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The vascular pattern of the renal glomerulus as revealed by plastic reconstruction from serial sections.

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THE VASCULAR PBTTERX
O F T H E RENAL GLOMERULIJS A S REVEALED BY
PLASTIC RECONSTRUCTION PROX
SERIAL SECTIONS
CHARLES C. BOYER
Department of Anatomy, University of Alabamu Medical Center,
Birmingham, Alabama
SIX FIGURER
INTRODUCTION
The present n7ork was fostered by an attempt to elucidate
the nature and pattern of the intercapillary space and the
arrangement of the glomerular basement me8mbrane. It is
obvious that the foundation for such a study is a clear understanding of the gross pattern of the vascular bed in the renal
unit. As conflicting opinions regarding this basic information
accumulated in recent literature, after a simple concept had
been generally accepted for over a century, it appeared desirable to re-investigate the glomerular pattern using more
modern techniques of reconstruction.
The term “rete mirabile” was used by Galen (Skinner,
’49) in describing the network of vessels about the base of
the brain and is generally purported to have been applied by
Malpighii to the vascular complex of the renal “glanduli.”
It does not appear, however, in his “De Renibus” (1686),
which nonetheless clearly describes a branching and anastomosing of vessels. McGregor ( ’29) has given an excellent
review of the literature on the structure of the renal glomerulus which therefore will not be repeated here. Suffice
it to say that, without frank disagreement, the details of the
pattern in the description given by Malpighii were largely
433
434
CHARLES C. ROYER
ignored by Bowman (1812), Vi’mtrup ( ’as),Zimmerman ( ’ 3 3 ) ,
MeManus ( ’48a), and many others intervening, all of whom
described the glomerulus as being characterized by a system
of capillary loops connecting afferent and efferent vessels,
with no anastomoses between loops. During this long period
dissenters were few. Johnston (18%) claimed numerous anastomoses, which claim was categorically denied by Vimtrup
( ’28). Trabucco and Marquez ( ’52) describe the glomerular
tuft as being formed by the evagination of one wall of an
arteriole, broken up into a digitated arrangement ending in
blind sacculations - “never in looping formations. ” Mueller
et al. ( ’55),although using the term “loops” throughout their
description, refer in their summary, perhaps inadvertently, to
the “capillary rete.”
With this as background, the present project of re-examination of the glomerular pattern by serial reconstruction was
undertaken.
TECHNIQUE AND PROCEDURE
The technique employed was essentially that previously
reported (Boyer, ’48) as applied to the study of embryonic
structure. Reconstructions attempted using routine autopsy
material proved completely unsatisfactory. Other attempts,
using animal kidneys or human kidney obtained at early autopsy and perfused with saline rendered better results but
left much to be desired. Good reconstructions of the renal
glomerulus can only be made from the freshest obtainttblc
kidneys. Our best results were obtained by perfusing whole
kidneys with saline, using gravity flow at a pressure not exceeding 100mm Hg. Animal kidneys were perfused in situ.
As soon as the emerging perfusion appeared completely clear,
filtered, diluted India ink was substituted for the saline at the
same pressure. Rather quickly the entire kidney becomes
black. The renal artery and vein were then tied and the kidney
fixed in Bouin’s solution, After blocking and embedding, 2 p
serial sections were cut in a refrigerated room (35°F.).
VASCULAR P A T T E R N
- RENAL
GLOMERULUS
435
The mounted sections, stained with a modification of McManus’ (’48b) PAS technique, present a very clear pattern
of the capillary bed filled with India ink with the glomerular
basement membrane well defined (fig. 1). Camera lucida
drawings of all sections of a particular glomerulus were made
at approximately 1000 x. The outline of the glomerulus and
the capillary pattern were then traced onto wax plates, then
the wax cut away with a short needle, producing plates as
Fig. 1 Section through glomerulus perfused with India ink and stained with
PAS. In any section glomeruli can be found running the gamut from completely
free of ink to completely full. Only those completely filled (or very nearly so)
are suitable for accurate reronstruction. X 450.
shown in figure 2. These plates were then assembled in order,
all edges, both internal and external, being fused with a hot
needle as each plate was added, to the preceding. When all
plates have been assembled the result is a complex mold of
the glomerulus with two openings, representing the afferent
and eff crent vessels.
Into this mold is then poured a liquid plastic. Ward’s
“ B i o p l a ~ t i cor
~ any comparable product works well. Trapped
436
C€L44RLES C. BOYER
air is removed in a vacuum chamber and additional plastic
added until the mold is full. After hardening of the plastic,
which is expedited by keeping the preparation in an oven at
a temperature just below the melting point of the wax mixture used for about 48 hours, the mold is removed by raising
the temperature to slightly above the melting point of the
wax. Excessive heat is to be avoided at this stage since 1213
Fig. 2 Sample wax plates t o which camera lucida drawings of scctioiis of
glomeruli similar to that showu in figure 4 have been transferred. CapillarT
lumina have been cut away. When a n entire series has been prepared in this
fashion and stacked in order a n accurate mold of the glomerulus is complete.
plastic is still malleable. When the plastic cast is fully exposed final removal of the residual wax film is accomplished
by immersion in xylol. The preparation may be kept here indefinitely or may be dried at room temperature.
The resultant preparation, shown in figure 6, is an accurate,
permanent duplication of the capillary pattern of the glomerlus. Because of the irregularity of its surface with its mass
of light reflecting facets, it is best studied immersed in a fluid
medium. Water or xylol may be used with good results. Be-
VASCULAR P A T T E R R - R E N A L GLOMERULUS
43’7
cause of the optical properties mentioned above, photography
of the plastic is difficult. Therefore a description of the technique successfully employed here appears warranted.
F o r the preparation of figure 6 we are indebted to Mr. J.
E. Mineo, Chief of the Medical Illustration Section, VA Hospital, Eirminghani. The picture was made in total darkness
using a fine grain panatomic X film in a view camera set at
f.11 and at 1: 1 magnification. The shutter was set for a 7
second exposure. During this exposure period the object was
literally “painted” with a moving beam of ultra-violet light
supplied by a Wood’s “ Black-Light. ” Spectacular color
transparencies were also made using the same technique of
illumination but using Ectachrome daylight film and 15 seconds exposure.
DISCUSSION
The capillary pattern diagrammed by Virntrup ( ’28) and
shown in figure 3 might be considered to embody the classical
concept of glomerular structure. A system such as this has
been described or tacitly accepted by virtually all workers in
the field of renal histology since Bowman (1842), who, followiiig injection of the vessels, described {he renal tuft as
being composed of a series of capillary loops held together
by their own interlacement. Most of the worlrers since that
time have been concerned with finer details of histology of
the organ. It is apparent that scant attention was paid to
the vascular pattern, a s such, during these years. Of the few
whose studies were specifically directed toward the capillary
arrange.nient, Johnston (1899) described numerous anastomosee, - this based on reconstructions from serial sections;
Vimtrup (’28) categorically denied the existence of anastomoses, or indeed, any deviation from the loop pattern, -this
based on injection and maceration studies ; Trabucco and
Rlarquez (’52) found no loops but instead, the unique arrangement of sacculations shown i n figure 5. Their interpretation
was based on the result of studies employing injection, corrosion, and serial sections with and without ink injection.
438
CIIAELES C. BOYER
No reconstructions were made and corrosion preparations
were compressed and teased for study. Hall ( '55a) finds numerous anastomoses, rarely loops, and this based on study of
serial sections in conjunction with latex injected, macerated,
dissected and compressed glomeruli.
It is interesting to note that among those employing careful study of serial sections and reconstructions there has been
general agreement on the absence of loops and existence of anastomoses. Conversely, results obtained by maceration, dissection, compression, corrosion, o r any other technique involving serious disturbance of the normal glomerulus, have
Fig. 3 Classical concept of glonierular pattern as described by Eowmaii and
generally accepted since 1842. (Redrawn a f t e r Vimtrup.)
Fig. 4 Glomerular pattern (schematic) as revealed by reconstruction froin
s e r i d sections, showing niultiple anastomoses arid n o siiriple loops. This is R
true rete, comparable in pattern t o capillary beds found everywhere i n tho body.
Fig. 5 Concept of glomerulsr pattern propounded by Trabucco axid Marquez
showing no loops, b u t instead a series of interdigitated sacculations. (Redrawn
from Trabucco and Marquez.)
been the subject of continuing controversy. One cannot but
recognize the fragility of the glomerular capillary net. Excessive pressure in injection, compression under a cover glass
for examination, or other manipulation, should be strongly
suspected of disrupting tlie norgmal arrangement. Thin serial
sections, coupled with careful reconstruction, continue to offer
the soundest basis for the elucidation of any complex morphological, microscopic pattern. This technique, as described,
shows very clearly the absence of simple capillary loops and
VASCULAR PATTERN - RENAL GLOlMERULUS
439
the presence of multiple anastomoses. Indeed, as our preliminary results had indicated (Boyer, ’3),
the capillary bed
in this site is a true rete (fig. 4), essentially no different
in pattern from capillary nets found elsewhere in the body
except for its adaptation to its location. This same view
has been arrived at by Hall (’55b) with whose general concept of glomerular pattern we are in complete agreement,
except that our preparations do not show the lobulations described by him. Sections through a glomerulus show apparently clear-cut lobuIes. Reconstructions from these sections,
however, show anastomotic vessels bridging these gaps so
that it is not possible to define any areas completely separable
down to the vascular pole. It is suggested that the lobulation
described by Hall may be an artifact resulting from uncontrolled injection pressure or the procedure he describes as
follows :
. . So, w e renaoved an individual latex injected glomerulus from the kidney, dissected off Bowman’s capsule, separated, u n f o l d e d , and arranged the lobules f o r maximum visibility, and then flattened the preparation with a cover glass
for photography . . .” (italics mine).
((.
We a r e however, in full support of the position that the classical concept of capillary loops as the basis of the glomerular
pattern is no longer tenable.
SUMMARY
1. A technique is described in detail for the preparation of
reconstructions in plastic from serial sections. The method
is especially useful in the study of complex vascular o r other
tubular patterns. It has been applied to the renal glomerulus.
Its application to the study of embryonic structure has been
previously reported.
2. The glomerulus is found to be composed of a capillary
network, comparable to all other capillary beds in the body,
adapted to the spatial arrangement of its unique location.
440
CHARLES C. BOYER
3. The classical concept of capillary loops without anastomoses between loops or between limbs of the same loop has no
foundation in fact and must be abandoned.
4. Clarification of the gross morphology of the glomerulus
now makes possible a sounder study of the arrangement of the
basement membrane and other fine details of glomerular histology.
L I T E R A T U R E CITED
BOWMAN,
W. 1842 IV. On the structure and use of the malpighian bodies of
the kidney with observations on the circulation through t h a t gland.
Phil. Tr. Roy. Soc., London, I : 57-80.
BOYER,C. C. 1948 The use of plastic in reconstructions from serial sections.
Anat. Rec., 100: 191-200.
1955 Plastic reconstruction of the human renal glomerulus from
serial sections. Anat. Rec., 121: 427 (abstract).
HALL, B. V. 1955a The organization of the renal glomerulus into independent
lobular systems of intercommunicating anastomosing capillaries. Anat.
Rec., 282: 433 (abstract).
195513 Further studies of the normal structure of the renal glomerulus. Proc. 6th Ann. Conf. on Nephrotic Syndrome (1954) pp. 1-40.
JOHNSTON,
W. B. 1899 Reconstruction of a glomerulus of the human kidney.
Anat. Anz., Ed. 16.
MCGREGOR,
L. 1929 The finer histology of the normal glomerulus. Am. J.
Path., 5: 545-558.
MCMANUS, J. F. A. 1948a Structure of the glomerulus of the human kidney.
Am. J. Path., 2 4 : 1259-1269.
194813 The periodic acid routine applied to t h e kidney. Am. J.
Path., 2 4 : 643-654.
MALPIQHII,MARCELLI 1686 De Renibus. Opera Omnia. IAonclon,pp. 87-100.
MUELLER,C. B., A. D. M ~ S O NJR.
,
A N D D. G. S T O U T 1955 Anatomy of the
glomerulus. Am. J. Med., 2 8 : 267-276.
SKINNER,
H. A. 1949 The origin of medical terms. The Williams and U’ilkins
Co., Baltimore, 1’111
379.
TRABUCCO,
A., AND F. MARQPEZ 1952 Structure of the gloinerulnr tuft. J.
Urol., 67: 235-255.
VIMTRUP,B. J. 1928 On the number, shape, structure, and surface area of the
glomeruli in the kidneys of man and mammals. Am. J. Anat., 41:
123-151.
ZINMERNAN,K. W. 7933 Glomerular structure in mammals. Ztschr. f. 1nic.r.anat. Forsch., 32: 176-278.
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