Neringa Pauziene et al- Morphology of human intracardiac nerves: an electron microscope study

8/3/2019 Neringa Pauziene et al- Morphology of human intracardiac nerves: an electron microscope study

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J. Anat. (2000) 197, pp. 437459, with 15 figures Printed in the United Kingdom 437

Morphology of human intracardiac nerves: an electronmicroscope study

N E R I N G A P A U Z I E N E 1, 2, D A I N I U S H . P A U Z A 2 AND RIM VYDAS S TROPUS 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)

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 differ 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

fibres 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 fibres showed normal ultrastructure,

although a number of profiles displayed a variety of different 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.

The pivotal significance of the intracardiac nervous

system in influencing cardiac rate, atrial and ven-

tricular refractoriness, coronary artery blood flow,

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 inflammation 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 ratherlimited (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.


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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 fibroblasts, 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 fine morphology of the

intracardiac nerves in relation to their topography

and that of the intracardiac ganglia because recent

neuroanatomical findings indicate that they may be

related (Pauza et al. 1999).

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 difference 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 coronarysinus were taken in order to compare them with those

that were situated epicardially. However, since major

ultrastructural differences 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 m): 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 fixative containing 2% paraformaldehyde and

2n5% glutaraldehyde in 0n1 cacodylate buffer (pH

7n4) for at least 4 h at room temperature or overnight

at 4 mC. Afterwards, the samples were postfixed for

2 h with 1% osmium tetroxide solution in 0n1

cacodylate buffer (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 films.

Terminology

The terminology of Pannese (1994) was used to

describe the intrinsic neural structures. According to

this terminology, a nerve fibre (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 fibres

was not surrounded by perineurium and epineurium.

An assemblage of nerve bundles surrounded byconnective tissue sheaths, i.e. endoneurium, peri-

neurium or\and epineurium was denoted as a nerve.

The fine meshwork of collagen fibrils that closely

surrounds a nerve fibre 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 m or more

were classed as thick. For myelinated fibres, the g

ratio (axon diameter:total fibre diameter) was used,

438 N. Pauziene, D. H. Pauza and R. Stropus


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A

B

89

7

6

Right inferior pulmonary veinSuperior vena cava

Inferiorvenacava

n = 12n = 10

n = 62

n = 19

Left inferiorpulmonaryvein

4n = 5

3

5

2

1n = 5n = 7

n = 14n = 4

Right superior pulmonary veinSuperior vena cava

Left inferiorpulmonaryvein

Left atrial neural fold

Fig. 1. Drawings of ventral (a) and dorsal (b) views of thepressure-inflated 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 circumflex

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 nervediameter and the number of perineurial cell layers was

analysed using linear regression (Microcal Origin v.

4n00).

Intracardiac nerve sheaths

Epineurium

Thick epicardial nerves were regularly enveloped by

well-developed epineurium that was mostly composedof collagen fibrils (Fig. 2a). Epineurial collagen fibrils,

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 fibres were occasionally

located (Fig. 2 a, c). Thin fibrils were also regularly

distributed in the epineurium.

In the thin nerves, either epicardial or myocardial,

the epineurium was sparse, both the bundles of

collagen fibrils and solitary collagen fibrils 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 layerswere present (Fig. 3a). In general, the number of

perineurial cell layers was related to nerve diameter

(Fig. 4 a). The interstices between perineurial cell

layers was occupied by collagen fibrils that were

longitudinally, obliquely or circularly oriented and

flattened against the basal laminae of perineurial cells,

including their invaginations (Fig. 3 a). Between the

bundles of collagen fibrils, isolated fibrils and elastic

fibres intervened. Some perineurial cells located in

adjacent layers were linked or separated by very few

Human intracardiac nerves 439


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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 fibres that are longitudinally (LC) and obliquely (OC) oriented compose the bundles. These

collagen bundles are separatedinto 4 layersby processesof the epineurial fibroblasts (EpF),between which a fewelastic fibres (E) arelocated.



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collagen fibrils (Fig. 3a). Occasionally, capillaries and

nerve fibres were located between the perineurial cells,

but no contacts between nerve fibres with neig-

hbouring cells were identified (Fig. 3bd).

The perineurium of the thin nerves that were less

than 100 m in diameter consisted of 12 cell layers

(Figs 4, 5 a). 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 classified as bundles of nerve fibres.

However, the presence of perineurial cells was not

strictly dependent on nerve diameter. Occasionally,

we detected nerves containing only a few un-

myelinated nerve fibres 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 wascommonly wide and occupied by irregularly dis-

tributed collagen fibrils, capillaries and fibroblasts

(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. 5 a).

Endoneurium

Endoneurial collagen fibril density was variable. In

most nerves, fibrils were more or less evenly dis-

tributed around nerve fibres (Fig. 2a). In some

intracardiac nerves examined bundles of fibrils were

widely separated by gaps containing solitary fibrils

(Figs 5a, 6b). In third type, the endoneurium was

extremely compact, the collagen fibrils being tightly

packed between nerve fibres (Fig. 6 a). These 3 types

of endoneurium were frequently encountered in nerves

that were adjacent to each other. A correlation be-

tween the pattern of the endoneurium and 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 fibrils was

similar to that for the perineurium and ranged

(b) Epineurium of thin nerve containing very few collagen fibres that intermingle with surrounding connective tissue. (c) Epineurium of an

epicardiac nerve with the clearly visible elastic fibres (E). Bars, 1 m. Abbreviations for this and subsequent figures: Ad, adipose cell; AER,

agranular endoplasmic reticulum; Ax, axon; Bl, basal lamina; CC, circularly orientated collagen fibrils; E elastic fibres; EnC, endoneurial

collagen fibrils; En, endoneurium; Ep, epineurium; EpF, epineurial fibroblasts; Er, erythrocyte; F, fibroblast; GER, granular endoplasmic

reticulum; LC, longitudinally oriented collagen fibrils; m, microfibrils; Ma, macrophage; MC, mast cell; MNF, myelinated nerve fibre;

Nu, cell nucleus; OC, obliquely oriented collagen fibrils; P, perineurial cells; Pe, pericyte; Sch, Schwann cell; SF, subperineurial fibroblast;

UNF, unmyelinated nerve fibre; VL, vascular lumen; AxV, axon varicosity.

approximately from 40 to 60 nm. The Plenk-Laidlaw

meshwork was well developed only around myelinated

nerve fibres, whereas it was indefinite around un-

myelinated fibres. In contrast to solitary macrophages

and mast cells, fibroblasts 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

identified. The number of intraneural capillaries

increased with nerve diameter (Fig. 4b). Epicardial

nerves that appeared flattened 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

fibres varied and ranged from 1:3 to 1:41.

Blood vessels of the intracardiac nerves

The outer diameter of the endoneurial capillariesranged from about 4 mt o 13 m (mean7n6p0n4 m;

nl28). Pericytes, completely or partly ensheathing

the capillaries, were also very common (Figs 3b, 8 a).

The fine 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 ; nl 13) 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, 8 a,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,



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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 filled by the longitudinally (LC), obliquely (OC) or circularly (CC) oriented collagen fibrils

while some perineurial cells from adjacent layers are linked (arrows). (b) A capillary and (c, d) nerve fibres 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.



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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, coefficient

of regression; n, the number of nerves from which the data were

obtained.

they sometimes possessed fine external processes that

came into contact with pericytes (Fig. 8c). Conversely,

on a few occasions we observed fine 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 wereextremely thin and\or possessed fenestrations (Fig.

8 d, e). Mostly, these fenestrations were clustered

unilaterally on one side of the capillary which

appeared as if closed by a very thin diaphragm (Fig.

8 e).

Unmyelinated nerve fibres

An analysis of unmyelinated nerve fibres (UNFs) or

Remak fibres revealed that the distribution of axons

within these fibres 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

significantly 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 individualcontained abundant UNFs with 2030 axons that

were very rare or entirely absent in the other specimens

examined (Fig. 10). Profiles of these multi-axon UNFs

were unusually irregular and their Schwann cells

contained collagen pockets (Fig. 12 b). In addition,

in a few thick nerves from the 22-y-old subject we

identified the largest UNF profiles that contained

more than 200 axons surrounded by entangled

Schwann cell processes. Although in the profiles of

such UNFs as many as 56 Schwann cell nuclei were

sometimes seen, this whole unmyelinated nerve fibre

complex was regularly enveloped by a common basallamina (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 few Schwann cell processes (Fig.

11a, b), while UNFs containing 58 axons were

regularly enclosed by 56 processes (Fig. 11c, d).

Profiles 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 UNFsof 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 fibre 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



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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 fibroblasts (F) in (b) which encircle unmyelinated nerve fibres

(UNF) with their processes and resemble perineurial cells. Bars, 1 m.



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Fig. 6. Electron micrographs showing different patterns of density of UNFs within endoneurium of human intracardiac nerves. ( a) Compact

pattern of distribution of UNFs with restricted endoneurial space that is filled with collagen fibrils. ( b) Sparse distributions pattern of UNFs,

in which bundles of endoneurial collagen fibrils are separated by wide gaps. Bars, 1 m.



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Fig. 7. Electron micrographs of a macrophage (a) and mast cell (b) within human intracardiac nerves. Bars, 1 m.

UNF (Figs 12a, 14 d, 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 endoneurium formed 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 fibrils 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 filaments

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 fibres

Myelinated nerve fibres (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 fibres was 0n60n7 (Fig. 13a). In

spite of this, thin nerve fibres 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 fibres varying from 0n73 m to 0n85 m in outer

diameter with axons thinner than 0n15 m were also

observed (Fig. 13c). These fibres had very low g ratio

(0n180n2) and therefore, appeared similar to thoseidentified as beaded by Ochs & Jersild (1987). The

structure and periodicity of the myelin sheath in such

fibres 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 neurofilaments. With certain exceptions,

described below, most transverse sections of axons

contained a few mitochondria and cisternae of

agranular ER that 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 neurofilaments of most axons were evenly

distributed throughout the cross section, but some

axons contained neurofilaments that were clustered



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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



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Fig. 9. Percentage distribution of axons (nl 3392) within un-

myelinated nerve fibres with differing 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 portionof the axon profile (Fig. 14 b, c). When bundles of

neurofilaments were present the axon profiles 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. 14 b, c).

Microtubules were usually evenly distributed within

clear axoplasm in UNFs (Fig. 14a) but some axons

differed both by having a higher density of the

axoplasmic matrix and more numerous microtubules

(Figs 13a, 14 eg). Commonly, these axons were very

irregularly shaped in transverse section. Some hadlong wing-like protuberances or were very flattened

(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 neurofilaments

that were present were difficult 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 definite

cardiac location. Occasional axons with an ordinaryinternal 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 from 1 m t o 5n2 m and containing

a variety of organelles. Some were richly filled with

neurofilaments, microtubules, mitochondria, cisternae

of agranular ER, and dense-core and clear vesicles

(Fig. 15 a, f). Varicosities of another type were larger

in diameter and incompletely enclosed by Schwann

cells (Fig. 15b). The axoplasm of 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. 15 c, d, f). The density of

the latter structures varied from low (Fig. 15c) to very

high (Fig. 15 d). When axoplasmic density was low,

most components of the varicosity were aggregated in

its central part, while neurofilaments 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 epicardialnerves occasionally possessed MNFs with axons that

were filled with abundant mitochondria, vesicles and

lamellar bodies (Fig. 13d).

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 orientedepineurial collagen fibrils are significant for nerve

protection from the effects 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 fibres.

The bundles of epineurial collagen fibrils were

820 m in width, this corresponding to that ofbundles of epineurial collagen in the rat facial and

sciatic nerves (Ushiki & Ide, 1990; Ishii & Takeuchi,

1993). Although the exact orientation of the collagen

fibrils 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



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Fig. 10. Transverse section of the intracardiac nerve from a 22-y-old individual in which unmyelinated nerve fibres involving a large number

of axons were frequently evident. Dashed lines frame the adjacent unmyelinated nerve fibres. Bar, 1 m.



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Fig. 11. Electron micrographs showing the structural organisation of unmyelinated nerve fibres 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.



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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 gapsbetween its collagen fibres were evident. Elastic fibres

and blood capillaries were absent within this thin

epineurium. Bundles of the epineurial collagen fibres

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 flat cells, between which zones of collagen

fibres are included (Ushiki & Ide, 1990). The thicknessof 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 adiffusion 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 fibres distributed between layers of

the perineurial cells form a dense and flexible

meshwork that not only provides mechanical support

for the nerve fibres, but also restrains the intra-

fascicular contents which are under greater pressure

than the extraneural environment (Ushiki & Ide,

1986, 1990; Ishii & Takeuchi, 1993). The findings of

the present study demonstrate that the thickness of

the perineurium in the human intracardiac nervesvaries 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



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Fig. 13. Electron micrographs showing the structural organisation of myelinated nerve fibres within human intracardiac nerves. (a) Two

typical MNFs with the g ratio of 0 n60n7. Note the UNFs that contain the dark axons (dark arrowheads). (b) MNF with a comparatively

thin myelin sheath. (c) MNF that resembles a beaded fibre 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 fibres 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 neurofilaments (clear arrowheads). (b, c) Axons of increased diameter filled with

numerous neurofilaments. (d) Irregularly shaped axon (Ax1) with a protuberance extending towards the endoneurium (En). Another axon

(Ax2) is normalin appearance. (eg) 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 particularfeatures compared with those that have been described

in a range of peripheral nerves (Thomas, 1963;

Gamble, 1964; Cumasov, 1975; Ushiki & Ide, 1986,



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Fig. 14. For legend see opposite.



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Fig. 15. Electron micrographs illustrating a variety of axon varicosities (AxV) in human cardiac nerves. The clear arrowheads indicate the

clearvesicles; blackarrowheads, largeand\or smalldense-core vesicles; smallarrows, glycogen particles; black arrows, multivesicularbodies;

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.



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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 fibroblasts,

while mast cells and macrophages are less common.

The present study confirms 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 collagenfibres and their bundles that are organized into 2

meshworks, coarse and fine (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 fibres that

follow the course of nerve fibres. Internal to this, the

fine or Plenk-Laidlaw meshwork of thin collagen

fibres lies in intimate contact with the nerve fibres.

Fibres of the Plenk-Laidlaw meshwork are irregularly

oriented. In the intrinsic nerves of the human heart,

the fine Plenk-Laidlaw meshwork is well developed

only around myelinated nerve fibres, while it is less

evident on unmyelinated fibres. 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 fibres 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 ddemonstrating 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 differed 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 flattened at their margins

and, because of this the shape of capillary lumena in

these nerves resembled irregular asterisks.

The findings of the present study provided evidence

that some of the intraneural capillaries in the humanheart are fenestrated. Although fenestrations were not

very abundant, they were typical in structure. This

finding 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 fibres

In some human peripheral nerves examined previously

(Gamble & Eames, 1964; Ochoa & Mair, 1969 a, b),Schwann cell profiles associated with unmyelinated

axons commonly were related to 12 axons. In the

human greater auricular nerve 2248% of the profiles

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, thinhuman nerves, in contrast to thick ones, had no UNFs

with more than 8 axons. Since these differences 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



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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 findings demonstrate that profiles

consisting of stacks of flattened 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 profile 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 differences

in their frequency was apparent in the humanintracardiac nerves examined, although they were

more common within large and complicated UNFs

than in smaller ones. Although the exact significance

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 fibres

The g ratio (axon diameter:total fibre diameter) is an

important measure in relation to myelinated fibre

function (Gillespie & Stein, 1983; Tuisku & Hilde-

brand, 1992 ; Pannese, 1994). Optimal conduction

occurs in myelinated fibres 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, fibres with g ratio

values of 0n5 as well as 0n80n9 were also occasionallypresent in the human intracardiac nerves. Excepting

the thin MNFs of the trigeminal alveolar nerve from

the cichlid fish 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 fibres 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 from 1: 78 to 1: 93. In the human

intracardiac nerves examined by us, the ratio between

the number of capillaries and MNFs varied sig-

nificantly 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 specific features

distinct from typical thin peripheral MNFs were

detected.

Internal structure of axons

In the literature, sensory nerve terminals have mostly

been described as fibres 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 filled with

numerous neurofilaments, 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 fibres (Chiba & Yamauchi, 1970; Novotny et al.

1995) that the latter varicosities should be considered

as belonging to sensory nerve fibres. Therefore, it isconcluded 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



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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 reflect their

functional differences 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

affecting the human heart.

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 ofClinical Neurosciences, Royal Free and University

College Medical School (London) as well as toProfessor David A. Hopkins from the Department ofAnatomy 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 StudiesFoundation (No. 364).

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Neringa Pauziene et al- Morphology of human intracardiac nerves: an electron microscope study

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