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Three large dissectable rat glomerular models reconstructed from wide-field electron micrographs.

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THE ANATOMICAL RECORD 196:431-440 1980)
Three Large Dissectable Rat Glomerular Models
Reconstructed from Wide-Field Electron Micrographs
GRACE C. H. YANG AND ASHTON B. MORRISON
Department of Pathology, Rutgers Medical School, College of Medicine and
Dentistry of New Jersey, Piscataway, New Jersey 08854
ABSTRACT
Three very large Styrofoam models of glomerular capillary tufts
have been built, based on serial ultrathin sections which cut through three complete rat glomeruli isolated from all regions of renal cortex. The images of
glomeruli are enlarged on 2 0 x 24" wide-field electron micrographs, from which
each capillary is traced on Styrofoam discs and then individually connected. These
models can be disassembled to measure the width and length of each capillary
segment and to map the complicated glomerular vascular arrangement. Each of
the three randomly selected glomeruli has three arteriolar openings passing
through the Bowman's capsulw.g., an afferent opening and two closely situated
efferent openings. Each of the three glomeruli consists of three major lobules, e.g.,
two larger lateral lobules and a smaller central lobule. Assuming the model is
positioned so that the vascular pole is superior and the afferent arteriole is closer to
the observer than the two efferent vessels, the lobule occupying the frontal right
portion originates from the right major branching of the afferent arteriole and
reconverges a t the right efferent opening. The lobule occupying the frontal left
portion originates from the left major branching of the afferent arteriole and
reconverges a t the left efferent opening. The lobule occupying the central portion
originates from the central major branching and connects both lateral lobules with
a few interlobular anastomoses. The diameter and the length of interlobular
anastomoses, and the volume of major lobules are listed. The technique of the
isolation and reembedment of glomeruli and the construction of models is described
in detail.
In seeking to study the arrangement of vascular arrangement are as follows: 1) the
glomerular capillary tufts, a few models have glomerulus consists of a n anastomosing plexus
been built; those include 1) a plastic model re- of capillaries, the lobulation is a technical arconstructed from serial light microscopic sec- tifact (Boyer, '56);2) the glomerular capillaries
tions of 2 pm thickness (Boyer, '56).This model form a complicated network divided into
adequately shows the outside of the vascular lobules which communicate with each other by
arrangement, but it cannot be taken apart to a few interbridging capillaries (Murakami et
reveal the internal structure because of the ri- al., '71).
The objective of the present work is to take
gidity of the plastic. 2) An ironwire model from
serial light microscopic sections of 0.5 pm advantage of the newly developed technique of
thickness (Aeikens et al., '77). This model wide-field electron microscopy (Yang and Morshows a network of wires without portraying rison, '75) to build glomerular models which are
large enough to keep track of the smallest capilthe actual shapes of the capillary.
Murakami et al. ('71) have shown scanning lary, accessible for the measurement and mapelectron micrographs of stretched corrosion ping of the complicated capillary network, and
casts of r a t glomeruli. Excellent as these finally to observe the vascular arrangement of
stretched corrosion casts are in showing the the glomerulus.
lobular arrangement, they are too small to be
measured or mapped.
Received June 5, 1979, accepted September 18, 1979.
Other early studies on the glomerular vascular arrangements include Johnston, 1899;
Address Reprint Requests to Grace C.H. Yang, M.S. Dept of AnatVimtrup, '28; Wilmer, '41; Trabucco and Mar- omy, CMDNJ-Rutgers Medical School, Piscataway, N.Y. 08854.
A.B. Morrison, M.D., Ph.D., is Dean and Vice President for Academic
quez, '52; Hall, '55;Zlabek, '57; and Lewis, '58. Affairs,
Eastern Virginia Medical School, P.O. Box 1980. Norfolk, Va.
23501.
The more recent views on the glomerular
000-3276X/80/1964-0431$02.000 1980 ALAN R. LISS. INC.
43 1
432
GRACE C.H. YANG AND ASHTON B. MORRISON
MATERIALS AND METHODS
A kidney of a normal male Sprague-Dawley
rat, weighing 191g was perfused via abdominal
aorta with 1%glutaraldehyde and 1.5%tannic
acid in 0.1 M phosphate buffer (pH 7.2). The
cortex was cut into 1.5 mm thick slices, which
were then postosmicated and then stained en
bloc with WOuranyl acetate in maleate buffer
(pH 6) a t 60°C overnight. After rinsing in
maleate buffer (pH 5.4) twice, the tissue was
dehydrated in graded ethanols and embedded
in Epon-Araldite at the flat end of a BEEM
capsule.
The isolation and reembedment of glomeruli
After polymerization, the cortical tissue was
cut half way with a razor blade. The tissue was
then fractured at the cut. Consequently, the
glomeruli encapsulated in Bowman's capsules
were exposed (Fig. la). One end of a finepointed tweezers was used to pick out the exposed glomeruli which were then separated
from the rest of the tissue a t their vascular
poles without the macula densa. Several
glomeruli were thus isolated from all regions of
the cortex and collected in a BEEM capsule
(Fig. lb). A sharpened applicator stick, dipped
in fresh Epon-Araldite mixture, was used to
draw a thin line on the flat cap of another
BEEM capsule, where four randomly picked
glomeruli were then aligned. The fresh Epon in
the BEEM capsule was polymerized before it
was filled with more resin mixture to avoid
disturbing the linear arrangement of the
glomeruli. Finally, a block containing four
glomeruli in a straight line was obtained. It
was then trimmed into a thin block face and
was ready to be sectioned (Fig. Id). The above
procedures were done under a dissecting microscope.
The preparation of serial sections
Every effort was made to reduce the tremendous workload to a more manageable level. Two
steps were taken to avoid the staining ofmasses
of grids and to eliminate the danger of section
breakage during staining. First, kidney was
stained en bloc with uranyl acetate prior to
embedment. Secondly, ultrathin sections
thicker than usual were used. They are of dark
gold interference color a t a n estimated thickness of 100 nm (Yang and Shea, '75). Therefore,
once the sections were mounted on slot grids
coated with plain 0.5% Formvar, they could be
photographed immediately without additional
treatment. The serial sections were made with
a diamond knife mounted on a LKB Ultratome
111. The entire process of sectioning took five
days, during which time it was necessary to use
extreme caution to avoid damaging either the
diamond knife or the block face. The entire
apparatus was left overnight on the ultramicrotome to minimize handling and to avoid
shifting cutting planes which would result in
the discontinuity of glomerular images. The
block face was trimmed several times while
still mounted on the ultramicrotome, thus reducing the number of grids carrying the serial
sections. Consequently, the necessity for
changing grids in and out of the electron microscopic column was reduced to a minimum. The
whole glomerular images were recorded by a
Philips 300 electron microscope at x 420 using
half mask, so that each negative could record
two whole glomerular images.
The selection of negatives
Though serial sections were cut, it was found
to be unnecessary to record every section. Each
negative was compared to the adjacent negative under a dissecting microscope which was
placed on top of a narrow illuminated viewer.
Only negatives showing the changes in the
capillary lumina were chosen, so that it was
possible t o delete unnecessary films and yet
still follow the pathways of every capillary.
Fewer negatives near the top or bottom portions of the glomeruli were selected, and more
negatives were used from the central portion
where small capillaries appeared more frequently and were more tortuous in their
courses.
Nearly 150 films were chosen for each
glomerulus. Hundreds of 2 0 x 2 4 wide-field
electron micrographs were then obtained
(Yang and Morrison, '75). The final magnification was x 3,909.
The height of the model was compensated for
the unselected sections as follows: first, the
theoretical height of the glomerulus was obtained by multiplying the section thickness by
the total number of sections occupying the entire glomerulus; then the theoretical height
was divided by the number of selected sections;
in this manner, the use of Styrofoam discs of 3
mm thickness was determined and custom ordered from the manufacturer.
The construction of the models
Each print was given a section number in the
order of its sequence. The outlines of all the
capillary lumina were traced on 2 0 x 24"
transparent gel sheets (photographic supply
store) with a marking pen. A micrograph with
LARGE DISSECTABLE GLOMERULAR MODELS
433
Fig. 1. The isolation, reembedment, and sectioning of glomeruli. a) A piece of osmicated tissue fractured from an Epon
block, exposing a glomerulus (arrow). b) Several osmicated and plasticized glomeruli isolated from tissue. c) Four glomeruli
aligned in a row. d) The trimmed block face ready to be sectioned. e) Serial sections showing 32 images of glomeruli.
0 Close-up view of the serial sections showing glomeruli encapsulated in Bowman’s capsule.
the most complicated capillary lumina from the
center part of the glomerulus was chosen as the
standard for orientation. First, it was traced on
a gel sheet, then given a center axis on which
subsequent tracings were aligned. This was
necessary because the glomerular images on
the micrographs were not printed at the exact
same angles. A ball pen was used to press down
the outlines of all the capillary lumina from the
gel sheet onto styrofoam sheets of 3 mm thick-
ness (Tekniplex, Inc. Somerville, N.J.).
Styrofoam discs of various shapes were then cut
using a n X-act0 knife, and labeled with the
section number and a n arrow showing the
orientation. The discs were attached to the
matching outlines on the gel sheets by pieces of
double-stick tape. Using the centering arrow on
the gel sheet as a guide, the discs were removed
from the gel sheet, pasted in the correct position
with Elmer’s glue, and reinforced by small pins.
434
GRACE C.H. YANG AND ASHTON B. MORRISON
The micrographs were checked frequently to
ensure the accuracy of various connections. The
interlobular anastomoses were determined by
gently pulling the lobules away from each other
to locate the connecting points. These were
then separated and fastened with Velcro tapes
(fabric store). This enabled the models to be
pulled apart to reveal the internal details and
reconnected to show the entire capillary tuft.
Each of the major lobules was weighed, and the
volume was calculated from the density of the
Styrofoam. Only three glomerular models were
built, because one glomerulus was damaged at
its vascular pole.
Though the pieces of the model were glued
together, the models could be easily dissected
because they were made of Styrofoam discs,
which did not absorb the glue as thoroughly as
discs made of wood or other material. The
thicker capillary segments, e.g., the primary
afferent branchings, were dissected by a
jeweler’s saw. Other segments were dissected
by simply inserting a knife between the discs
and moving it up and down until the discs separated. The dried glue could be easily peeled
away and the pins could be removed by forceps.
RESULTS
Each of the three glomerular models has
three arteriolar openings passing through the
Bowman’scapsule verified on the original electron micrographs (Fig. 5)-e.g., an afferent
opening (identified by the J G granules on its
arteriolar wall), and two closely situated efferent openings (Figs. 2a, 3a, 4a). Each of the
three models can be easily separated into three
lobules-two larger lateral lobules and a
smaller central lobule (Figs. 2b, 3b, 4b). The
lobule occupying the frontal right portion originates from the right major afferent branching
and reconverges a t the right efferent opening.
The lobule occupying the frontal left portion
originates from the left major afferent branching and reconverges a t the left efferent opening. The lobule occupying the central portion
originates from the central major branching as
a separate unit, but later connects both lateral
lobules with a few interlobular anastomoses.
No direct connection is observed between two
lateral lobules, although they are next to each
other in the frontal plane with their own efferent arteriolar openings situated close together
as a pair. Each lobule consists of a very complicated anastomosing network of capillaries, and
no “blind-end” was found. The classically described simple capillary “loops”were not found
in any of the three glomerular models.
The diameter of the capillaries ranges from
about 2 pm to 22 pm; most of them measure
between 5 pm to 10 pm. The course of the pathways is quite tortuous. A capillary turns a t
sharp angles in short distances and isjoined by
other capillaries at various points. It was found
that the diameter of a capillary may vary a
great deal within a segment. Therefore, it could
be said that a single red blood cell must travel
extensively before it passes through the maze
of the capillary network.
The diameter of three openings, the diameters of the major branchings of the three openings, the length and diameter of each interlobular anastomosis, and the volume of the capillary lumina of the major lobules are illustrated
in diagrams showing simplified versions of the
glomerular capillary tufts (Figs. 5,6).
Model #2 has been completely dissected to
measure and map the complex capillary network. The data have been mathematically
analyzed and their physiological significance
have been discussed (Shea, ’79).
DISCUSSION
In all three models, the glomerulus divides
into three major lobules, with the center
smaller lobule acting as the bridge between the
two larger lateral lobules. Our findings are
somewhat similar to Murakami et al. (’71).
However, all three models show double efferent
openings extending out of Bowman’s capsule,
whereas only 11out of 1,200rat glomeruli have
true double efferent arterioles (Murakami et al.
’71). This discrepancy may be due to a difference in technique. In their corrosion cast
method, the Bowman’s capsule is corroded
away and the location of its borderline cannot
be determined. With our isolation technique,
the glomeruli are severed from their vascular
poles. By combining observations from these
two different approaches, it is possible that
after the two exiting arterioles pass through
the Bowman’s capsule, they join together to
become the single efferent arteriole found in
most of the rat glomeruli in Murakami’s corrosion study. Of course, it is also possible that
those two exiting arterioles are true double efferent arterioles.
The isolation and reembedment of the
glomeruli is a crucial step in obtaining its complete ultrastructural images. Serial tissue sections are only adequate in building glomerular
models based on light microscopic images. The
semithin “survey” tissue section may locate
many glomeruli, but only after the top parts of
their capillary tuft are removed. In order to be
LARGE DISSECTABLE GLOMERULAR MODELS
435
Fig. 2. Glomerular model X1. a) Frontal view showing an afferent arteriolar opening (A) and a pair of efferent openings
(E);b) Dorsal view showingthree lobules:two larger lateral lobules, a right (R) and a left (L), and a smaller central lobule (C).
(Reduction: x 4.5).
436
GRACE C.H. YANG AND ASHTON B. MORRISON
Fig. 3. Glomerular model #2. a) Frontal view showingthree openings:two (E) and one (A). b) Dorsal view showing three
lobules: right (R), center (C), and left (L).
LARGE DISSECTABLE GLOMERULAR MODELS
437
Fig. 4. Glomerular model X3. a) Frontal view showing three openings:two (E)and one (A).b) Dorsal view showing three
lobules: right (R),center 0,and left (L).
438
GRACE C.H. YANG AND ASHTON B. MORRISON
Fig. 5. Two wide-field electron micrographs chosen from glomerulus 6 3 . a) Seetion X128 showing af€erent opening (A);
b) Section X331 showing double efferent openings (ELBowman’s capsules are indicated by arrows.
439
LARGE DISSECTABLE GLOMERULAR MODELS
GLOMERULUSSl
Center to Left
Anastomoses
Diam.
9.9pm
7.4
6.6
10.7
6.6
6.1
8.2
Center to Right
Ana stornoses
Lensth
11.5pm
17.9
21.7
9
9
1 7.9
14
Diam.
6.1p
93
86
Length
35.8p.m
64
16.9
GLOMERULUS S 2
9.6pn
9.6
5.9
2
9
6.6
1 1 . 3 ~ 1~m
4.9
9.7
10.1
38.6
20.7~
20
9
6.1
39.1
1
GLOMERULUSB 3
14.9
6 . 9 ~ 7 14.lpm
16.6
7.4
@
; .6
I5.5P
xl$ .47
1.69
x ~
~ 5
None
Volume
1 0.9
10.9~
Fig. 6. Three simplified and stretched glomerular diagrams on which the following measurements are listed: the
volume of left (L),right (R),and center (C)lobules; the diametersof afferent openings (El, and their major hranchings; and
the diameters and the lengths of all the interlobular anastomoses.
440
GRACE C.H. YANG AND ASHTON B. MORRISON
recorded, an ultrathin section has to be supported on a grid where the largest opening a t
present is only 1 mm x 2 mm. Of course, a
complete glomerulus might be encountered by
very long serial sectioning. However, such a
probability on a section of lmm x 2 mm
through a tissue slice of 1.5 mm thickness is,
indeed, very low, not to mention the wear-andtear on the diamond knife by aimless sectioning.
The efficiency of sectioning is further improved by embedding three glomeruli in a row;
by placing the glomeruli close to the cutting
surface, and by lengthwise cross sectioning associated with oval glomeruli being placed on a
flat surface.
Styrofoam is an ideal material in building
complicatedmodels. It has very low density and
therefore can be built into a complex network
without falling apart a t weak points. The
finished models have a capacity for bouncing,
i.e., a part of the lobule can be pushed away and
it will bounce back. This is very helpful in locating the interlobular anastomoses. Styrofoam
sheets are also cheaper and easier to work with
than wood or other material.
In Boyer’s model, the lobulation of the capillary tuft is indistinct, perhaps because the
adhesion of the neighboring lobules is unavoidable since liquid plastic is poured into stacks of
wax plates with many holes representing capillary profiles. In our glomerular models, the pattern of lobulation is quite distinct because there
is no random adhesion of the neighboring
lobules, even though they might be touching
each other in the finished model. If the connection cannot be verified on the micrographs, no
pin or glue is applied.
The reason why the parietal layer of the
Bowman’s capsule stays attached to the
glomeruli in all cases using this technique (Fig.
5, Fig. lc) is that it has been reinforced by the
polymerized Epon. When the fracture forces the
plasticized cortex apart, the surrounding
tubules separate, leaving the intact renal corpuscle exposed, The mapping of capillary network can be done with much assurance. Not
only each connection has been checked on the
original electron micrographs, but also the
model is so large that even the smallest capillary of 2 pm in width has a diameter of 7.8 mm
in our model.
Though the construction of these models took
very much time and effort (from January, 1975
to June, 19761, it nevertheless led to a better
understanding of the vascular arrangement of
the rat glomerulus, and it is the only way at
present to measure and map the complex network of the glomerulus.
ACKNOWLEDGMENTS
It is a pleasure to thank Dr. Stephen Shea for
suggestions and support during the work. The
authors also wish to thank Miss Eloise Ten
Eyck for her artistic help in the construction of
models, and Miss Robin Paulmeno for her most
diligent work in printing and tracing of hundreds of micrographs.
This study was supported by U.S. Public
Health Service grant No. AM-12495 (Principal
Investigator: Dr. Shea) and CMDNJ-Rutgers
Medical School General Research grant No.
9642.
LITERATURE CITED
Aeikens, B., A. Eenboom, W. Plate, L. Fronhold, and A.
Bohle (1977)The construction of models intending spatial
representation of complex vessel structures demonstrated
by means of the mammalian glomerulus. (Ger.) Microsc.
Acta, 79: 12CL-126.
Boyer, C.C. (1956) The vascular pattern of the renal
glomerulus as revealed by plastic reconstructions from
serial sections. Anat. Rec., 125:43%441.
Elias, H. (1957) De structura glomeruli renalis. Anat. Am.,
104:26-36.
Johnston, W.B. (1899 A reconstruction of the human kidney. Anat. Am., Bd. 16.
Hall, B.V. (1955) The organization of the renal glomerulus
into independent lobular systems of intercommunicating
anastomosing capillaries. Anat. Rec., 121,433 (Abstr.).
Lewis, O.J. (1958) The vascular arrangement of the mammalian renal glomerulus as revealed by a study of its
development. J. Anat. (Lond.), 92,433.
Murakami, T., M. Miyoshi, and T. Fujita (1971) Glomerular
vessels of the rat kidney with special reference to double
efferent arterioles. A scanning electron microscope study
of corrosion casts. Arch. Histol. Jap., 33: 179-198.
Shea, S.M. (1979) Glomerular hemodynamics and vascular
structure. The pattern and dimensions of a single rat
glomerular capillary network reconstructed from ultrathin section. Microvasc. Res., 17 (In press).
Trabucco, A,, and F. Marquez (1952) Structure of the
glomerular 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;12%151.
Wilmer, H.A. (1941)The arrangement ofthe capillary tuft of
the human glomerulus. Anat. Rec., 80:507.
Yang, G.C.H., and A.B. Morrison (1975) Wide-field electron
microscopy. A rapid method for the study of histologic
material that provides a bridge between light and electron
microscopy. Am. J. Clin. Pathol., 64:648-654.
Yang, G.C.H., and S.M. Shea (1975) The precise measurement of the thickness of ultrathin sections by a “resectioned section” technique. J. Microsc., 103:385392.
Yang, G.C.H., A.B. Morrison, and S.M. Shea (1976)Three’ dimensional models of the glomerulus. XI Congress of the
International Academy of Pathology. Washington, D.C.,
October 17-23 (Exhibit).
Yang, G.C.H., and A.B. Morrison (1977) The application of
wide-field electron microscopy.Microsc. Acta, 79:277-279.
Zlabek, K. (1957) The arrangement of the intraglomerular
blood vessels in the human kidney. Rev. Szech. Med. 111,
41:l-12.
Zlabek, K. (1973)Uber dunne intercapillare anastomosen in
nierenglomerulus der rat. Acta. Anat. (Basel), 85: 177189.
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