J. Anat. (2000) 197, pp.

437459, with 15 gures Printed in the United Kingdom 437

Morphology of human intracardiac nerves: an electron
microscope study
NERINGA PAUZIENE
1, 2
, DAINIUS H. PAUZA
2
AND RIMVYDAS STROPUS
2
" Laboratory of Electron Microscopy, Kaunas University of Medicine, and # Laboratory of Neuromorphology,
Department of Human Anatomy, Kaunas University of Medicine, Kaunas, Lithuania
(Accepted 18 April 2000)
:ns1r:c1
Since many human heart diseases involve both the intrinsic cardiac neurons and nerves, their detailed
normal ultrastructure was examined in material from autopsy cases without cardiac complications obtained
no more than 8 h after death. Many intracardiac nerves were covered by epineurium, the thickness of which
was related to nerve diameter. The perineurial sheath varied from nerve to nerve and, depending on nerve
diameter, contained up to 12 layers of perineurial cells. The sheaths of the intracardiac nerves therefore
become progressively attenuated during their course in the heart. The intraneural capillaries of the human
heart dier from those in animals in possessing an increased number of endothelial cells. A proportion of
the intraneural capillaries were fenestrated. The number of unmyelinated axons within unmyelinated nerve
bres was related to nerve diameter, thin cardiac nerves possessing fewer axons. The most distinctive feature
was the presence of stacks of laminated Schwann cell processes unassociated with axons that were more
frequent in older subjects. Most unmyelinated and myelinated nerve bres showed normal ultrastructure,
although a number of proles displayed a variety of dierent axoplasmic contents. Collectively, the data
provide baseline information on the normal structure of intracardiac nerves in healthy humans which may
be useful for assessing the degree of nerve damage both in autonomic and sensory neuropathies in the
human heart.
Key words: Intracardiac nervous system; peripheral nervous system; vasculature.
i N1robic1i oN
The pivotal signicance of the intracardiac nervous
system in inuencing cardiac rate, atrial and ven-
tricular refractoriness, coronary artery blood ow,
valvular function and atrial natriuretic peptide se-
cretion, as well as in causing the development of
hazardous disorders of human cardiac activity, has
been established in a range of studies (Forssmann,
1989; Neely & Urthaler, 1992; Gulbenkian et al.
1993; Bernardi et al. 1994; Ehlert et al. 1994;
Ferguson & Mark, 1994; Mitrani & Zipes, 1994; Oki
et al. 1994; van de Borne et al. 1994; Zabel et al. 1994;
Baumgart & Heusch, 1995; Esler et al. 1995;
Schuessler et al. 1996; Zucker, 1996; Beaulie &
Lambert, 1998; Chiou & Zipes, 1998; Stevens et al.
Correspondence to Associate Professor D.-H. Pauza, Laboratory of Neuromorphology, Department of Human Anatomy, Kaunas Uni-
versity of Medicine, A. Mickeviciaus Street 9, Kaunas LT-3000, Lithuania. Fax: (370 7) 220733; e-mail : daipau!kma.lt
1998; Wen et al. 1998; Armour, 1999). Likewise, there
are increasing indications that structural alterations in
the sensory and autonomic nerves may be a predictor
of tissue inammation and\or dysfunction of tissue
homeostasis (Saria et al. 1983; Coderre et al. 1989;
Wadhwani et al. 1989; Weerasuriya & Hockman,
1992; Bush et al. 1993). In spite of this, the electron
microscope investigations devoted to the human
intracardiac nerves and ganglia have been rather
limited (Chiba & Yamauchi, 1970; Ellison & Hibbs,
1976; Kyosola et al. 1976; Shvalev & Sosunov, 1985;
Sosunov et al. 1988; Armour et al. 1997) and, in
general, have dealt with the ultrastructure of neurons
and\or nerve terminals in the human myocardium or
within intrinsic ganglia. The ultrastructure of the
human intracardiac nerves has received no attention.
However, neuropathological alterations involve not
only the neurons or their axons, but peripheral nerve
lesions maybe also involve their sheaths, vasculature
and\or intraneural cells such as broblasts, macro-
phages, Schwann or mast cells (Illanes et al. 1990;
Korthals et al. 1991; Latker et al. 1991; Wang et al.
1992; Reynolds & Heath, 1995; Maeda et al. 1999).
An accurate morphological assessment of the intra-
cardiac nerves should therefore include all intraneural
components. Since the ultrastructure of the human
intracardiac nerves has so far not been described in
detail, the aim of the present study was to investigate
this in healthy humans. In addition, an attempt has
been made to analyse the ne morphology of the
intracardiac nerves in relation to their topography
and that of the intracardiac ganglia because recent
neuroanatomical ndings indicate that they may be
related (Pauza et al. 1999).
x:1rri :is :Nb xr1nobs
Hearts or portions of hearts from 7 individuals
without cardiac complications were obtained at
autopsy performed no more than 8 h after death at the
Kaunas mortuary for forensic medicine. Two hearts
were from females aged 50 and 55 y, while the
remainder were from males aged 22, 40, 55, 70 and
80 y. Since dierence between sexes were not observed,
the data were combined. The collection of the hearts
conformed to the ethical requirements of the insti-
tution from which they were obtained.
In contrast to earlier electron microscope examin-
ations of the human heart (Chiba & Yamauchi, 1970;
Ellison &Hibbs, 1976; Kyosola et al. 1976; Shvalev &
Sosunov, 1985; and others), most samples in this
study were taken from the epicardial nerves, the
courses of which are indicated schematically in Figure
1. Altogether we examined 106 tissue samples ultra-
structurally containing more than 160 epicardial
nerves from the 9 sites (Fig. 1). In addition, a few
tissue samples with the nerves from the left atrial
endocardial neural network close to the coronary
sinus were taken in order to compare them with those
that were situated epicardially. However, since major
ultrastructural dierences between diversely located
nerves were not detected in this study, the data
obtained from endocardial, myocardial, and epi-
cardial nerves were mostly analysed together.
For whole hearts, the organ was perfused by a
syringe with saline at room temperature via both
coronary arteries. The composition of the saline (pH
7n3) was (in mx) : NaCl 170; KCl 4n7; CaCl
#
2n5;
MgCl
#
1n2; NaHCO
$
2n5; glucose, 11n5. Following
perfusion, the intracardiac neural structures were
visualised by supravital staining with methylene blue
in a special chamber. The methylene blue solution
(0n002%; Merck, Darmstadt) had been prepared
with the aforementioned saline (pH 7n3). Staining of
the nerves, nerve bundles and ganglion cells, usually
taking 3040 min at room temperature, was moni-
tored by a dissecting microscope. Following visu-
alisation of the contours of the intrinsic nerves, the
methylene blue solution was replaced with the saline
and tissue samples of "1i1 mm were excised from
the heart using microsurgical instruments. Sub-
sequently the extirpated tissue samples were immersed
in a xative containing 2% paraformaldehyde and
2n5% glutaraldehyde in 0n1 x cacodylate buer (pH
7n4) for at least 4 h at room temperature or overnight
at 4 mC. Afterwards, the samples were postxed for
2 h with 1% osmium tetroxide solution in 0n1 x
cacodylate buer (pH 7n4), dehydrated through a
graded ethanol series and embedded in a mixture of
Epon 812 and Araldite. Semithin sections (1 m) were
stained with methylene blue according to Ridgway
(1986) and examined with an Axiomat (Zeiss,
Germany) microscope. Ultrathin sections, obtained
with an LKB-IV (Sweden) ultratome, were contrasted
with uranyl acetate and lead citrate, and examined
with a Tesla BS 500 (Czechoslovakia) or Philips (The
Netherlands) electron microscope using Svema
(Ukraine) or Agfa (Belgium) electron image lms.
Terminology
The terminology of Pannese (1994) was used to
describe the intrinsic neural structures. According to
this terminology, a nerve bre (either myelinated or
unmyelinated) was considered as a unit consisting of
one or more axons and associated Schwann cells, sur-
rounded by a basal lamina. The term nerve bundle was
used when an assembly of one or more nerve bres
was not surrounded by perineurium and epineurium.
An assemblage of nerve bundles surrounded by
connective tissue sheaths, i.e. endoneurium, peri-
neurium or\and epineurium was denoted as a nerve.
The ne meshwork of collagen brils that closely
surrounds a nerve bre was referred to as a Plenk-
Laidlaw meshwork, according to Pannese (1994). In
the present study, intracardiac nerves that were no
more than 100 m in diameter were considered as
thin, whereas those with a diameter of 100 mor more
were classed as thick. For myelinated bres, the g
ratio (axon diameter: total bre diameter) was used,
438 N. Pauziene, D. H. Pauza and R. Stropus
A
B
8
9
7
6
Right inferior pulmonary vein
Superior vena cava
Inferior
vena
cava
n = 12
n = 10
n = 62
n = 19
Left inferior
pulmonary
vein
4
n = 5
3
5
2
1
n = 5
n = 7
n = 14
n = 4
Right superior pulmonary vein
Superior vena cava
Left inferior
pulmonary
vein
Left atrial neural fold
Fig. 1. Drawings of ventral (a) and dorsal (b) views of the
pressure-inated human heart to illustrate 9 (19) locations of the
epicardiac nerves sampled for the present electron microscopic
study. n indicates the number of nerve samples taken from the
following locations: 1, over the root of the right coronary artery; 2,
over the root of the left coronary artery; 3, at the bifurcation of the
left coronary artery into the anterior interventricular and circumex
branches; 4, over the anterior interventricular branch of the left
coronary artery; 5, below the left atrial nerve fold; 6, between the
left and right inferior pulmonary veins; 7, over the coronary sinus
on the dorsal left atrium; 8, at the coronary sinus and crux of the
heart ; 9, over the dorsal right coronary groove. Location and
course of the sampled epicardiac nerves are schematically depicted
by shadowing. Dotted lines indicate the limits of the heart hilum.
as in earlier studies (Gillespie & Stein, 1983; Tuisku &
Hildebrand, 1992; Pannese, 1994).
Statistical analysis
The relationship between nerve diameter and the
number of capillaries, as well as that between nerve
diameter and the number of perineurial cell layers was
analysed using linear regression (Microcal Origin v.
4n00).
rrsii1s
Intracardiac nerve sheaths
Epineurium
Thick epicardial nerves were regularly enveloped by
well-developed epineurium that was mostly composed
of collagen brils (Fig. 2a). Epineurial collagen brils,
the diameter of which ranged from 40 to 100 nm, were
longitudinally or obliquely oriented and organised
into bundles 820 m in width and "7 m thick.
These bundles were arranged in 35 layers, between
which capillaries and elastic bres were occasionally
located (Fig. 2a, c). Thin brils were also regularly
distributed in the epineurium.
In the thin nerves, either epicardial or myocardial,
the epineurium was sparse, both the bundles of
collagen brils and solitary collagen brils being
mostly absent external to the perineurial cells (Fig.
2b). The thin intracardiac nerves of the human were
therefore separated from adjacent connective tissue
only by the sheath of perineurial cells.
Perineurium
Thick epicardial nerves that were more than 100 m
in diameter were commonly surrounded by 24 layers
of perineurial cells (Fig. 2a). However, at the heart
hilum, where some of the thickest epicardial nerves
were located, as many as 812 perineurial cell layers
were present (Fig. 3a). In general, the number of
perineurial cell layers was related to nerve diameter
(Fig. 4a). The interstices between perineurial cell
layers was occupied by collagen brils that were
longitudinally, obliquely or circularly oriented and
attened against the basal laminae of perineurial cells,
including their invaginations (Fig. 3a). Between the
bundles of collagen brils, isolated brils and elastic
bres intervened. Some perineurial cells located in
adjacent layers were linked or separated by very few
Human intracardiac nerves 439
Fig. 2. Electron micrographs demonstrating the structure of epineurium from the human intracardiac nerves in their transverse sections. (a)
Epineurium of thick nerve, in which collagen bres that are longitudinally (LC) and obliquely (OC) oriented compose the bundles. These
collagen bundles are separated into 4 layers by processes of the epineurial broblasts (EpF), between which a fewelastic bres (E) are located.
440 N. Pauziene, D. H. Pauza and R. Stropus
collagen brils (Fig. 3a). Occasionally, capillaries and
nerve bres were located between the perineurial cells,
but no contacts between nerve bres with neig-
hbouring cells were identied (Fig. 3bd).
The perineurium of the thin nerves that were less
than 100 m in diameter consisted of 12 cell layers
(Figs 4, 5a). When intracardiac nerves were "50 m
or less in diameter, they were enclosed by a single
layer of perineurial cells or a perineurial sheath was
lacking (Fig. 5b). When perineurial cells were absent,
the nerves were classied as bundles of nerve bres.
However, the presence of perineurial cells was not
strictly dependent on nerve diameter. Occasionally,
we detected nerves containing only a few un-
myelinated nerve bres and with diameter 5 m
that were surrounded by a perineurial cell sheath. In
other instances, comparatively thick cardiac nerves
were enveloped by a single layer of perineurial cells.
The subperineurial space in the thick nerves was
commonly wide and occupied by irregularly dis-
tributed collagen brils, capillaries and broblasts
(Fig. 2a). Infrequently, mast cells, eosinophils and
macrophages were also present in the subperineurial
space (Fig. 7). The subperineurial space of the thin
nerves was comparatively narrow and indistinct
(Fig. 5a).
Endoneurium
Endoneurial collagen bril density was variable. In
most nerves, brils were more or less evenly dis-
tributed around nerve bres (Fig. 2a). In some
intracardiac nerves examined bundles of brils were
widely separated by gaps containing solitary brils
(Figs 5a, 6b). In third type, the endoneurium was
extremely compact, the collagen brils being tightly
packed between nerve bres (Fig. 6a). These 3 types
of endoneuriumwere frequently encountered in nerves
that were adjacent to each other. A correlation be-
tween the pattern of the endoneuriumand the location
of nerves or their diameter was not detected. Nerves
with a varying endoneurial pattern were equally
common both in the atria and ventricles.
The thickness of endoneurial collagen brils was
similar to that for the perineurium and ranged
(b) Epineurium of thin nerve containing very few collagen bres that intermingle with surrounding connective tissue. (c) Epineurium of an
epicardiac nerve with the clearly visible elastic bres (E). Bars, 1 m. Abbreviations for this and subsequent gures: Ad, adipose cell ; AER,
agranular endoplasmic reticulum; Ax, axon; Bl, basal lamina; CC, circularly orientated collagen brils; E elastic bres; EnC, endoneurial
collagen brils; En, endoneurium; Ep, epineurium; EpF, epineurial broblasts; Er, erythrocyte; F, broblast ; GER, granular endoplasmic
reticulum; LC, longitudinally oriented collagen brils; m, microbrils; Ma, macrophage; MC, mast cell ; MNF, myelinated nerve bre;
Nu, cell nucleus; OC, obliquely oriented collagen brils; P, perineurial cells; Pe, pericyte; Sch, Schwann cell ; SF, subperineurial broblast ;
UNF, unmyelinated nerve bre; VL, vascular lumen; AxV, axon varicosity.
approximately from 40 to 60 nm. The Plenk-Laidlaw
meshwork was well developed only around myelinated
nerve bres, whereas it was indenite around un-
myelinated bres. In contrast to solitary macrophages
and mast cells, broblasts were the typical cells
encountered in the endoneurial space (Figs 5, 6).
The sheaths of thick intracardiac nerves contained
plentiful capillaries. No other type of blood vessel was
identied. The number of intraneural capillaries
increased with nerve diameter (Fig. 4b). Epicardial
nerves that appeared attened in transverse section
contained fewer capillaries than those that had an
oval shape. Nevertheless, abundant thin cardiac
nerves with a diameter 50 m were mostly devoid
of capillaries. The ratio of capillaries to myelinated
bres varied and ranged from 1: 3 to 1: 41.
Blood vessels of the intracardiac nerves
The outer diameter of the endoneurial capillaries
ranged fromabout 4 mto 13 m(mean 7n6p0n4 m;
nl28). Pericytes, completely or partly ensheathing
the capillaries, were also very common (Figs 3b, 8a).
The ne meshwork corresponding to the Plenk-
Laidlaw sheath was readily observable around endo-
neurial capillaries (Fig. 8b). In thinner nerves,
capillaries were mostly situated subperineurially (Fig.
8a). The vascular lumen of endoneurial capillaries
was surrounded by 210 (2n9p0n9; nl29) endo-
thelial cells (Figs 3b, 8a, b, f ). In this study, we did not
encounter intraneural capillaries with a lumen en-
closed by a single endothelial cell. Occasionally, the
thickness of the endothelial cells reached 6 m
(3n6p0n2 m; nl13) at cell perikarya, while it
ranged from 0n7 m to 4 m (1n6p0n2 m; nl28) at
cell peripheries. At the site of apposition of contiguous
endothelial cells, these cell were mostly of 0n21n3 m
(0n6p0n05 m; nl28) in thickness. The lumen of
capillaries thus resembled an irregular asterisk (Figs
3b, 8a, b, f ). The borders of adjacent endothelial cells
came into mutual contact via simple apposition or by
overlapping each other. Along the interface, the 2
margins of the adjacent endothelial cells formed
interdigitations or junctions similar to a zonula
occludens (Fig. 8g). The external surface of the
endothelial cells that faced the basal lamina was
comparatively smooth in most instances. However,
Human intracardiac nerves 441
Fig. 3. Electron micrographs demonstrating the structure of perineurium from the human intracardiac nerves in transverse section.
(a) Perineurium of thick nerve, perineurial cells of which are disposed in 12 layers that are indicated by the Roman numbers. Note that
interstices between many perineurial cells are lled by the longitudinally (LC), obliquely (OC) or circularly (CC) oriented collagen brils
while some perineurial cells from adjacent layers are linked (arrows). (b) A capillary and (c, d) nerve bres intervening between perineurial
cells. Note that some axons (arrows) are not surrounded by the Schwann cell and contain the small clear vesicles. Bars, 1 m.
442 N. Pauziene, D. H. Pauza and R. Stropus
Fig. 4. Relationship between intracardiac nerve diameter (in m)
with the number of layers of perineurial cells (a) and number of
intraneural capillaries (b). Data are given as means (solid circles),
standard error (short bars), and standard deviation (long bars). The
straight lines indicate the linear regression of the data. R, coecient
of regression; n, the number of nerves from which the data were
obtained.
they sometimes possessed ne external processes that
came into contact with pericytes (Fig. 8c). Conversely,
on a few occasions we observed ne pericyte processes
that extended into delicate invaginations of the
endothelial cells. Compared with the external pro-
cesses, the internal ones were more numerous,
especially near contact sites between adjacent en-
dothelial cells (Fig. 8f, g). Lipofuscin granules were
consistently detected in the endothelial cells.
The walls of many endoneurial capillaries were
extremely thin and\or possessed fenestrations (Fig.
8d, e). Mostly, these fenestrations were clustered
unilaterally on one side of the capillary which
appeared as if closed by a very thin diaphragm (Fig.
8e).
Unmyelinated nerve bres
An analysis of unmyelinated nerve bres (UNFs) or
Remak bres revealed that the distribution of axons
within these bres was associated with nerve thickness
(Fig. 9). Out of 925 axons counted within UNFs in
thin intracardiac nerves, 28% were individually
ensheathed by the Schwann cell (Figs. 5, 9). In thick
cardiac nerves, UNFs with a single axon were
signicantly rarer and, according to an assessment of
2467 axons located within UNFs in such nerves, they
comprised only 8% (Fig. 9). Likewise, UNFs with 2,
3 or 4 axons were more common in thin than in thick
nerves (Fig. 9). In thick cardiac nerves, however,
UNFs with 5 or more axons were more plentiful,
while UNFs with more than 8 axons were entirely
absent in thin human intracardiac nerves (Fig. 9).
Moreover, variations in the number of axons within
UNFs was in general not age-related, although in
nerves of younger individuals (2240 y) we frequently
detected UNFs with 2025 axons that were not
observed in nerves from older subjects (5580 y).
The intracardiac nerves of a 22-y-old individual
contained abundant UNFs with 2030 axons that
were very rare or entirely absent in the other specimens
examined (Fig. 10). Proles of these multi-axon UNFs
were unusually irregular and their Schwann cells
contained collagen pockets (Fig. 12b). In addition,
in a few thick nerves from the 22-y-old subject we
identied the largest UNF proles that contained
more than 200 axons surrounded by entangled
Schwann cell processes. Although in the proles of
such UNFs as many as 56 Schwann cell nuclei were
sometimes seen, this whole unmyelinated nerve bre
complex was regularly enveloped by a common basal
lamina (Fig. 10).
A very distinctive feature of human UNFs was
lamination of Schwann cell processes. This lamination
was greater in large UNFs, especially of older subjects
(Fig. 11). Usually, UNFs with 1 or 2 axons were
surrounded by only a fewSchwann cell processes (Fig.
11a, b), while UNFs containing 58 axons were
regularly enclosed by 56 processes (Fig. 11c, d).
Proles of laminated Schwann cell processes un-
associated with axons but enveloped by a common
basal lamina were also present (Fig. 11b, e).
A large majority of mesaxons, especially in UNFs
of older subjects, were elongated, meandering and
spiralled around the axons (Fig. 11f ) but others were
short. Since the axons of UNFs were frequently
associated with processes from many Schwann cells,
some of them within this type of nerve bre had a
few mesaxons (Fig. 11c, d, f ). This formation was
more usual for axons of smaller diameter. Some axons
were only partially surrounded by Schwann cells,
portions of their plasma membranes being in direct
contact with the basal lamina surrounding the whole
Human intracardiac nerves 443
Fig. 5. Electron micrographs illustrating 2-layered perineurium of a thin intrinsic cardiac nerve (a) and its absence in an epicardiac nerve
bundle (b). Note the close relationship of the perineurial cells in (a) and the broblasts (F) in (b) which encircle unmyelinated nerve bres
(UNF) with their processes and resemble perineurial cells. Bars, 1 m.
444 N. Pauziene, D. H. Pauza and R. Stropus
Fig. 6. Electron micrographs showing dierent patterns of density of UNFs within endoneurium of human intracardiac nerves. (a) Compact
pattern of distribution of UNFs with restricted endoneurial space that is lled with collagen brils. (b) Sparse distributions pattern of UNFs,
in which bundles of endoneurial collagen brils are separated by wide gaps. Bars, 1 m.
Human intracardiac nerves 445
Fig. 7. Electron micrographs of a macrophage (a) and mast cell (b) within human intracardiac nerves. Bars, 1 m.
UNF (Figs 12a, 14d, f, g). Absence of a mesaxon was
more characteristic of younger individuals as in nerves
of the 22, 40, and 80-y-old subjects the UNFs with
axons open to endoneuriumformed respectively 38%,
6%, and 4% of all (nl3392) axons examined. All
examined UNFs possessed single Schwann cell
troughs containing solitary axons. Collagen pockets
were occasionally very large and the bundles of
collagen brils that they contained were separated
from the Schwann cell by its basal lamina (Fig. 10).
The Schwann cell nuclei had no indentations and
heterochromatin was present as centrally located
clumps and in a peripheral band. The perikarya of the
Schwann cells contained well-developed granular and
agranular endoplasmic reticulum (ER), polysomes,
Golgi apparatus, mitochondria and dark inclusions.
Centrioles were observed in a few instances (Fig. 12a).
Cisternae of the granular endoplasmic reticulum, free
polysomes, mitochondria, microtubules and laments
were evenly distributed in Schwann cell processes.
Lipofuscin granules were also observed sporadically
within the cytoplasm of the Schwann cells (Fig.
12c, d).
Myelinated nerve bres
Myelinated nerve bres (MNFs) were unevenly
distributed within the intracardiac nerves and varied
in their morphology.
contiguous endothelial cells. A pericyte (Pe) and its processes in (a) envelops the endothelial cells and, as seen in enlarged area (c) that
is boxed in (a), forms a few contacts (arrows) with the endothelial cells. (d, e) Electron micrographs demonstrating the fenestrations (clear
arrowheads) in wall of capillaries from the human cardiac nerves. (g) Enlarged area that is boxed in (b) showing the interdigitations
(arrowheads) between the endothelial cells. Bars, 1 m.
In most intracardiac nerves examined, the g ratio of
the thin myelinated bres was 0n60n7 (Fig. 13a). In
spite of this, thin nerve bres with somewhat thicker
myelin sheaths (g ratio 0n5) as well as others with thin
sheaths (g ratio 0n80n9) were occasionally en-
countered (Fig. 13b). In addition, small myelinated
nerve bres varying from 0n73 m to 0n85 m in outer
diameter with axons thinner than 0n15 m were also
observed (Fig. 13c). These bres had very low g ratio
(0n180n2) and therefore, appeared similar to those
identied as beaded by Ochs & Jersild (1987). The
structure and periodicity of the myelin sheath in such
bres was normal.
Internal structure of axons
Both the myelinated and unmyelinated axons usually
displayed typical ultrastructural appearances. Their
clear axoplasm contained evenly distributed micro-
tubules and neurolaments. With certain exceptions,
described below, most transverse sections of axons
contained a few mitochondria and cisternae of
agranular ERthat were mostly oriented longitudinally
(Fig. 14a). In a few cases, cisternae of agranular ER
that were perpendicularly or circularly oriented within
axons were observed (Fig. 14c).
The neurolaments of most axons were evenly
distributed throughout the cross section, but some
axons contained neurolaments that were clustered
446 N. Pauziene, D. H. Pauza and R. Stropus
Fig. 8. Electron micrographs showing structural features of capillaries from human intracardiac nerves. Walls of the capillaries are
formed by 6 (a), 7 (f ), and even 10 (b) endothelial cells. Note that the asterisk-like vascular lumen of these capillaries (VL) is caused by
internally protruding endothelial cells both at the level of cell nucleus (Nu) and the cell periphery. Arrowheads indicate the junctions between
[continued opposite
Human intracardiac nerves 447
Fig. 9. Percentage distribution of axons (nl3392) within un-
myelinated nerve bres with diering numbers of axons in thin
(squares) and thick (circles) human intracardiac nerves. The number
of axons in thin and thick nerves was 925 and 2467, respectively.
The percentage for axons is expressed as the mean.
into bundles occupying the whole or a greater portion
of the axon prole (Fig. 14b, c). When bundles of
neurolaments were present the axon proles were
obviously larger than those of their neighbours. Large
dense-core vesicles, cisternae of agranular ER, mito-
chondria and microtubules were also present in such
axons (Fig. 14b, c).
Microtubules were usually evenly distributed within
clear axoplasm in UNFs (Fig. 14a) but some axons
diered both by having a higher density of the
axoplasmic matrix and more numerous microtubules
(Figs 13a, 14eg). Commonly, these axons were very
irregularly shaped in transverse section. Some had
long wing-like protuberances or were very attened
(Fig. 14eg). Their wing-like protuberances were
often unsheathed by the Schwann cell and were
directly in contact with the surrounding basal lamina.
The matrix of these axons was packed with numerous
microtubules and, because of this, the cisternae of
agranular ER, large clear vesicles and neurolaments
that were present were dicult to visualise. Generally,
these axons were darker than the others within UNFs.
The distribution of these dark axons varied from one
intracardiac nerve to another, but without any denite
cardiac location. Occasional axons with an ordinary
internal structure were also rather irregular in shape
because of long protuberances that extended between
the Schwann cell processes towards the endoneurium
(Fig. 14d).
Unmyelinated axons in the intracardiac nerves were
observed to exhibit varicosities (Fig. 15). The great
majority of varicosities were oval in shape with minor
diameters ranging from1 mto 5n2 mand containing
a variety of organelles. Some were richly lled with
neurolaments, microtubules, mitochondria, cisternae
of agranular ER, and dense-core and clear vesicles
(Fig. 15a, f ). Varicosities of another type were larger
in diameter and incompletely enclosed by Schwann
cells (Fig. 15b). The axoplasmof these varicosities was
clear and contained a larger number of mitochondria
and ER cisternae as well as a few small clear and large
dense-core vesicles. Nevertheless, the most prevalent
and largest were axon varicosities that contained
multiple mitochondria, small clear and large granular
vesicles, glycogen particles, and dense lamellar and
multivesicular bodies (Fig. 15c, d, f ). The density of
the latter structures varied from low (Fig. 15c) to very
high (Fig. 15d). When axoplasmic density was low,
most components of the varicosity were aggregated in
its central part, while neurolaments and microtubules
were distributed in the periphery (Fig. 15c, e).
In contrast to UNFs, the MNFs were more
restricted in the range of their axonal contents. Apart
from MNFs with typical axons, human epicardial
nerves occasionally possessed MNFs with axons that
were lled with abundant mitochondria, vesicles and
lamellar bodies (Fig. 13d).
bi scissi oN
Epineurium
It is widely known that the epineurium of the
peripheral nerves becomes considerably thinner in
their distal portions and interlaces with adjacent
connective tissue following entry into the target organs
(Peters et al. 1976). Thick and obliquely oriented
epineurial collagen brils are signicant for nerve
protection from the eects of longitudinal tension
(Ushiki & Ide, 1990; Ishii & Takeuchi, 1993). The
present study has revealed that human intracardiac
nerves are covered by epineurium, the thickness of
which was related to nerve diameter. Thick intra-
cardiac nerves, especially those that ran close to the
cardiac base, had epineurial coats up to 7 m in
thickness containing 35 layers of collagen bundles
together with blood capillaries and a few elastic bres.
The bundles of epineurial collagen brils were
820 m in width, this corresponding to that of
bundles of epineurial collagen in the rat facial and
sciatic nerves (Ushiki & Ide, 1990; Ishii & Takeuchi,
1993). Although the exact orientation of the collagen
brils was not determined in this transmission electron
microscope study, it is clear that thick intrinsic nerves
of the human heart are covered by epineurium typical
of extrinsic nerves.
Thin intrinsic nerves of the human heart that were
less than 100 m in diameter also possessed epi-
neurium, but this was rather indistinct compared with
448 N. Pauziene, D. H. Pauza and R. Stropus
Fig. 10. Transverse section of the intracardiac nerve from a 22-y-old individual in which unmyelinated nerve bres involving a large number
of axons were frequently evident. Dashed lines frame the adjacent unmyelinated nerve bres. Bar, 1 m.
Human intracardiac nerves 449
Fig. 11. Electron micrographs showing the structural organisation of unmyelinated nerve bres within the human intracardiac nerves. (a,
b) UNFs with solitary axons in which a few Schwann cell processes (Sch) form a laminated envelope. Note the column of the Schwann cell
processes (asterisks) in (b) that is devoid of any axons. (c, d) UNFs containing 48 axons that are wrapped by 46 Schwann cell processes.
450 N. Pauziene, D. H. Pauza and R. Stropus
Fig. 12. Electron micrographs of human intracardiac nerves demonstrating (a) centrioles (arrow) within the Schwann cell cytoplasm, (b)
Schwann cell collagen pockets (asterisks) and (c, d) the lipofuscin granules (arrowheads) within the Schwann cells. Note that the axons in
a and d that have very short mesaxons or are in direct contact with the basal lamina (clear arrowheads). Bars, 1 m.
the epineurium of thicker nerves. In general, the
epineurium of thin nerves was thinner and large gaps
between its collagen bres were evident. Elastic bres
and blood capillaries were absent within this thin
epineurium. Bundles of the epineurial collagen bres
barely reached 7n5 m in width and therefore were
unusually thin compared with those of typical epi-
neurium.
Perineurium
The perineurial coat of peripheral nerves is composed
of layers of at cells, between which zones of collagen
bres are included (Ushiki &Ide, 1990). The thickness
of the perineurium is positively correlated with nerve
diameter and a thin perineurium is characteristic of
intrinsic nerves (Cumasov, 1975; Ushiki & Ide, 1990).
Since perineurial cells are structurally organized as an
uninterrupted and many-layered structure around the
Note the axons that have several mesaxons (black arrowheads). (e) Columns of laminated Schwann cell processes (asterisks) that have no
axons, but are enveloped by a common basal lamina (clear arrowheads). (f ) Electron micrograph illustrating a spiral mesaxon (arrow) as
well as axons with several mesaxons (arrowheads). Bars, 0n5 m.
nerve fascicles and are linked by tight junctions
(Thomas, 1963), they are important in providing a
diusion barrier (Fukuhara et al. 1979; Eldridge et al.
1986; Bush & Allt, 1990; Gerhart & Drewes, 1990;
Bush et al. 1991, 1993; Allen & Kiernan, 1994; and
others). Collagen bres distributed between layers of
the perineurial cells form a dense and exible
meshwork that not only provides mechanical support
for the nerve bres, but also restrains the intra-
fascicular contents which are under greater pressure
than the extraneural environment (Ushiki & Ide,
1986, 1990; Ishii & Takeuchi, 1993). The ndings of
the present study demonstrate that the thickness of
the perineurium in the human intracardiac nerves
varies from nerve to nerve. The perineurium of the
thick epicardiac nerves is multilayered and en-
compasses 56 or occasionally even 1011 layers,
whereas their thinner branches are commonly coated
only by 12 layers. It can therefore be concluded that
Human intracardiac nerves 451
Fig. 13. Electron micrographs showing the structural organisation of myelinated nerve bres within human intracardiac nerves. (a) Two
typical MNFs with the g ratio of 0n60n7. Note the UNFs that contain the dark axons (dark arrowheads). (b) MNF with a comparatively
thin myelin sheath. (c) MNF that resembles a beaded bre due to its compressed axoplasm. (d) MNF with axoplasm containing plentiful
mitochondria (dark arrowheads), vesicles (small arrows) and lamellar bodies (clear arrowheads). Bars, 1 m.
in general the intrinsic nerves of the human heart, in
spite of their comparative thinness, have a well-
developed perineurium, the thickness of which, as in
all peripheral nerves, depends on nerve diameter.
Similarly, the ultrastructure of the perineurial cells as
Fig. 14. Electron micrographs demonstrating the typical (a) and atypical (bg) internal structure of nerve bres within human cardiac nerves.
(a) Typical axon (Ax) enveloped by a Schwann cell process (Sch) and containing a few mitochondria (large arrows), cisternae of agranular
ER (small arrows), microtubules (black arrowheads) and neurolaments (clear arrowheads). (b, c) Axons of increased diameter lled with
numerous neurolaments. (d) Irregularly shaped axon (Ax1) with a protuberance extending towards the endoneurium (En). Another axon
(Ax2) is normal in appearance. (e g) Dark axons (asterisks) with a dense axoplasmic matrix and multiple compactly arranged microtubules.
Note dark axons (asterisks) that have the wing-like protuberances (curved arrows) as well as axons that are in direct contact with the basal
lamina (clear arrowheads). Bars, 0n5 m.
well as the subperineurial space of the human
intracardiac nerves do not display any particular
features compared with those that have been described
in a range of peripheral nerves (Thomas, 1963;
Gamble, 1964; Cumasov, 1975; Ushiki & Ide, 1986,
452 N. Pauziene, D. H. Pauza and R. Stropus
Fig. 14. For legend see opposite.
Human intracardiac nerves 453
Fig. 15. Electron micrographs illustrating a variety of axon varicosities (AxV) in human cardiac nerves. The clear arrowheads indicate the
clear vesicles; black arrowheads, large and\or small dense-core vesicles; small arrows, glycogen particles; black arrows, multivesicular bodies;
clear arrows, mitochondria; curved arrows, lamellar bodies. (a, e) Axon varicosities with multiple clear vesicles, large and small dense-core
vesicles, and glycogen particles. (b) Axon varicosity that is in contact with a Schwann cell (asterisk) only for a small part of its circumference.
454 N. Pauziene, D. H. Pauza and R. Stropus
1990; Rechtland & Rapoport, 1987; Ghabriel et al.
1989; Bush & Allt, 1990; Ishii & Takeuchi, 1993;
Allen & Kiernan, 1994; and others).
Endoneurium
According to earlier investigations (Thomas, 1963;
Gamble, 1964; Gamble & Eames, 1964; Ushiki & Ide,
1986), the most frequent cells found within the
endoneurium of peripheral nerves are broblasts,
while mast cells and macrophages are less common.
The present study conrms this only in part, because
amongst cells in the endoneurium of the human
intracardiac nerves macrophages are also relatively
common. Moreover, the intrinsic nerves examined by
us contained eosinophilic granulocytes, this having
not previously been described in extrinsic nerves.
As has been shown by scanning electron micro-
scopy, the endoneurial stroma is formed by collagen
bres and their bundles that are organized into 2
meshworks, coarse and ne (Ushiki & Ide, 1986,
1990; Ishii & Takeuchi, 1993). The coarse meshwork,
sometimes referred to as the Key-Retzius meshwork,
is formed by thick wavy bundles of collagen bres that
follow the course of nerve bres. Internal to this, the
ne or Plenk-Laidlaw meshwork of thin collagen
bres lies in intimate contact with the nerve bres.
Fibres of the Plenk-Laidlaw meshwork are irregularly
oriented. In the intrinsic nerves of the human heart,
the ne Plenk-Laidlaw meshwork is well developed
only around myelinated nerve bres, while it is less
evident on unmyelinated bres. The thickness and
compactness of the collagen bundles in the Key-
Retzius meshwork varyied from one intracardiac
nerve to another, in some nerves being thick and
compact, in others thin and loose.
Intraneural blood capillaries
Peripheral nerves are well vascularised and possess
both an external and an internal circulation (Lund-
borg & Branemark, 1968; Lundborg, 1988;
Wadhwani & Rapoport, 1994). The external cir-
culation comprises epi- and perineurial arterioles,
venules and their anastomoses. All are linked with the
endoneurial capillaries that are oriented longitudinally
along nerve bres within the fascicles and range from
4 to 10 m in diameter (Melman, 1988; Olsson, 1990;
(c, d) Axon varicosities containing numerous mitochondria, small clear and large dense-core vesicles, dense lamellar and multivesicular
bodies, and glycogen particles. (f ) Enlarged view of the boxed area in d demonstrating more clearly the densely packed content of the axon
varicosity. Bars: 1 m in ae, 0n5 m in f.
Wadhwani & Rapoport, 1994). In the present study,
we did not identify either arterioles or venules within
the intracardiac nerves. Irrespective of nerve thick-
ness, all intraneural blood vessels were capillaries
varying in diameter from 4 to 7 m. The intraneural
capillaries of the human heart diered from typical
ones in the number of endothelial cells enclosing the
lumen. The lumen within previously examined per-
ipheral nerves was usually enclosed by 23 endothelial
cells (Olsson & Reese, 1971; Alabin, 1977; Melman,
1988; Olsson, 1990; Wadhwani &Rapoport, 1994) ; in
the human heart there were usually as many as 46
endothelial cells which, in addition, were thicker than
usual. Within the human intracardiac nerves, in
general these cells were not attened at their margins
and, because of this the shape of capillary lumena in
these nerves resembled irregular asterisks.
The ndings of the present study provided evidence
that some of the intraneural capillaries in the human
heart are fenestrated. Although fenestrations were not
very abundant, they were typical in structure. This
nding is in contrast with the situation in endoneurial
capillaries in limb nerves where nonfenestrated
capillaries contribute to the blood-nerve barrier
(Olsson & Reese, 1971; Pannese, 1994).
Unmyelinated nerve bres
In some human peripheral nerves examined previously
(Gamble & Eames, 1964; Ochoa & Mair, 1969 a, b),
Schwann cell proles associated with unmyelinated
axons commonly were related to 12 axons. In the
human greater auricular nerve 2248% of the proles
possessed 35 axons with a maximum number of 23
(Gibbels et al. 1994). The latter authors considered
that UNFs with multiple axons in the greater auricular
nerve could be a manifestation of the shortness of this
sensory nerve. In nerves from the human heart, we
found that the number of axons within UNFs is
mainly related to nerve diameter. Thus in thin cardiac
nerves, 13 axons were usual, while in thick nerves a
value of 58 was most common. Moreover, thin
human nerves, in contrast to thick ones, had no UNFs
with more than 8 axons. Since these dierences in
axon number were characteristic for individuals of
any age, we concluded that the number of axons
within UNFs is not strictly related to age. On the
other hand, cardiac nerves from the youngest subject
Human intracardiac nerves 455
examined contained the most notable UNFs that
possessed the largest number of axons (up to 200). It
is possible that this 22-y-old individual represents an
unexplained atypical variant.
The present ndings demonstrate that proles
consisting of stacks of attened Schwann cell pro-
cesses unassociated with axons and enclosed within a
common basal lamina were frequent in human
intracardiac nerves. Such laminated structures were
more common in nerves of elderly individuals, and the
number of laminated processes per prole also
increased with age. These appearances are also found
in the normal sural nerve of the elderly humans
(Ochoa & Mair, 1969a, b; Behse et al. 1975; Ochoa,
1978) and are likely to be the consequence of loss of
unmyelinated axons.
Collagen pockets in the Schwann cells of UNFs
were widely distributed and no age-related dierences
in their frequency was apparent in the human
intracardiac nerves examined, although they were
more common within large and complicated UNFs
than in smaller ones. Although the exact signicance
of collagen pockets remains unclear, Gamble &Eames
(1964), Gamble (1964) and Illanes et al. (1990)
considered that they may act as an exoskeleton for
large UNFs.
Except for microvilli, the ultrastructure of Schwann
cells in human UNFs corresponded completely with
descriptions from other mammalian peripheral nerves
(Peters et al. 1976; Illanes et al. 1990; Pannese, 1994).
Myelinated nerve bres
The g ratio (axon diameter: total bre diameter) is an
important measure in relation to myelinated bre
function (Gillespie & Stein, 1983; Tuisku & Hilde-
brand, 1992; Pannese, 1994). Optimal conduction
occurs in myelinated bres with g ratio values close to
0n7 (Tuisku & Hildebrand, 1992). In the human
intracardiac nerves, g ratio values for most MNFs
ranged from 0n6 to 0n7. However, bres with g ratio
values of 0n5 as well as 0n80n9 were also occasionally
present in the human intracardiac nerves. Excepting
the thin MNFs of the trigeminal alveolar nerve from
the cichlid sh in which similar variety of g ratio
values was found (Tuisku & Hildebrand, 1992), in all
nerves examined from the human and mice this index
varied extremely close to 0n7 (Friede & Beuche, 1985;
Little & Heath, 1994). Presumably the variable g ratio
values of MNFs in the human intracardiac nerves
suggest that these nerve bres are heterogeneous in
their origin and function.
It has been found that in peripheral nerves the ratio
between the number of intraneural capillaries and
MNFs is variable and depends both on species and
the topographic location of the nerve. In the feline
ulnar nerve, this ratio was 1: 25 (Marcarian & Smith,
1968), while in the canine nerves examined by Melman
et al. (1981) it ranged from1: 78 to 1: 93. In the human
intracardiac nerves examined by us, the ratio between
the number of capillaries and MNFs varied sig-
nicantly ranging from 1: 3 to 1: 41, and was evidently
related to nerve diameter: the thicker cardiac nerves
supplied by a larger number of capillaries also
contained more MNFs.
With respect to the ultrastructure of the MNFs
within the human cardiac nerves, no specic features
distinct from typical thin peripheral MNFs were
detected.
Internal structure of axons
In the literature, sensory nerve terminals have mostly
been described as bres with varicosities ranging from
1n5 m to 3 m in diameter and containing multiple
mitochondria, large granular vesicles, glycogen
particles, and lamellar bodies (Chiba & Yamauchi,
1970), or as varicosities with clear axoplasm and
plentiful mitochondria (Novotny et al. 1995). Axon
varicosities observed in the human intracardiac nerves
varied in their contents. Many were lled with
numerous neurolaments, microtubules, large dense-
core vesicles and cisternae of the agranular ER.
Others contained abundant small clear vesicles and
mitochondria in addition to sparse large dense-core
vesicles and ER cisternae. However, axon varicosities
with plentiful mitochondria, glycogen particles, dense
lamellar and multivesicular bodies as well as small
clear and large granular vesicles were the most usual
within the human cardiac nerves. We suggest on the
basis of generally recognised descriptions of sensory
nerve bres (Chiba & Yamauchi, 1970; Novotny et al.
1995) that the latter varicosities should be considered
as belonging to sensory nerve bres. Therefore, it is
concluded that the human intracardiac nerves pre-
sumably carry cardiac sensory innervation.
Concluding remarks
The present study shows that the ultrastructure of the
intracardiac nerves in healthy normal humans in-
cludes many features (axon varicosities, laminated
Schwann cell processes unassociated with axons, the
number and atypical morphometry of the capillary
456 N. Pauziene, D. H. Pauza and R. Stropus
endothelial cells, fenestration of the endoneurial
capillaries, etc.) that have not featured in previous
electron microscopic descriptions of the animal
nerves. The presence and complexity of the coats in
the human intracardiac nerves as well as the blood
supply of these nerves depend directly on nerve
diameter. The structural diversity of the axon contents
in the human cardiac nerves may reect their
functional dierences and is not associated either with
their location in the organ or with the diameter of the
nerve. Results reported here may be a useful frame-
work for assessing and interpreting the degree of
nerve damage in autonomic and sensory neuropathies
aecting the human heart.
:ciNovirbcrxrN1s
Special thanks are due to Mr Riciardas Strolia and
Mrs Rima Masiene for excellent technical assistance.
We are exceptionally grateful to Professor P. K.
Thomas and Edward Fenton from the Department of
Clinical Neurosciences, Royal Free and University
College Medical School (London) as well as to
Professor David A. Hopkins from the Department of
Anatomy and Neurobiology, Faculty of Medicine,
Dalhousie University (Halifax, Nova Scotia, Canada)
for giving of their time in careful reading of the
manuscript, constructive criticisms and friendly edi-
torial assistance. This study was supported in part by
a grant from the Lithuanian State Science and Studies
Foundation (No. 364).
rrrrrrNcrs
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