Three-Dimensional Aspects of the Structural Characteristics

and Kidney Angioarchitecture of Adult and Aged Wistar Rats:

A Scanning Electron Microscopy Study
CRISTINA DE SOUSA BOLINA,
1
REGINA DE SOUSA BOLINA-MATOS,
1
PAULO HENRIQUE DE MATOS ALVES,
1
DIEGO PULZATO CURY,
1
ADRIANO POLICAN CIENA,
1,2
* AND II-SEI WATANABE
1,2
1
Department of Surgery, Faculty of Veterinary Medicine and Animal Science, University of S~ ao Paulo, S~ ao Paulo, Brazil
2
Department of Anatomy, Institute of Biomedical Sciences-ICB III, University of S~ ao Paulo, S~ ao Paulo, Brazil
KEY WORDS electron microscopy methods; angioarchitecture; vascular corrosion casts;
MercoxVR
; cryofracture; renal morphology
ABSTRACT The aim of this study was to describe the structural morphological characteristics
and organization of the kidney angioarchitecture in adult and aged Wistar rats using different scan-
ning electron microscopy (SEM) methods. Twenty male Wistar rats (Rattus norvegicus) were divided
into two groups: the Adult Group (n510), which included animals at 3 months of age, and the Aged
Group (n510), which included animals at 24 months of age. Kidney samples from both groups were
analyzed using three distinct SEM methods: coronal sections, cryofracturing and vascular corrosion
casts/angioarchitecture. This study demonstrates the specicity of each technique for three-dimen-
sional morphological analyses of the kidney using SEM. In the coronal sections, it was possible to
view the structures on a given plane; the stratigraphy was best observed in the cryofracture method,
and the vascular corrosion casts showed the organization and arrangement of the angioarchitecture.
Thus, we concluded that the SEM methods together provide the best description of the morphologi-
cal characteristics of normal and pathological kidneys of adult and aged Wistar rats. Microsc. Res.
Tech. 76:538544, 2013. VC
2013 Wiley Periodicals, Inc.
INTRODUCTION
Biological materials have been widely studied using
scanning electron microscopy (SEM) methods to reveal
the structural aspects of surfaces in three-dimensions
(Joy, 1997).
Conventional SEM techniques visualize a structure
of interest, identify and describe adjacent structures.
The fundamental characteristic of identifying struc-
tures in relation to neighboring structure, called syn-
topy, allows for a description of vascularization relative
to organs and systems. The descriptions make it possi-
ble to perform relationship, ratio and arrangement
analyses of blood vessels with their adjacent structures
and target organ (Kronka et al., 2000).
Macroscopic descriptions of blood vessels can also be
obtained by combining various preparation techniques
such as vinylite injection, which can be used to achieve
a desired magnitude of visualization (Chen et al., 2010).
The analysis of the vascular arrangement, speci-
cally the microcirculation, by injecting resin (MercoxVR
)
permits the path and organization of blood vessels to be
visualized according to their different diameters and
densities, collateral circulation, terminal branches, and
anastomoses (Makiyama et al., 1998; Motoyama and
Watanabe, 2001; Kronka et al., 2006; Verli et al., 2008).
These aspects can illustrate morphological and/or
organizational changes that reect functionally rele-
vant changes that can be used for diagnosis and treat-
ment (Walocha et al., 2012).
The aim of this study was to describe the morpholog-
ical and structural organization of the kidney
angioarchitecture in adult and aged Wistar rats using
different SEM methods.
MATERIAL AND METHODS
Twenty male Wistar rats (Rattus norvegicus) were
divided into two groups: the Adult Group (n510),
which included animals at 3 months of age, and the
Aged Group (n510), which included animals at 24
months of age. The rats were provided by the animal
facility at the Institute of Biomedical Sciences, Univer-
sity of S~ ao Paulo-USP. The animals were housed in
polypropylene cages under a 12-h light/dark cycle at
room temperature (24 62

C) and were provided food

and water ad libitum. All the procedures were
approved by and were based on the Ethical Principles
of Animal Experimentation adopted by the Brazilian
Society of Animal Laboratory Science and by the
Ethics Committee on Animal Experiments at the Insti-
tute of Biomedical Sciences, USP.
*Correspondence to: Adriano Polican Ciena, Department of Surgery, Faculty
of Veterinary Medicine and Animal Science. University of S~ ao Paulo, S~ ao Paulo,
Brazil. E-mail: [email protected]
Received 24 January 2013; accepted in revised form 4 February 2013
Contract grant sponsor: School of Veterinary Medicine and Animal Science-
University of S~ ao Paulo.
DOI 10.1002/jemt.22197
Published online 28 February 2013 in Wiley Online Library
(wileyonlinelibrary.com).
V VC 2013 WILEY PERIODICALS, INC.
MICROSCOPY RESEARCH AND TECHNIQUE 76:538544 (2013)
SEM
Six animals from both groups were anesthetized in-
traperitoneally with ketamine (50 mg/kg) and xylazine
(10 mg/kg) perfused with a modied Karnovsky solu-
tion (2.5% glutaraldehyde and 2% paraformaldehyde
in a 0.1 M sodium phosphate buffer solution, pH 7.4)
according to the methodology reported by Watanabe
and Yamada (1983). The right and left kidneys were
removed and placed in the xative solution for 48 h at
4

C. After this period, half of the samples were sec-

tioned in the coronal plane and the remaining samples
were longitudinally cryofractured by freezing in liquid
nitrogen (2196

C). All the samples were postxed in

an aqueous 1% osmium tetroxide solution for 2 h at
4

C, washed with distilled water, and immersed in a

1% tannic acid solution for 1 h (Murakami, 1973;
Watanabe et al., 1995). The coronally sectioned and
cryofractured kidney samples were dehydrated in
increasing alcohol series (70% to absolute) and dried in
a critical-point drying apparatus (Balzers CPD-020)
using liquid CO
2
.
To study the kidney angioarchitecture, four animals
were used from both groups for the vascular corrosion
cast technique. Following the anesthesia as described
above, the animals were perfused with a heparinized
saline solution (30 mL) to inhibit blood clotting followed
by a saline solution (20 mL) to completely ush the vas-
cular bed. The samples were then ushed with a modi-
ed Karnovsky solution to x the vascular bed and
prevent resin leakage and avoid alterations in the mor-
phology patterns of the endothelial cells (Manelli et al.,
2007). Acrylic resin (10 mL of MercoxVR
CI-2 Red) con-
sisting of 25% methyl methacrylate monomer and 0.25
g of catalyst (benzoyl peroxide) was manually injected
via a cannula into the thoracic aorta until venous reux
was observed through the right atrium and the viscos-
ity of the resin increased. The cannula was removed
and ligatures were immediately inserted into the tho-
racic and abdominal aorta on both ends with a suture
thread (Catgut 4.0 chrome) to prevent backow of the
resin. The animals were maintained at room tempera-
ture, and after 1 h, both kidneys were removed and im-
mediately immersed in hot water (35

C) for 1 h to
complete the polymerization process. The polymerized
samples were immersed in aqueous 15% sodium hy-
droxide (NaOH) solution replaced daily for 6 days
(Ohtani, 1987; Kronka et al., 2000) for corrosion casting
of the tissue structures adjacent to the blood vessels.
Finally, the vascular casts were rinsed in distilled water
and dried at room temperature for 3 days.
The samples pertaining to the different methods
(coronally sectioned, cryofractured and vascular corro-
sion casts/angioarchitecture) were mounted on metal
stubs and were sputter coated with gold ions in an EMI-
TECH-K550 device (Duro et al., 2012). The mounted
samples were examined using a Leo 435 scanning elec-
tron microscope (Department of Surgery, FMVZ-USP).
RESULTS
SEMCoronally Sectioned
In the Adult Group, the outer layer surrounding the
organ and bundles of dense connective tissue forming
the brous renal capsule demonstrated a close relation-
ship with the cortex region (Fig. 1A). In the inner layer,
the medullary region was evident with emphasis on the
renal papilla followed by the renal pelvis (Fig. 1B).
At a greater magnication, the characteristics of the
transition region between the cortex, which is consti-
tuted by its condensed aspect, and the medulla, which
is distinguished by the longitudinal disposition of blood
vessels, were observed along with lymphatic vessels
and collecting ducts along the axis of the renal pyramid
(Fig. 1C). In the cortical region, the arrangement of the
glomerulus was observed. The proximal convoluted
tubules (PCT) and distal convoluted tubules (DCT)
were distributed around the capsular space (Fig. 1D).
In the Aged Group, there was a clear difference in
the tissue density in the cortical region compared with
the medullary region and the renal pelvis was located
medially (Fig. 1E). A qualitatively greater increase
was observed in a large quantity of renal corpuscles
and glomeruli distributed throughout the cortex (Figs.
1F and 1H). The transition between the medullary
region and the inner surface of the renal pelvis was
evident in the coronal section (Fig. 1G). Highlighted,
in the cortex, the glomeruli and the renal corpuscles
were present (Fig. 1H).
SEMCryofracture
In the Adult Group, the renal papilla was evident in
the medullary region, with an emphasis on the struc-
tures forming the papilla, which includes both the
lumen of blood vessels and the collecting ducts (Fig.
2A). The orice, longitudinal trajectory, and varying
diameters of the blood vessels were visible in the cortex
and medulla regions (Fig. 2B). An analysis of the corti-
cal region showed the arrangement of the PCT and
DCT. The disposition of the parietal and visceral layers
of the glomerular capsule were observed. The capsular
space and lumen of the glomerular capillaries were also
apparent (Fig. 2C). The podocytes and the primary
extensions together with the ltration barrier were no-
table by observing the glomerulus alone (Fig. 2D).
In the Aged Group, the general shape of the kidney
was observed, consisting of the glomeruli in the corti-
cal region and the spaces containing both the cortical
and juxtamedullary glomerular locations. The trajecto-
ries of different blood vessels in the cortical and medul-
lary regions were evident along with the renal papilla,
which was directed toward the renal pelvis and the
inner surface (Fig. 2E). Upon analyzing the internal
surface of the renal pelvis, tissue morphological
changes were observed on the epithelium lining the in-
terior of the renal pelvis (Fig. 2F).
In the cortical region, two glomeruli were observed
with parietal and visceral capsules and respective cap-
sular spaces. The location where the glomerulus was
accommodated was observed lateral to the glomeruli
due to its removal (Fig. 2G). In this region, the lumen
of the capillaries and the adjacent PCT and DCT were
observed in the glomerular cross-section (Fig. 2H).
SEMVascular Corrosion Casts
(Angioarchitecture)
In the Adult Group, the distribution and organization
of the vascular microcirculation in the renal regions
(cortical and medullary) were evident in three-dimen-
sions. A terminal branch of the renal artery was
A SCANNING ELECTRON MICROSCOPY OF THE KIDNEY 539
Microscopy Research and Technique
Fig. 1. Scanning Electron Photomicrographs - Coronally sectioned.
Adult Group: (A) Shows the renal brous capsule (arrow) in a close
relationship with the cortex (*). Scale bar: 1 mm. B: Shows the medul-
lary region (M) and the renal pelvis (P) is highlighted. Scale bar: 300
lm. C: At increased magnication, the transition between the cortex
(*) and medulla (M) is observed. Scale bar: 200 lm. D: In the cortex,
the glomerulus (G) and the proximal (arrow) and distal (arrow head)
convoluted tubules can be observed. Scale bar: 30 lm. Aged Group:
(E) The stratigraphy between the cortex (C), medulla (M) and renal
pelvis (P) can be observed. Scale bar: 1 mm. F: At increased magni-
cation, note the renal corpuscles (arrows) distributed in the cortex.
Scale bar: 300 lm. G: Note the longitudinal arrangement of the con-
ductive tubules (*) and the epithelial surface of the renal papilla
(arrow). Scale bar: 30 lm. H: In the cortex, the glomeruli (G) and re-
nal corpuscles (C) can be observed. Scale bar: 100 lm.
540 C. DE SOUSA BOLINA ETAL.
Microscopy Research and Technique
Fig. 2. Scanning electron photomicrographcryofracture. Adult
Group: (A) The technique reveal numerous orices in the lumen of
blood vessels and collecting ducts (arrow). Scale bar: 300 lm. B: In
the cortex (C) and medulla (M), longitudinal arrangement of the
arteries (arrow head) and veins (arrow) is observed. Scale bar: 300
lm. C: Featured in the cortex, note the arrangement of the proximal
(arrow) and distal (arrow head) convoluted tubules and the glomeru-
lus with a partial removal of the capsule (G). Scale bar: 100 lm. D: At
an increased magnication of a glomerulus, the podocytes (large
arrow) and primary extensions (smaller arrow) are observed. Scale
bar: 100 lm. Aged Group: (E) The general aspects of the renal stratig-
raphy show the glomeruli (arrows) and the arrangement of the blood
vessels (arrow heads). Scale bar: 30 lm. F: In the renal pelvis (P),
morphological changes with the tissue projections are observed (*).
Scale bar: 300 lm. G: In the renal cortex, the glomeruli (G) and a par-
tial view of the capsule (arrows) are present. Scale bar: 30 lm. H: At
a higher magnication, the sectioned glomerulus (G) and arrange-
ment of the proximal (arrow) and distal (arrow head) convoluted
tubules are observed. Scale bar: 30 lm.
observed in the renal sinus. The capillaries were
arranged to form a vast network in the direction of the
cortex and medullary region (Fig. 3A). An extensive
network of capillaries with several branches was noted
on the dorsal surface of the cortex (Fig. 3B). The juxta-
medullary region revealed trajectory and parallel orga-
nization of the blood vessels and glomerular capillaries
(Fig. 3C). Glomeruli and a dense vascular network con-
taining numerous capillaries were evident on the dorsal
surface of the cortical region (Fig. 3 D). The interlobar
arteries, afferent, and efferent glomerular arterioles
were observed (Fig. 3E). The entire network of capilla-
ries and adjacent arterioles were visualized by isolating
a single glomerulus at a higher magnication (Fig. 3F).
In the Aged Group, the renal angioarchitecture dem-
onstrated numerous capillaries organized and distrib-
uted throughout the medullary region and dense
capillaries in the cortical region (Fig. 4A). The capilla-
ries were distributed in networks with numerous
branches on the dorsal surface of the cortical region
(Fig. 4B). In the cortical region, an isolated glomerulus
was observed with capillaries distributed along the adja-
cent blood vessels (Fig. 4C). The analyses performed at a
lower magnication indicated a qualitatively higher
number of glomeruli. Blood vessels with different diame-
ters were observed surrounding the glomeruli. In con-
strast, small orices were observed, which suggested
intussusceptive forms of angiogenesis; however, a
Fig. 3. Scanning electron photomicrograph of the adult groupvas-
cular corrosion casts (angioarchitecture). A: The distribution and or-
ganization of the microvasculature (arrows) and the terminal branch
of a renal artery (*) are observed. Scale bar: 1 mm. B: On the dorsal
surface of the cortex, there is an extensive network of capillaries
(arrows). Scale bar: 100 lm. C: The juxtamedullary region shows the
trajectory and longitudinal organization of the blood vessels (arrows)
and the glomerular capillaries (*). Scale bar: 100 lm. D: In the cortex
region, the dense vascular network (arrows) and the glomeruli (*) are
observed. Scale bar: 100 lm. E: The interlobar artery (arrow), the
afferent arterioles (arrow heads) and the glomeruli (G) are clearly
visible. Scale bar: 30 lm. F: At a higher magnication, the dense glo-
merular capillary network (*) is observed. Scale bar: 30 lm.
542 C. DE SOUSA BOLINA ETAL.
Microscopy Research and Technique
similar appearance can produced by the incomplete ll-
ing of the resin in the vascular bed (Fig. 4D).
In the cortical region, the distribution of glomeruli
was heterogeneous, and some were located close to-
gether (Fig. 4E). At a higher magnication, the side
view of the glomerulus showed details of the distribu-
tion and arrangement of the capillaries (Fig. 4F).
DISCUSSION
The results of the present study showed the three-
dimensional (3D) aspects of the structural and organi-
zational characteristics of the renal angioarchitecture
in the cortical and medullary regions, changes in the
epithelial layer on the inner surface of the renal pelvis
of adult and aged rats.
In the analyses performed using SEM, the structural
characteristics of cortical and medullary regions of the
kidney revealed that the PCT and DCT were
distributed and organized adjacent to the glomeruli.
Glomeruli consist of an intricate network of blood capil-
laries typically covered by podocytes, which act as sup-
port processes, as described by Yu et al. (1998). A study
by Nyergaard and Bendtsen (1992) found that the num-
ber and volume of glomeruli decline with age; however,
Fig. 4. Scanning electron photomicrograph of the aged groupvas-
cular corrosion casts (angioarchitecture). A: General view of the
arrangement of the dense network of capillaries in the cortex (*) and
the longitudinal arrangement in the medulla (arrows). Scale bar: 1
mm. B: On the dorsal surface of the cortex, there is a wide distribu-
tion of capillaries (arrows) in the form of a network. Scale bar: 100
lm. C: The distribution of capillaries (arrows) in the cortical region
adjacent to the glomerular capillaries (*) is observed. Scale bar: 100
lm (D) At an increased magnication of the glomeruli, blood vessels
(arrows) with variable diameters and, highlighted, orices corre-
sponding to intussusceptive forms of angiogenesis can be clearly
observed (arrow head). Scale bar: 100 lm (E) The glomeruli (G)
arranged in close proximity can be observed. Scale bar: 30 lm (F) At
an increased magnication, the entanglement of the glomerular
capillaries (*) is observed. Scale bar: 30 lm.
A SCANNING ELECTRON MICROSCOPY OF THE KIDNEY 543
Microscopy Research and Technique
the aim of our study was to describe the characteristics
of various SEM techniques in evaluating renal morphol-
ogy instead of conducting quantitative analyses.
Podocytes, which are highly differentiated epithelial
cells, were attached to the glomerular basement mem-
brane (GBM) (Macconi et al., 2006). According to Appel
et al. (2009), in the postmitotic phase, podocytes do not
undergo complete cell division and are unable to regen-
erate with primary and secondary extensions.
The cryofracture technique was the most efcient
method to perform the glomeruli analyses and view
the capsules and glomerular space. The technique
revealed the thickness of the GBM, which is composed
of three layers: lamina rara externa, lamina densa,
and lamina rara interna. The last layer contains tubu-
lar microbrils, collagen bers and the long processes
of mesangial cells (Ojeda et al., 2003).
The vascular corrosion cast technique is indicated for
analyzing and describing the 3D organization of the
angioarchitecture (Giuv ar asteanu, 2007; Minnich and
Lametschwandtner, 2010). Several researchers have
used this technique on various types of tissues and
organs. Xu et al. (1994) studied the kidney microvascu-
lature in pigs. Naccarato et al. (1999) analyzed the
nasal septum of the Mongolian gerbil. Kronka et al.
(2000) and Silva et al. (2006) studied the palatal mucosa
of rabbits and gerbils, respectively. Kronka et al. (2001)
studied the palatal gingiva of young rabbits. Kronka
et al. (2006) studied the palatal mucosa of aged rabbits
subjected to an experimental model of diabetes.
Our data revealed that the capillaries organized
throughout the cortical and medullary regions of the
kidney form a dense network containing various types
of anastomoses. Although we were analyzing the capil-
lary networks, we noticed small openings in some en-
dothelial cell walls. The openings appeared to be
formed by intussusceptive forms of angiogenesis or
incomplete vascular lling of the resin, which have
also been reported by Wei et al., (2006) in a study on
autosomal dominant polycystic kidney disease.
This study demonstrates the specicity of each tech-
nique used for 3D morphological analyses of the kid-
ney using SEM. Using methods such as coronal
sections, it was possible to observe the structures in a
given plane. Cryofracture was the best method for vis-
ualizing the stratigraphy and the vascular corrosion
casts showed the organization and arrangement of the
angioarchitecture. We concluded that a combination of
these methods provide the best description of the nor-
mal and pathological morphological characteristics of
the kidney in adult and aged Wistar rats.
ACKNOWLEDGEMENTS
The authors like to thank the Rose Eli Grassi Rici of
the Electron Microscopy Laboratory, for technical help
in capturing the images.
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