
metabolite that locally regulates blood flow
through the red pulp of the erythropoietic
murine spleen. has stimulated an effort to
define the mechanism through which functional or active hyperemia may occur in
this organ. Before attempting to characterize the mechanism of action of this substance, in vivo microscopic studies were
conducted to identify the receptors and
innervation in the microvasculature of the
spleen, since these were poorly understood.
In the present study. neurohistochemical
techniques were used to determine the
distribution of the innervation in the spleen
and the identity of the neurotransmitters.
Prior pharmacologic and nerve stimulation studies demonstrated the presence of
7 and p-adrenergic receptors and of sympathetic innervation in arterioles and “arterial” capillaries. ( Reilly and McCuskey .
‘74. ’76a,b). The fluorescence technique
confirmed the presence of adrenergic innervation of the arterioles in the red and
white pulp of the spleen. In addition, arteries in the splenic white pulp, hilum and
trabeculae also were innervated. The fluorescent product in these nerves was applegreen in color, which is characteristic of
the neurotransmitter, norepinephrine and/
or dopamine. These results were consistent
with our previous pharmacologic and
physiologic data in the mouse. and with
neurohistochemical data in the cat (Fillenz, ‘70) and the dog (Dahlstr6m and
Zetterstrom, ’65).
Topical administration of norepinephrine
in vivo was found to cause dose dependent
arteriolar constriction and decreases in the
linear velocity of blood flow within these
vessels. Furthermore. electrical stimulation
of the nerves on the splenic artery near
the hilum resulted in constriction of only
arteries, arterioles, and “arterial” capillaries. This response could be abolished by
prior blockade of 9-receptors with phentolamine (Reilly and McCuskey, ’74, ’76a,b),
Veins, venules. the channels in the red
pulp, trabeculae. and the splenic capsule
lacked specific fluorescence. This was consistent with previous pharmacologic and
neurophysiologic data which suggested that
innervation was absent in these sites
(Reilly and McCuskey, ’74, ’76a,b). In contrast to the mouse, the smooth muscle of
avascular trabeculae and splenic capsule
of dog (Dahlstrom and Zetterstrom, ’65)
and cat (Fillenz. ’70) are innervated. In
these species, this direct innervation has
been suggested to be responsible for the
rapid onset of splenic contraction following sympathetic nerve stimulation and during the hypovolemic period of hemorrhagic
Green to yellow-green autofluorescence
was present in all sections of the spleen. advice, assistance and critical evaluation
This background fluorescence, due to of the neurohistochemical methods, and
elastic connective tissue, was most notice- Gladys Barton and Nancy McNeal for their
able in the capsule and internal elastic technical assistance.
lamina of arteries and arterioles in locaLITERATURE CITED
tions other than the splenic red pulp. The
fluorescence was distinguished easily from Corrodi, H., N. Hillarp and G. Jonsson 1964
Fluorescence methods for the histochemical
the catecholamine specific fluorescence
demonstration of monoamines. 111. Sodium
since it was resistant to sodium borohyborohydride reduction of the fluorescent conipounds as a specific test. J. Histochem. Cytodride reduction (Corrodi et al., '64), to
chem., 12: 582-586.
depletion by reserpine, and was present in
A. B., and B. E. M. Zetterstrom 1965
tissue sections treated with and without Dahlstrom,
Noradrenaline stores in nerve terminals of the
spleen: Changes during hemorrhagic shock.
Science, 147: 1583-1585.
The modified acetylcholinesterase technique (El-Badawi and Schenk, '67) re- El-Badawi, A., and E. A. Schenk 1967 Histochemical methods for separate, consecutive and
vealed a lack of this enzyme in association
simultaneous demonstration of acetylcholinwith the vascular system of the mouse
esterase and norepinephrine i n cryostate section. J. Histochem. Cytochem., 15: 580-588.
spleen. This observation was in agreement
with the previous pharmacologic and phys- Falck, B., and C. Owman 1965 A detailed methodological description of the fluorescence
iologic data which suggested the presence
method for the cellular demonstration of bioof sparse cholinergic receptors in arterioles
genic monoamines. Acta Univ. Lund, 2 : 5-23.
of the spleen (Reilly and McCuskey, '74, Fillenz, M. 1970 The innervation of the cat
spleen. Proc. Roy. SOC.(London) B, 174: 459'76a). When stimulated, these receptors
caused vasoconstriction, were pilocarpine Karnovsky,
M. J., and L. Roots 1964 A "directsensitive, could be antagonized with atrocoloring" thiocholine method for cholinesterpine, and were not innervated. The paraases. J. Histochem. Cytochem., 12: 219-221.
sympathomimetic agonist carbachol ad- Koelle, G. B. 1951 The elimination of enzymatic
diffusion artifacts in the histochemical localiministered both topically and systemically
zation of cholinesterase and a survey of their
elicited no vascular response. Electrical
cellular distributions, J. Pharm. Exp. Therap.,
stimulation of nerves present on the splenic
103: 153-171.
artery concomitant with 3-blockade did McCuskey, R. S . , and H. A. Meineke 1973 Studies of the hemopoietic microenvironment. 111.
not elicit microvascular constriction. The
Differences in the splenic microvascular system
splenic trabeculae and capsule also lacked
and stroma between SI/Sl" and W / W anemic
aceylcholinesterase positive fibers, a findmice. Am. J. Anat., 137: 187-198.
ing consistent with these previous obser- McCuskey, R. S . , 11. A . Meineke and S . M. Kaplan
197213 Studies of the hemopoietic microenvirvations.
onment. 11. Effect of erythropoietin on the
There was a complete absence of acetylsplenic microvasculature of polycythemic CF1
cholinesterase activity associated with the
mice. Blood, 39: 809-813.
splenic vasculature after prolonged incu- McCuskey, R. S., 13. A. Meineke and S . F. Townsend 1972a Studies of the hemopoietic microbation with substrate. Since both Koelle
environment. I. Changes i n the microvascular
('51) and SchIaepfer and Torack ('67)
system and stroma during erythrocyte regenerhave reported specific acetylcholinesterase
ation and suppression in the spleen of CF1
mice. Blood, 39: 697-712.
activity in low concentrations in sensory
F. D., and R. S. McCuskey 1974 Regulaneurons of the dorsal root and nodosal gan- Reilly,
tory mechanisms in the splenic microvascular
glion, it is suggested that there may be an
system of mice. Fed. Proc., 33: 393.
absence of general visceral afferent fibers
1976a Studies of hemopoietic microenvironment. VI. Humoral regulatory mechanisms
in the murine spleen. This lack of specific
in the splenic microvascular system of mice.
cholinesterase in association with splenic
Microvas. Res., in press.
vascular and capsular smooth muscle was
197613 Studies of hemopoietic microenconsistent with observations in the cat
vironment. VII. Neural mechanisms i n microvascular regulation in the spleens of mice.
( Fillenz, '70 ) ,
The authors wish to thank Drs. James
L. Hall and Carol Brownscheidle for their
Microvas. Res., i n press.
Schlaepfer, W. W., and R. M. Torack 1967 The
ultrastructura1 localization of cholinesterase activity i n the peripheral nervous system of rats.
J. Neuropath. Exp. Neural., 26: 137.
1 Fluorescing adrenergic nerves (arrows) i n the adventitia of a hilar
artery ( A ) of the mouse spleen. Note the autofluorescence of the
internal elastic lamina ( I ) of this vessel. Falck-Hillarp. x 160.
Fluorescing adrenergic nerves (arrows) in the red (R) and white ( W )
pulp of the mouse spleen. Note the absence of nerves adjacent to the
splenic capsule ( C ) which is autofluorescent. Falck-Hillarp. x 160.
Fluorescing adrenergic nerve (arrows) i n the red pulp ( R ) of the
mouse spleen. Note the beaded appearance ( b ) of this catecholamine
containing fiber, and the fluorescent bodies ( B ) distributed randomly
i n this region. Falck-Hillarp. x 400.
Venule ( V ) and channels ( C ) of the red pulp. Note the absence of
fluorescent nerve fibers adjacent to these structures, and the presence
of a fluorescing adrenergic nerve (arrow) in the red pulp. FalekHillarp. x 160.
F. D. Reilly. R . S . McCuskey and H. A . Meineke
Capsule (C), trabeculae (T),and red pulp ( R ) of the mouse spleen.
Note the presence of acetylcholinesterase-positive megakaryocytes ( M )
i n the red pulp and the absence of enzyme-specific nerve fibers adjacent to the splenic capsule and the trabeculae. Acetylcholinesterase.
x 160.
A hilar artery ( A ) of the mouse spleen. Note the presence of acetylcholinesterase containing megakaryocytes ( M ) and the absence of
nerve fibers containing this enzyme in the adventitia of this vessel.
Acetylcholinesterase. x 160.
7 Central arteriole ( A ) and red pulp venules (V) of the mouse spleen.
Note the presence of a n acetylcholinesterase containing megakaryocyte ( M ) in the red pulp and the absence of enzyme containing nerve
fibers adjacent to these microvessels. Acetylcholinesterase. x 160.
F. D. Reilly, R . S. McCuskey and H. A. Meineke
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andrenergic, spleen, murine, microenvironment, hemopoietic, studies, innervation, viii, cholinergic
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