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The fine structure of the vasa recta and associated nerves in the rabbit kidney.

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The Fine Structure of the Vasa Recta and
Associated Nerves in the Rabbit Kidney
JOHN A. GOSLING AND JOHN S. DIXON
Department of Anatomy, University of Manckester,
Munchester, 13, England
ABSTRACT
The fine structure of the vasa recta and associated nerves has been
studied i n the rabbit. Vasa recta were first identified in groups of two or three and
were accompanied by small groups of axons. A number of these axons contained
granulated, agranular and dense-cored vesicles. As the vasa recta subdivided, the
muscle coat consisted of a single Iayer of smooth muscle cells. Additional large axon
bundles were observed accompanying this segment of the vasa recta. Some of these
axons also contained granulated, agranular and dense-cored vesicles in varying proportions. Further towards the medulla, smooth muscle cells were replaced by others
which had irregular collections of filaments within their cytoplasm. These i n turn
were replaced by cells having a characteristic band of filaments on their luminal
aspects. Nerves were only identified adjacent to those parts o f the vasa recta which
included smooth muscle cells.
Factors which influence the diameters
of the vasa recta are of importance in the
control of renal medullary blood flow and
nerves related to those vessels are of possible significance. Techniques designed to
reveal sites of cholinesterase activity have
been successfully employed to demonstrate
nerves accompanying the vasa recta in
a number of species (Williams, '61;
McKenna and Angelakos, '68a; Gosling,
'69). Using electron microscopy, Moffat
('67) identified nerves adjacent to the
proximal segments of the vasa recta and
suggested a cholinergic role for them in
contrast to nerves which accompany other
intrarenal vessels. However, Doleiel ('67)
and McKenna and Angelakos ('68b) have
demonstrated monoaminergic nerves adjacent to the vasa recta which have a distribution cIosely resembling those observed
using cholinesterase techniques. It is now
generally accepted that morphological differences between cholinergic and adrenergic nerve terminals may be demonstrated
using electron microscopy (Hokfelt, '68).
Thus, in the present study of the vasa
recta, particular attention has been paid
to the associated nerves in an attempt to
correlate some of the observations of previous workers.
MATERIALS AND METHODS
The material used in this investigation
was obtained from nine adult rabbits of
ANAT. REC., 165: 503-514.
both sexes. Each animal wa.s anaesthetised
using intravenous sodium pentobarbital
and both kidneys were exposed. The renal
vessels were clamped and the kidneys divided from upper to lower pole. Icecold 1% osmium tetroxide buffered with
veronal acetate at pH 7.5 was dripped onto
the exposed surface for 2-3 minutes. A
wedge of tissue 1 cm in length and 1 mm
wide was excised at right angles to, and
including, the cortico-medullary region.
The tissue strip was placed in a small quantity of fresh fixative and subdivided into
ten approximately equal tissue blocks. During this procedure, care was taken to maintain the correct order of blocks obtained
from the original tissue strip. Each block
was fixed separately for one hour, dehydrated in ascending concentrations of
ethyl alcohol and embedded in Araldite.
Sections were cut on a LKB Ultramicrotome
using glass knives, mounted on uncoated
copper grids and double stained using alcoholic uranyl acetate and lead citrate
(Reynolds, '63). The sections were examined using a Philips EM 300 electron
microscope.
OBSERVATIONS
Injection studies of renal vasculature
have shown the descending vasa recta to
be arranged as a number of subdividing,
Received
M a y 1. '69. Accepted July 10, '69.
503
504
JOHN A. GOSLING A N D JOHN S. DIXON
closely related, vessels (Trueta et al., ’47;
Ljungqvist and Lagergren, ’62; Fourman
and Moffat, ’64). This characteristic arrangement has been used in the present investigation as the main criterion for their
identification. The following is a description of the vasa recta as they descend from
the cortico-medullary region towards the
medulla proper.
The descending vasa recta are first observed in groups of two or three, each having a diameter of 10-20 p (fig. 1 ) . Each
vessel has a relatively thick endothelium
which is separated from the smooth muscle
cells of the media by a basal lamina. An
internal elastic lamina is never observed.
The media consists of a single layer of
smooth muscle cells which often overlap
one another. The plasma membrane of
each cell is covered by a basal lamina except over small regions where adjacent
cells make intimate contact with each
other.
Each vessel is accompanied by one or
two small groups of non-myelinated axons
lying in the connective tissue between the
vessels (figs. 1, 2). Axons are never observed to penetrate between muscle cells.
Each group contains from one to six axons
partly or completely invested in Schwann
cell cytoplasm. These axon-Schwann cell
complexes are surrounded by a basal lamina (fig. 2). Frequently however, two or
three axons are observed in close contact
without intervening Schwann cell cytoplasm; these groups of axons may or may
not be covered by a basal lamina. Completely naked axons are occasionally observed lying at a distance of 700-1200A
from the outer surface of a smooth muscle
cell (fig. 3). In such instances the intervening gap always contains the basal lamina of the muscle cell.
The axons contain numerous membranebounded vesicles, small mitochondria and
neurotubles possessing central dense cores
when cut in cross-section. Mitochondria
are most often observed in those parts of
the axons where vesicles occur. The vesicles may be divided into three types depending on their fine structure. Some have
diameters of 300-500A and contain a
small dense inclusion of approximately
200 A diameter (fig. 3 ) which may be
spherical, oval or rod-shaped. This type
will be referred to as dense-cored vesicles.
Similar vesicles but without the dense inclusion will be referred to as agranular
vesicles. In addition, there are larger vesicles with diameters of 800-1000 A which
contain a spherical dense granule approximately 500 A in diameter (fig. 2). This type
will be referred to as granulated vesicles.
All three types of vesicle may be present
in a single axon but the agranular and
dense-cored vesicles are by far the most
common.
Further towards the medulla, the vasa
recta break up into groups of 3-5 vessels,
each accompanied by 2-4 nerve bundles
having chaicacteristics similar to those accompanying more proximal segments of
the vasa recta. In addition, larger nerve
bundles containing twenty or more tightlypacked axoins are occasionally encountered
in this region (fig. 4). The axons included
in these larger bundles contain granulated,
agranular and dense-cored vesicles in varying proportions.
In the vessels so far described the endothelium is non-fenestrated and consists of
a single layer of cells which bulge into the
lumen especially in the region of the nucleus (fig. 1). Many cells possess basal
processes which interlock with those of
adjacent cells, or project out towards the
underlying smooth muscle. The endothelial
basal lamina may be single or double but
always folllows the irregular contours of
the endothelial cell basal processes. Frequently the spaces between these basal
processes contain cytoplasmic projections
from the ulnderlying smooth muscle cells.
Some of the smooth muscle projections
possess a bulbous ending which lies in a
depression in the adjacent endothelial cell
without intervening basal lamina. These
correspond to the “peg and socket” junctions described by Moffat (’67). Very occasionally a close junction is observed between a srnooth muscle cell and an endothelial cell. Over this region a gap of
200 A separates the adjacent plasma membranes and the basal laminae of both the
endotheliall and smooth muscle cells are
discontinuous.
Vessels of a different character are frequently observed in close proximity to those
described above. These possess a thin
fenestrated endothelium and associated
FINE STRUCTURE O F VASA RECTA AND NERVES
basal lamina but are devoid of muscle
cells (fig. 5).
At deeper levels in the medulla, the muscular media of the vasa recta appears as
an incomplete layer of smooth muscle cells
surrounding each vessel (fig. 5 ) . The
smooth muscle cells are characterised by
large numbers of myofilaments and micropinocytotic vesicles which tend to accumulate at the plasma membrane.
Gradually the even distribution of filaments in the smooth muscle cells becomes
irregular. These cells are in turn replaced
by others containing a narrow band of filaments on their luminal aspect; numerous
micropinocytotic vesicles collect on the
opposite side of the cell (fig. 6). Cells of
this type have been named "perivascular
cells" by Moffat ('67). These cells occur
more and more frequently as deeper levels
of the vasa recta are examined. The numbers of axons accompanying the vasa recta
diminish markedly and none are detected
when all the cells associated with these
vessels are of the "perivascular" type.
DISCUSSION
Vasa recta are continuations of either
juxtamedullary efferent arterioles or aglomerular vessels (Trueta et al., '47; Ljungqvist
and Lagergren, '62; Fourman and Moffat,
'64 ). Because consecutive serial sections
of this region have not been prepared in
the present investigation, it has not been
possible to establish the origin of individual
vasa recta. When first identified, each vas
rectum is associated with nerves containing relatively few axons. Further towards
the medulla however, additional large axon
bundles (some containing more than 25
axons) can be identified running with the
vasa recta (fig. 4). Nerves, some of relatively large diameter, have been demonstrated passing directly from arcuate arteries to the vasa recta (Mitchell, '51;
McKenna and Angelakos, '68a; Gosling,
'69); the large axon bundles observed in
the present study could well be derived
from nerves accompanying adjacent major
vessels.
Agranular and large granulated vesicles
have been described in both adrenergic and
cholinergic nerve terminals (Grillo and
Palay, '62; Ishii et al., '65; Dixon, '66;
505
Grillo, '66). Small dense-cored vesicles of
approximately 500 A diameter are believed
to be specific to adrenergic nerve terminals;
they are reduced following administration
of drugs known to deplete tissue stores of
nonadrenalin (Bondereff, '65; Bloom and
Barrnett, '66; Hokfelt, '66; Farrell, '68).
In the present study similar dense-cored
vesicles observed in axons accompanying
the vasa recta suggest that a significant
number of these nerves are adrenergic
(fig. 2 ) . In contrast, Moffat ('67) postulated a cholinergic role for nerves running
alongside the vasa recta in the rat. He was
unable to demonstrate monoaminoxidase
jn these nerves, although he did observe a
few granulated vesicles with the electron
microscope. It is noteworthy that both
Doleiel ('67) and McKenna and Angelakos
('68b) have demonstrated monoaminergic
nerves close to canine vasa recta.
"Perivascular" cells have been identified
by Moffat ('67) surrounding the vasa recta
and, in support of this author's observations, the present study has failed to demonstrate nervous tissue in relation to similar cells in the rabbit. Nerves can only
be detected in regions where smooth muscle cells surround the vasa recta.
Both nerve-muscle relationships and the
vesicular content of axons related to vasa
recta have been compared with those of
muscular arterioles observed in the renal
cortex during the present study. These
observations together with those of other
workers (Lever and Esterhuizen, '61; Appenzeller, '64; Lever et al., '65; Simpson
and Devine, '66) indicate that the vasa
recta are innervated in a manner morphologically similar to other arterioles. In this
context, a possible vasoconstrictor role for
these nerves is worthy of speculation. Mild
renal nerve stimulation reduces cortical
blood flow and increases perfusion through
outer medullary peritubular capillaries
(Pomeranz et al., '68). Dilatation of the
vasa recta has been proposed to account
for these observations. As an alternative
explanation, constriction of the muscular
segments of the vasa recta during nerve
stimulation could result in diverting blood
through more proximal capillary branches
of the vasa recta and through accessory
juxtamedullary efferent capillaries.
506
JOHN A . G O S L I N G A ND JOHN S . D I X O N
ACKNOWLEDGMENTS
The authors wish to thank Professor
G. A. G. Mitchell for his encouragement
and advice and Miss E. White for her
skillful technical assistance.
LITERATURE CITED
Appenzeller, 0.
1964
Electron microscopic
study of the innervation of the auricular artery
of the rat. Am. J. Anat., 98: 87-91.
Bloom, F. E., and R. J. Barrnett 1966 Fine
structural localization of noradrenalin i n vesicles of autonomic nerve endings. Nature, London, 210: 599-601.
Bondareff, W. 1965 Submicroscopic morphology of granular vesicles i n sympathetic nerves
of rat pineal body. Z. Zellforsch., 67: 211-218.
Dixon. J. S. 1966 The fine structure of parasympathetic nerve cells in the otic ganglion of
the rabbit. Anat. Rec., 156: 239-252.
Doleiel, S. 1967 Monoaminergic innervation of
the kidney. Aorticorenal ganglion-a
sympathetic monoaminergic ganglion supplying the
renal vessels. Experientia, 23: 109-111.
Farrell, K. E. 1968 Fine structure of nerve
fibres i n smooth muscle of the vas deferens i n
normal and reserpinized rats. Nature, London,
21 7: 279-28 1.
Fourman, J., and D. B. Moffat 1964 Observations on the fine blood vessels of the kidney.
Symp. 2001. SOC.,Lond., 11: 57-71.
Gosling, J. A. 1969 Observations on the distribution of intrarenal nervous tissue. Anat Rec.,
163: 81-88.
Grillo, M. A. 1966 Electron Microscopy of sympathetic tissues. Pharmacol. Rev., 18: 387-399.
Grillo, M. A., and S. L. Palay 1962 Granulecontaining vesicles in the autonomic nervou3
system., V Internat. Cong. for Electron Microscopy, 2: u-1.
Hokfelt, T. 1966 The effect of reserpine o n the
intra-neuronal vesicles of the rat vas deferens.
Experientia, 22: 5G63.
1968 In vitro studies on central and peripheral monoamine neurones at the ultrastruc-
ture level. Z. Zellforsch. mikrosk. Anat., 91:
1-74.
Ishii, S., N. Shimizu, M. Matsuoka and R.
Imaizumi 1965 Correlation between catecholamine content and numbers of granulated vesicles in rabbit hypothalamus. Biochem. PharmaC O ~14:
. , 183-192.
Lever, J. D., M. Ahmed and G. Irvine 1965
Neuromuscular and intercellular relationships
i n the coronary arterioles. A morphological and
quantitativte study by light and electron microscopy. J. Anat., 99: 829-840.
Lever, J. D., and A. C. Esterhuizen 1961 Fine
structure of the arteriolar nerves in the guinea
pig pancreas. Nature, London, 192: 566567.
Ljungqvist, A . , and C. Lagergren 1962 Normal
intrarenal arterial pattern i n adult and aging
human kidney. J. Anat., 96: 285-300.
McKenna, 0. C., and E. T. Angelakos 1968a
Acetylcholinesterase containing nerve fibres in
the canine kidney. Circulation Res., 23: 645651.
19681, Adrenergic innervation of the
canine kidney. Circulation Res., 22: 345-354.
Mitchell, G. A. G. 1951 The intrinsic renal
nerves. Acia anat., 13: 1-15.
Moffat, D. B . 1967 The fine structure of the
blood vessels of the renal medulla with particular reference to the control of the medullary
circulation. J. Ultrastruct. Res., 19: 532-545.
Pomeranz, B. H., A. G. Birtch and A. C. Barger
1968 Neural control of intrarenal blood flow.
Am. J. Physiol., 215: 1067-1081.
Reynolds, E. S. 1963 The use of lead citrate as
a n electron opaque stain in electron microscopy.
J. Cell Biol., 17: 208-213.
Simpson, F. O., and C. E. Devine 1966 The fine
structure of autonomic neuromuscular contacts
i n arterioles of sheep renal cortex. J. Anat.,
100: 127-137.
Trueta, J., A. Barclay, P. M. Daniel, K. J. Franklin and M. M. L. Pritchard 1947 In: Studies
of the Renal Circulation. Blackwell Scientific
Publications, Oxford.
Williams, T. H. 1961 Some aspects of kidney
‘neurility’ as studied in the cat. I n : Cytology
of Nervous Tissue. Taylor and Francis, London.
Note added in proof: Recently Newstead and Munkacsi (’69) have obtained similar results using the rat ( 2 . Zellforsch., 97: 465-490).
PLATES
Abbreviations
A, thin-walled vessel
AV, agranular vesicle
AX, axon
DV, dense-cored vesicle
GV, granulated vesicle
N, nerve bundle
P, perivascular cell
SM, smooth muscle cell
VR, vas rectum
PLATE 1
EXPLANATION OF FIGURE
1
508
A survey micrograph of vasa recta, each surrounded by a single layer
of smooth muscle cells ( S M ) . The arrow indicates a small group of
axons. x 7,300.
FINE STRUCTURE OF VASA RECTA AND NERVES
John A. Gosling and John S. Dixon
PLATE 1
509
PLATE 2
EXPLANATION O F FIGURES
2
A small group of axons containing granulated (GV , agranular (AV)
and dense-cored vesicles (DV). X 41,000.
3
A naked axon (AX) lying close to a smooth muscle cell ( S M ) .
Arrows indicate dense-cored vesicles. X 56,000.
4 A large nerve bundle ( N ) containing twelve or more tightly packed
axons lies in close proximity to a vas rectum (VR) which is surrounded by an incomplete layer of smooth muscle (SM). X 20,500.
510
FINE STRUCTURE OF VASA RECTA AND NERVES
John A. Gosling and John S. Dixon
PLATE 2
511
PLATE 3
EXPLANATION OF FIGURES
5
Large gaps separate the individual smooth muscle cells ( S M ) surrounding a vas rectum ( V R ) . Note the thin fenestrated endothelium
of the vessel ( A ) towards the top of the field. X 28,000.
6
A “perivascular” cell ( P ) partially encircling a vas rectum ( V R ) . The
arrows indicate a characteristic band of filaments immediately beneath the plasma membrane. A thin-walled vessel ( A ) is also present.
x 18,200.
512
FINE STRUCTURE OF VASA RECTA AND NERVES
John A. Gosling and John S . Dixon
PLATE 3
513
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