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ORIGINAL ARTICLE

Morphology of the Non-Sensory Tissue Components in RatAging Vomeronasal OrganS. A. Eltony* and S. A. Elgayar

Address of authors: Department of Histology, Faculty of Medicine, Assiut University, Assiut 71515, Egypt

Introduction

The vomeronasal organ (VNO) is a chemosensory organ

that detects environmental pheromones and is essential

for mammalian reproduction activity (Keverne, 1999).

Pheromones are chemical signals between members of the

same species that convey behavioural or neuroendocrine

information (Smith et al., 2001). Odorants present in

adult life significantly impact animal behaviour later in

life in a manner analogous to the way that visual and

auditory stimuli via ‘imprinting’ influence later social

behaviours of some vertebrates (Doty, 2010). In addition,

the VNO and its primordium may play a role in brain

development. Hypothalamic neurons that produce gona-

dotropin-releasing hormone ‘GnRH-ir cells’ originate in

the VNO primordium (Takami, 2002).

The VNO is a bilateral simple tubular structure with a

crescent-shaped lumen (Vaccarezza et al., 1981). They are

located in the rostral region of the nose along each side

of the nasal septum and enclosed in a bony or cartilaginous

capsule (Wysocki, 1979). The duct vomeronasal duct

(VND) of the VNO is lined medially with sensory epithe-

lium (SE) and laterally with ‘non-sensory’ epithelium

(NSE) (Adams and Wiekamp, 1984; Farbman, 1992;

Williams et al., 1995). The soft tissue components of the

lamina propria of NSE include vomeronasal glands (VNGs),

nerves, vessels, smooth muscles and connective tissue. VNG

secretions are essential for the transport of pheromones

to chemoreceptors of the VNO (Mendoza, 1986).

The ‘non-sensory’ part of the VNO has received little

attention as it contains no receptors and does not partici-

pate directly in the sensory process (Breipohl et al.,

1979). However, as a major component of the VNO, its

smooth muscle of the lamina propria and T muscularis of

blood vessels (vomeronasal pump) moves the VNG secre-

tions out of the VNO to pick up the pheromones (Men-

doza, 1986; Meredith, 1994) and back into the VNO to

be exposed to the SE (Breipohl et al., 1979).

*Correspondence:

Tel.: +02(0101031153); fax: +2(088)2343703;

e-mail: [email protected]

With 30 figures, 3 histograms and 3 tables

Received April 2010; accepted for publication

January 2011

doi: 10.1111/j.1439-0264.2011.01064.x

Summary

The vomeronasal organ (VNO) is a chemosensory organ that detects environ-

mental pheromones. The morphology of the ‘non-sensory’ epithelium (NSE) of

the VNO and its lamina propria, as well as how it relates to ageing has received

little attention. Histological, histochemical, morphometric and ultrastructural

techniques were used to study the morphological structure of the rat NSE in

five adult (3 months old) and five aged (2–2.5 years old) male albino rats. In

adult rats, the NSE contained dark and light columnar cells with predominance

of the latter. The surface of the epithelial cells was covered with microvilli and/

or cilia. The lamina propria contained serous vomeronasal glands (VNGs),

smooth muscles with numerous variable-sized mitochondria, vessels including

lymphatic capillaries and nerve bundles. The following changes were detected

in aged rats. The NSE exhibited an increase in number of dark columnar cells.

Some cells revealed a prominent cell coat, dense aggregation of filaments in the

luminal cytoplasm and appearance of multinucleated cells. Their surface

revealed malformed configuration. Large mitochondria (2 lm), formed by

fusion, were frequently observed in the smooth muscle cells of the lamina pro-

pria. Lipid droplets were frequently detected both in the VNGs acini and in

the lymphatic endothelium. Ageing affected both the cells of the tissues and

the extracellular matrix.

Anatomia Histologia Embryologia

ª 2011 Blackwell Verlag GmbH • Anat. Histol. Embryol. 40 (2011) 263–277 263

Chemosensory dysfunctions of taste and olfactory sen-

sation affect the nutritional well-being of ageing patients,

as well as the safety and quality of life (Huntenbrink,

1995; Schiffman, 1997). Although many studies have

described the histology of the young and adult VNO, few

have focused on how the VNO is affected during ageing.

The aim of this study was to document the age-related

histological, histochemical and ultrastructural changes of

the NSE and the soft tissue components of the VNO.

Material and Methods

Five mature male albino rats (3 months old) and five aged

male albino rats (2–2.5 years old) (Ohta and Ichimura,

2000; Robinson et al., 2002) were used. The rats were anes-

thetized with ether and their thorax opened to expose the

heart, which was used to perfuse the appropriate fixative.

Light microscopy: Two rats from each animal group

were perfused intracardially with 10% formaldehyde solu-

tion. After perfusion, the rostral part of the skull contain-

ing the intact nasal cavity was removed and immersed

into 10% formaldehyde to continue fixation 2 more days.

Next, the specimens were decalcified in 13% formic acid

for 3–7 days (Drury and Wallington, 1980). Then, the

decalcified specimens were processed for the preparation

of paraffin blocks. Paraffin sections (5–7 lm) were cut in

a transverse plane, mounted on glass slides, and every

10th section was stained with haematoxylin–eosin stain.

In addition, selected sections were processed for histo-

chemical demonstration of polysaccharides using Alcian

blue (AB) at pH 2.5 for acid mucosubstances and Peri-

odic acid Schiff (PAS) method for neutral mucosubstanc-

es. Orcein stain was used for elastic fibres and Masson’s

trichrome for collagen fibres. Processing and staining

techniques were performed according to Drury and

Wallington (1980).

Electron microscopy (EM): Three rats from each group

were used and perfused intracardially with 4% glutaralde-

hyde in cacodylate buffer (pH 7.4). After perfusion, both

VNOs were dissected free. One was used for transmission

EM and the other used for scanning EM.

Transmission electron microscope (TEM): The speci-

mens were immersed in glutaraldehyde cacodylate fixative

for 24 h and post-fixed in 1% osmium tetroxide in phos-

phate buffer for 2 h. Tissues were rinsed in the same buf-

fer, dehydrated with alcohol, cleared with propylene oxide

and embedded in epon 812. Semi-thin sections (0.5–

1 lm) were cut and stained with toluidine blue (Gupta,

1983) for examination on a light microscope. Ultrathin

sections (500–800A) were cut from selected areas of the

blocks and contrasted with uranyl acetate and lead citrate

(Reynolds, 1963). These sections were observed with the

transmission electron microscope (Jeol E.M.-100 CX11;

Japanese electron optic laboratory, Tokyo, Japan) and

photographed at 80 kV.

Scanning electron microscope (SEM): After dissection,

the lateral side (portion lined by NSE) of the remaining

VNO was obtained by cutting the tissue along the longi-

tudinal axis under a dissecting microscope and the NSE

portions were immersed in the fixative for several hours.

A graded series of alcohols was used for dehydration, and

liquid carbon dioxide was used to dry the specimen.

Dried specimens were mounted on aluminium stubs,

fixed in place with colloidal silver and sputter coated with

gold (Naguro and Breipohl, 1982). A Joel (J.S.M-5400

LV; Japanese electron optic laboratory) was used to view

the specimens. Photographs were taken at 15 kV.

Morphometry

Using computerized assisted image analysis, the number of

NSE dark and light columnar cells per field was counted

on semi-thin sections (toluidine blue stain) viewed using a

100· oil immersion lens. Thirty fields were counted using

the modified touch method (Vohra et al., 2002). Nuclear

area was measured using the same lens configuration

(Crocker et al., 1983; Watkins and Cullen, 1988). Four

hundred nuclei per animal group were measured for the

two types of columnar cells (light and dark cells).

Statistical analysis

The morphometric data of each animal group were statis-

tically analysed. The student’s t-test was employed to

compare the studied animal groups. P < 0.05 was consid-

ered significant.

Results

In adult rats, the non-sensory components of the VNO were

the NSE, VNGs, smooth muscle, vessels and nerves. The

NSE was pseudo-stratified columnar epithelium. In semi-

thin sections, two types of columnar cells were identified,

dark and light with predominance of the latter (Fig. 1). The

number of light columnar cells in adult NSE ranged from 7

to 16 (11.4 mean ± 2.3) (Table 1 and Histogram 1). Dark

columnar cells in adult NSE ranged from 1 to 4 (2.45

mean ± 0.8), which represents only 16.3% of the columnar

cells (Table 2 and Histogram 2a). Flat to elongated basal

cells were interspersed between the columnar cells (Fig. 1).

None of the cells were PAS or AB positive; however, a thin

layer of PAS-positive material coated the apices of the NSE.

Nuclear area of NSE ranged from 18 to 59 lm2

(32.2 mean ± 6.7 lm2) (Table 3 and Histogram 3).

Ultrastructurally, the perikaryon of the columnar cells

reached the basement membrane basally where they were

Morphology of the Non-Sensory Tissue Components S. A. Eltony and S. A. Elgayar

264 ª 2011 Blackwell Verlag GmbH • Anat. Histol. Embryol. 40 (2011) 263–277

Fig. 1. Photomicrograph, adult VNO, ‘non-sensory’ epithelium, semi-thin section. Dark (DK) and light (Lt) columnar cells, basal cells (B). Toluidine

blue (·1000).

Fig. 2. TEM micrograph, adult VNO, ‘non-sensory’ epithelium. Interdigitated lateral cell membranes ( ‹ ), basal cell (B), basement membrane

(››), leucocyte (L) (·4000).

Fig. 3. TEM micrograph, adult VNO, ‘non-sensory’ epithelium. Apical microvilli (Mv), mitochondria (mt), SER (S), Golgi apparatus (G), lysosome

(Ly), interdigitations ( ‹ ) (·5200).

Fig. 4. TEM micrograph, adult VNO, ‘non-sensory’ epithelium. Microvilli and cilia (C), basal bodies (›), tight junctions (>) (·8700).

Table 1. Dark and light columnar cell numbers/field in ‘non-sensory’

epithelium (NSE) of adult and aged rat VNO

Adult NSE Aged NSE

Dark columnar cells

Mean ± SD 2.45 ± 0.8 7.75 ± 1.6

Range 1–4 5–11

P-value 0.001

Light columnar cells

Mean ± SD 11.4 ± 2.3 6.4 ± 1.4

Range 7–16 4–9

P-value 0.000

0

2

4

6

8

10

12

Mea

n nu

mbe

r of d

ark

& li

ght c

olum

nar c

ells

/ fie

ld

Adult Aged

DarkLight

Histogram 1. Number of dark and light columnar cells/field in ‘non-

sensory’ epithelium of adult and aged rat VNO.

S. A. Eltony and S. A. Elgayar Morphology of the Non-Sensory Tissue Components


narrow, and apically the cells were broad (Fig. 2). The

cytoplasm of the light cells contained numerous polarized

mitochondria, Smooth endoplasmic reticulum (SER), few

strands of RER and Golgi complex. Occasionally, groups

of lysosomes and multivesicular bodies could be seen

(Figs 2 and 3). The cytoplasm of the dark cells contained

numerous polarized mitochondria, endoplasmic reticulum

and ribosomes. Adjacent cell membranes interdigitated

(Fig. 3). The apical parts of the lateral cell membranes had

junctional complexes (Fig. 4). The luminal surface had

apical microvilli with no prominent cell coat and short

cilia, which were connected to basal bodies. The luminal

cytoplasm contained dispersed filaments with no peculiar

terminal web (Figs 3 and 4). Basal cells had variable elec-

tron density, dark or light. Their cytoplasm was rich in

free ribosomes (Fig. 2). The lateral aspect of the basal cells

showed interdigitating folds (Fig. 2). Leucocytes were

observed among the lining cells of NSE (Fig. 2).

In aged rats, the non-sensory components of the VNO

were comprised of the NSE, VNGs, smooth muscle, ves-

sels and nerves. The NSE was pseudo-stratified columnar

epithelium. In semi-thin sections, two types of columnar

cells were also identified, light and dark types (Fig. 5).

However, the dark cells were predominant and ranged

from 5 to 11 (7.7 mean ± 1.6) and represented 52.4%

from the columnar cells (Table 2 and Histogram 2b),

while the number of light cells in the aged NSE ranged

from 4 to 9 (6.4 mean ± 1.4) (Table 1 and Histogram 1).

Nuclei were enlarged in aged NSE and ranged from 18

to 69 lm2 (52.1 mean ± 17.5 lm2) (Table 3 and Histo-

gram 3). There were numerous light and dark multinucle-

ated cells containing two to four nuclei (Fig. 5). Flat to

elongated basal cells were interspersed between the

columnar cells. None of the cells were PAS or AB posi-

tive. A thick layer of PAS-positive material coated the

apices of the NSE.

Ultrastructurally, the perikaryon of the columnar cells

reached the basement membrane where they were narrow,

and apically the cells were broad (Fig. 6). The cytoplasm

of the light cells contained numerous mitochondria, SER,

few strands of RER and Golgi complex (Fig. 6). The

mitochondria had a marked polarization in the juxtalu-

minal region. Occasionally, grouping of lysosomes and

multivesicular bodies was observed. The cytoplasm of the

Table 3. Nuclear area (lm2) of columnar cells in ‘non-sensory’ epithe-

lium (NSE) of adult and aged rat VNO

Adult NSE Aged NSE

Mean ± SD 32.2 ± 6.7 52.1 ± 17.5

Range 18–59 18–69

P-value 0.001

Table 2. Percentage of dark columnar cells in ‘non-sensory’ epithe-

lium (NSE) of adult and aged rat VNO

Sum of dark and

light columnar cells

Number of dark

columnar cells

% of dark

columnar cells

Aged NSE 296 155 52.4

Adult NSE 300 49 16.3

Adult

12

Aged

12

(a)

(b)

Histogram 2. (a) Per cent of dark(1) columnar cells in adult ‘non-sen-

sory’ epithelium (NSE). (b) Per cent of dark(1) columnar cells in aged

NSE.

0102030405060

Mea

n ar

ea

(um

2) o

f col

umna

rce

llnuc

lei

Adult Aged

Histogram 3. Columnar cell nuclear area (lm2) ‘non-sensory’ epithe-

lium of adult and aged rat VNO.



dark and some light cells characteristically contained sev-

eral mitochondrial profiles with structural changes: dam-

aged cristae; abnormal forms of mitochondria with a

dense matrix or formation of rings; fusion of adjacent

mitochondria, as well as autosomal formation (Figs 7 and

8A). The adjacent cell membranes interdigitated (Fig. 7).

Fig. 5. Photomicrograph, aged VNO, ‘non-sensory’ epithelium, semi-thin section. Multinucleated columnar cells (b). Toluidine blue (·1000).

Fig. 6. TEM micrograph, aged VNO, ‘non-sensory’ epithelium. Aggregation of mitochondria (mt) (·2700).

Fig. 7. TEM micrograph, aged VNO, ‘non-sensory’ epithelium. Prominent microvillar coat (.), dense filaments and terminal web (Tw), degradated

mitochondria (›) (·5200).

Fig. 8. (A) TEM micrograph, aged VNO, ‘non-sensory’ epithelium (NSE). Autosome (>), fusing mitochondrial profiles ( fi ) (·2700). Left inset:

High magnification of the autosome (·8000). Right inset: High magnification of fusing mitochondria (·8000). (B) TEM micrograph, aged VNO,

NSE. Intranuclear inclusion (›) (·8700).

Fig. 9. TEM micrograph, aged VNO, ‘non-sensory’ epithelium. Dilated cilia (C), microvilli (Mv), basal bodies ( ), mitochondria (mt) (·5000).



The apical parts of the lateral cell membranes had junc-

tional complexes. In some cells, the surface microvilli

exhibited a prominent cell coat and an increase in the

underlying filaments (terminal web) (Fig. 7). The luminal

surface of other cells had apical microvilli and dilated

cilia, which were connected to basal bodies and contained

disorganized internal structure (Fig. 9). Multinucleated

cells were observed (Fig. 8A) with intranuclear inclusions.

The inclusions were formed of circular filaments that

contained granules (Fig. 8B). The basal cells had variable

electron density, dark or light. Their cytoplasm was rich

in free ribosomes. The lateral aspect of the basal cells

showed interdigitating folds. Leucocytes were observed

among the lining cells of NSE.

With SEM, in adult rats, the surfaces of the cells lining

NSE, SE and the transitional region were shown at low

power (Fig. 10). The majority of the NSE cells surfaces

were well demarcated and covered with short unbranched

microvilli and/or cilia (Fig. 11). Some cells had long

microvilli, while other cells were covered exclusively with

cilia (Fig. 12). The surface of the SE was covered with

long disoriented microvilli (Fig. 13). In the transitional

region between the sensory and non-sensory epithelium

(i.e. the area around the ducts), the cell surface revealed

Fig. 10. SEM micrograph, adult VNO, ‘non-sensory’ epithelium, SE, transitional region (TR), duct (D) (·2000).

Fig. 11. SEM micrograph, adult VNO, ‘non-sensory’ epithelium. Cells surfaces demarcation (*), short microvilli (.), cilia ( fi ) (·3500).

Fig. 12. SEM micrograph, adult VNO, ‘non-sensory’ epithelium. Long microvilli (c), cilia ( fi ) (·3500).

Fig. 13. SEM micrograph, adult VNO, SE (·5000).

Fig. 14. SEM micrograph, adult VNO, Transitional region. Dense radially aggregated microvilli (fl), short microvilli (.), duct of VNG (D) (·5000).



dense radially directed short micovilli with indistinct cell

demarcations (Fig. 14).

In aged rats, individual cell demarcation was more pro-

nounced by the short, unbranched microvilli and/or cilia

at the periphery of the cell (Fig. 15). The central portion

of the cell surface appeared deformed and covered with

swollen projections (Fig. 15). These changes were focal in

distribution. Some cells had long microvilli (Fig. 16), while

other cells were covered exclusively with cilia. The surface

of the SE was covered with long widely separated disori-

ented microvilli. In the transitional region between the

sensory and non-sensory epithelium (i.e. the area around

the ducts), the cells were either covered with microvilli of

variable lengths (Fig. 17) or covered with cilia and short

microvilli with distinct cell demarcation (Figs 18). Secre-

tory product was observed in the duct (Fig. 18).

Fig. 15. SEM micrograph, aged VNO, ‘non-sensory’ epithelium. Irregular swollen projections (v), short microvilli (fl) (·3500).

Fig. 16. SEM micrograph, aged VNO. Duct of a VNG (D), short microvilli and swollen cilia (C) (·2000).

Fig. 17. SEM micrograph, aged VNO. Transitional region. Duct of a VNG (D) (·3500). Inset: Low magnification of the duct (fl) and the surround-

ing area. (X 350).

Fig. 18. Higher magnification of the previous figure. Cilia (C) and secretion (<) in the duct (D) (·5000).



VNGs

In adult rats, the VNGs had the typical morphological

features of serous acini. The secretory cells had round to

oval basal nuclei (Fig. 19). The acini revealed a strong

PAS-positive reaction and negative AB reaction. Using

Masson’s trichrome stain, only the apices of the secretory

cells yielded a green colour. Ultrastructurally, the luminal

surface of the acini had microvilli (Fig. 19). The lateral

cell membranes were joined by juxtaluminal junctional

Fig. 19. TEM micrograph, adult VNO, acinus of VNG. Lumen (lu), basal nuclei (N), secretory granules (Sg), interdigitations (›), intra-epithelial

nerve ending (*) (·3500). Left inset: Photomicrograph, secretory acinus, semi-thin section. Toluidine blue (·1000). Right inset: TEM micrograph.

Apical microvilli (mv), junctional complexes (·10 000).

Fig. 20. TEM micrograph, adult VNO, duct of VNG. Lumen (Lu), microvilli (Mv) and interdigitations ( ‹ ) (·4000). Inset: Photomicrograph, duct

of VNG, semi-thin section. Toluidine blue (·1000).

Fig. 21. TEM micrograph, aged VNO, VNG. Golgi bodies (c) (·8700).

Fig. 22. TEM micrograph, aged VNO, VNG. Lipid droplets (L) (·6700).

Fig. 23. TEM micrograph, aged VNO, duct of VNG. Enlarged mitochondria (fl), SER, lysosomes (Ly) (·4000).



complexes and interdigitating folds (Fig. 19). Their cyto-

plasm characteristically contained numerous basal RER

and apical dense secretory granules of variable sizes

(Fig. 19). Intra-epithelial nerve endings were observed

(Fig. 19). The ducts of the VNGs had wide lumina, lined

by cuboidal or columnar epithelium with round to oval

nuclei and pale cytoplasm (Fig. 20). The main duct

opened into the area between SE and NSE both dorsally

and ventrally. Ultrastructurally, the luminal surface of the

duct had microvilli. The adjacent lining cells had apical

tight junctions and interdigitations (Fig. 20). The cyto-

plasm characteristically contained vesicular SER in addi-

tion to the usual cell organelles.

In aged rats, the secretory acinar cells had round to

oval basal nuclei. The acini revealed a strong PAS-positive

reaction and negative AB reaction. However, in some sec-

tions, there was an intense green staining of the cyto-

plasm of the acinar cells with Masson’s trichrome stain.

Ultrastructurally, some secretory cells revealed collapse of

the Golgi cisternae (Fig. 21) and numerous lipid droplets,

particularly basally (Fig. 22). The ducts of the VNGs

exhibited numerous SER, lysosomes and enlarged mito-

chondria in the cytoplasm of the lining cells (Fig. 23).

Smooth muscle

In adult rats, smooth muscle cells arranged individually

or in groups were present throughout the lamina propria.

In the area between the NSE and the VNGs, smooth mus-

cle cell orientation was perpendicular to the luminal axis

of the VNO. Whereas between acini of VNGs, individual

or groups of smooth muscle cells were present at their

base parallel to the basement membrane of the acini of

VNGs. Their numerous mitochondria varied in size

(Fig. 24). In aged rats, some smooth cells contained only

a few mitochondria compared with adult. Some mito-

chondrial profiles appeared enlarged and formed of sev-

eral segments, which gave the appearance of fusion of

several mitochondrial profiles (Figs 25 and 26). Enlarged

mitochondria contained vacuoles, dense inclusions and/or

tubular cristae (Fig. 26). Occasionally, some of the

smooth muscle cells that surrounded the acini exhibited

apoptotic changes.

Vessels and nerves

In adult rats, the lamina propria surrounding the VND

contained different types of vessels (arteriole, venule,

sinusoid, blood and lymphatic capillaries) and nerve bun-

dles. The lymphatic capillaries were comprised of a single

endothelial cell layer with an incomplete basal lamina.

The endothelial cells were non-fenestrated, greatly attenu-

ated with overlapping intercellular junctions. Their cyto-

plasm contained dense granules, mitochondria and

centrioles (Fig. 27). In aged rats, the lymphatic capillaries

consisted of attenuated non-fenestrated endothelial cells

with overlapping intercellular junction and discontinuous

basal lamina. However, their cytoplasm contained numer-

ous dense granules in the endothelial lining cells. Some

lymphatic dense granules were observed in contact with

the luminal surface (Fig. 28). Some lymphatic capillaries

with lipid droplets in their endothelium were observed in

direct contact with secretory cells of VNGs. The secretory

acinar cells also contained lipid droplets in their cyto-

plasm (Fig. 29). Desmosomal junctions between adjacent

endothelial and acinar cells were also observed (Fig. 30).

The endothelial cells demonstrated luminal dissolution of

the endothelial cell membrane overlying lipid droplets

(Fig. 30). The blood capillaries revealed a thick basal lam-

ina.

Connective tissue

In adult rats, the connective tissue in the lamina propria

of NSE contained elastic fibres and minimal collagen

fibres, whereas in aged rats, there was deposition of both

elastic and collagen fibres (Fig. 28). In adult rats, the con-

nective tissue around the VNGs had nerve bundles that

contained both myelinated and non-myelinated nerve ter-

minals arranged singly or in groups and surrounded by

endoneurium containing collagen fibres. In aged rats, the

nerve bundles exhibited numerous unstained collagen

fibres in the endo and perineurium.

Discussion

The non-sensory portion of the VNO lies in the lateral

wall of its duct and in the surrounding associated struc-

tures in the lamina propria: VNGs, vessels, nerves,

smooth muscles and connective tissue. Non-sensory epi-

thelium (NSE) is the term used for the epithelium lining

the lateral surface of the VND in this work. NSE and the

surrounding structures do not participate directly in the

chemoperception (Halpern, 1987; Keverne, 1999). NSE is

also called receptor-free epithelium (Breipohl et al., 1979)

and respiratory epithelium (Cooper and Bhatnagar,

1976).

Age changes are known to be most prominent in post-

mitotic cells such as neurons and cardiac myocytes,

whereas in proliferating cell populations, only minor or

undetectable changes occur (Comfort, 1979). In this

study, the NSE of adult rats revealed two types of colum-

nar cells (light and dark) with predominance of the light

type. Breipohl et al. (1979) reported the presence of light

and dark cells in the NSE of adult rat VNO. However,

dark cells were considered to be old and worn out, and



we observed numerous organelles in the dark cells, which

indicate active cells. In support of this view, the presence

of structurally variable light and dark olfactory sensory

neurons has been reported in adult bandicoot (Kratzing,

1978). These olfactory cells represented several popula-

tions at different stages of development. The dark olfac-

tory cells being containing more organelles they were

considered as more developed cells (Kratzing, 1978). In

addition, dark cells were observed in the vomeronasal

sensory epithelium following the transection of vomero-

nasal nerve (Ichikawa et al., 1998).

In aged rats, the number of light cells was statistically

lower than dark cells (as P < 0.05) suggesting that light

cells are young cells in a continuous process of turnover

Fig. 24. TEM micrograph, adult VNO, smooth muscle cell (›). Mitochondria (mt), intra-epithelial nerve ending (b) (·5200).

Fig. 25. TEM micrograph, aged VNO, smooth muscle cell. Enlarged mitochondria (hatched arrow), fusion of mitochondria (circles), nucleus (N)

(·10 000). Inset: Site of fusion of mitochondria (c) and intramitochondrial inclusion (›) (·18 200).

Fig. 26. TEM micrograph, aged VNO, smooth muscle cell. Enlarged mitochondria (hatched arrow), intramitochondrial inclusions (fl), vacuoles (v),

fusion of mitochondria (b) and mitochondria with tubular cristae (thick arrow) (·20 000).



as suggested by Breipohl et al. (1979). This reduced num-

ber of light cells is likely attributed to their transforma-

tion into dark type with age with a reduction in the

process of their renewal from the basal cells. As cells age,

they become less able to divide and reproduce (Tollefsbol,

2007).

Using EM, light columnar cell cytoplasm of aged NSE

revealed several degenerative mitochondrial profiles: dam-

aged cristae, dense matrix and formation of rings with

mitochondrial fusion. Mitochondrial decay with ageing

has been reported in both animals (rat liver) and human

(brain and liver) (Shigenaga et al., 1994). It is suggested

that sustained damage inflected by endogenously pro-

duced oxidants is the likely cause of the age-related defi-

cits in mitochondria (Shigenaga et al., 1994).

Multinucleated cells (both light and dark), as well as

increased nuclear size, were characteristically observed in

aged NSE. Similar findings have been reported in ageing

testes in Sertoli cells (Schulze and Schulze, 1981), in Ley-

dig cells (Paniagua et al., 1986) and in spermatocytes

Fig. 27. TEM micrograph, adult VNO, lymphatic capillary. Lumen (Lu), endothelial nucleus (N), cytoplasmic dense granules (v), centriole (C), mito-

chondria (mt), junction between overlying endothelial cells (.), discontinuous basal lamina (››) (·10 400).

Fig. 28. TEM micrograph, aged VNO, lymphatic capillary. Endothelial cell nucleus (N), lumen (Lu), cytoplasmic dense granules (v), a dense granule

in contact with the luminal surface ( fi ), discontinuous basal lamina (flfl) deposited collagen (C) and elastic (E) fibres (·10 400).

Fig. 29. TEM micrograph, aged VNO, acinus of VNG and a lymphatic capillary. Capillary lumen (Lu), lipid droplet (L) in the secretory acinus, lipid

droplet (*), endothelium (En), secretory granule (Sg), Golgi (G) (·4000).

Fig. 30. TEM micrograph, aged VNO, parts of an acinus of VNG and a lymphatic capillary. Lipid droplet (*), dissolution of the overlying mem-

brane (b), endothelium (En), lumen (Lu), desmosome (D), interdigitation ( ‹ ), an acinar cell (Ac) (·8700).



(Miething, 1993) from elderly men. As ageing process is

accompanied by degenerative changes in NSE, we can

postulate that these nuclear changes in light and dark cells

might enable cells to function as phagocytes. This sugges-

tion is supported by the finding of multinucleated fibro-

blastic cells, which possessed many phagosomes

containing intact collagen fibrils in the periodontal liga-

ment of aged rats (Sasaki and Garant, 1993). These

authors suggested that these multinucleated cells might

function as phagocyte.

Other structural changes were detected in some aged

NSE cells. These include a prominent cell coat overlying

the apical microvilli, pronounced PAS reaction and dense

luminal aggregation of filaments and mitochondria. Nor-

mally, the cell coat and the filaments in the luminal cyto-

plasm function as a barrier. Therefore, we suggest that

the presence of these structures in excess might potentiate

the epithelial barrier function (of NSE), and the accumu-

lating mitochondria might provide the necessary energy.

The potentiation of the barrier function is necessary

because nasal mucosa is continuously exposed to airborne

pathogens and other immunogens. These along with age-

ing exert significant effects on cells of the innate immune

system including numbers, function and early stages of

activation (Plackett et al., 2004; Gomez et al., 2005; Sol-

ana et al., 2006; Agrawal et al., 2008).

Leucocytes observed among the lining cells of NSE in

adult and aged groups were similarly detected by Adams

(1986) in adult bovine VNO. It is hypothesized that the

operation of the vomeronasal pump induces repeated epi-

sodes of transient focal ischaemia followed by reperfusion

of NSE, which results in the release of neutrophil chemo-

attractants and the modulation of adhesion factors that

regulate the extravasation and migration of neutrophils

into the NSE (Getchell and Kulkarni, 1995). The VN

pump is the pumping mechanism of the VNO. Its action

is based on the contraction and dilatation of the cavern-

ous body and other blood vessels in the lateral wall of the

VNO and distensible elastic fibres under the NSE and the

VN capsule (Meredith, 1994). The cavernous body is the

tissue beneath the NSE and includes venous sinuses,

smooth muscle cells, myelinated and non-myelinated

nerve fibres (Taniguchi and Mochizuki, 1983). During

vasodilatation, the lumen of the VND is compressed

whereby glandular secretions are transported from the

lumen of the VND to the base of the nasal cavity. In con-

trast, during vasoconstriction, the secretions are trans-

ported in the opposite direction from the nasal cavity

into the VND (Wysocki et al., 1980; Singer et al., 1988).

SEM of NSE in adult rats revealed variations in the

surface configurations: short or long microvilli and or

cilia. These findings are in agreement with those reported

in the NSE of adult mouse (Naguro and Breipohl, 1982);

however, the goblet cell–like protrusions found in the

mouse were not detected in rat NSE in this study. In NSE

of the aged rat group, the cells that exhibited deformed

swollen projections can be correlated with swellings in the

cilia detected in this study by TEM.

The presence of a strong PAS-positive and negative AB

reaction in VNGs acini in this work in adult rats indicates

the presence of neutral glycoprotein. These findings con-

tradict those reported by Bojsen-Moller (1964) who

noticed the presence of both neutral and acidic glycopro-

tein in adult rat and Nunez-Chichet et al. (2007) who

reported a weak AB reaction in 15- to 35-day-old rats.

However, Garrosa et al. (1986) found no AB stain posi-

tivity in pre- and post-natal rats (up to 42 days). This

controversy may lead one to suggest the possible presence

of seasonal variation in the composition of the secretion

of the VNGs. The previously mentioned ages are either

adult or young adult (35 or 42 days old).

Occasionally, the acini of ageing VNGs revealed intense

green–stained cytoplasm in some sections with Masson’s

trichrome stain. While in adult, only the apices of the

secretory cells yielded green colour. Therefore, change in

the composition of the secretory material might occasion-

ally occur with age. Intense green staining with Masson’s

trichrome stain is an indicative of the presence of mucin

that is a heavily glycosylated protein (Drury and Walling-

ton, 1980). Ultrastructurally, reduced Golgi cisternae and

the presence of lipid droplets in aged rats’ VNGs indicate

that ageing might be associated with altered ability to

synthesize glycoprotein as well as reduced level of secre-

tory activity of the acinar cells. Similarly, Draper et al.

(2003) reported reduction in Golgi apparatus in acinar

cells of ageing lacrimal glands. While the presence of lipid

droplets was reported in the acinar cells of aged rat paro-

tid gland by Kim (1984) and Mahay et al. (2004).

Presence of numerous SER and lysosomes detected in

the cytoplasm of the cells lining ducts of aged VNGs in

this work might represent an enhancement of the barrier

function exerted against invading pathogens. An abun-

dance of SER in other cellular systems has been associated

with several functions including detoxification (Ponzio,

1996). Moreover, Ferrari et al. (1998) hypothesized that

the great development of SER in the apical location of

the supporting cells of armadillo olfactory epithelium is

related to nasal xenobiotic metabolism. As these ducts are

in continuity with the surface epithelium, they share in

its barrier function.

The presence of giant mitochondria with structural

abnormalities as vacuoles and dense inclusions within the

matrix has been reported in ageing non-renewing tissues

as in senescent spinal ganglion neurons (Vanneste and

Bosch de Aguilar, 1981) and cardiomyocytes (Coleman et

al., 1987). The presence of a few mitochondria with age-



ing has been reported in skeletal muscles (Menshikova

et al., 2006).

Smooth muscle cells in the connective tissue surround-

ing the VNGs and finding a few mitochondria with the

frequent presence of enlarged profiles that appeared to be

formed as a result of fusion are significant. An increase in

size of mitochondria could be considered as a compen-

sation for the decrease in its number as suggested by

Solmi et al. (1994). The enlarged mitochondrion would

lead to the appearance of giant mitochondria as enlarged

mitochondria may be less likely to be autophagocytosed

(Terman et al., 2004).

The difference in the effect of ageing on the mitochon-

dria of the NSE and the smooth muscles could be attrib-

uted to the difference in the nature of each tissue

regarding their rate of proliferation.

Lymphatic vessels are normally detected in the adult

rat VNO. Similar findings have been reported by other

investigators (Salazar and Sanchez Quinteiro, 1998; Soler

and Suburo, 1998). The increased number of dense gran-

ules detected in this study in the lymphatic endothelium

in aged rat group might indicate an increase in chemo-

kines to compensate for the reduced activity of the cells

of the innate immune system occurring with ageing as

reported by Agrawal et al. (2008). Lymphatic endothelial

cells are known to produce chemokines (Mancaradi et al.,

2003) that may regulate cell migration from extravascular

tissues into the lymphatic network. Lymphatic capillaries

containing lipid droplets in their endothelium have been

observed in a direct apposition with acinar cells of VNGs,

which also contained lipid droplets in their cytoplasm.

The endothelial lipid droplets appeared transporting to

the lumen of the lymphatic capillary. These findings coin-

cide with the general function of lymphatic vessels, which

include tissue drainage, fat transport and immune func-

tion.

Presence of unstained collagen fibres in the bundles of

nerve terminals of aged rats coincide with the work of

Bahcelioglu et al. (2008). These authors found a signifi-

cant decrease in collagen type IV immunoreactivity with

age in rat oculomotor nerve. They reported that the

peripheral nerve is surrounded by a weak barrier in the

old age group when compared to the young age group.

Disclosure

No conflicts of interest have been declared. The Assiut

University is the source of funding for this research.

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