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Permeability barriers to cytochrome-c in nerves of adult and immature rats.

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Permeability Barriers to Cytochrome-C in Nerves of
Adult and Immature Rats
LESLIE T. MALMGRE" AND JOHN J. BRINK
Department of Biology, Clark University, Worcester,
Massachusetts 01610, U.S . A.
ABSTRACT
Nerves in the tongues of adult and immature rats were examined
with respect to their permeability to exogenous cytochrome-c (mol wt 12,000)
injected into the tongue. The distribution of cytochrome-c was determined in
cryostat sections on the basis of the peroxidase activity of this protein. Nerves
of 14-day-old rats were permeable to injected cytochrome-c. The larger nerves of
older animals showed only localized accumulations of cytochrome-c reaction
product both between and within axons adjacent to endoneurial blood vessels.
Reaction product was not found, however, in association with blood vessels penetrating nerves of the tongue that were not within the limits of tracer spread. In
the smallest nerve branches, thin linear strands of reaction product filled the
interstices between the nerve fibers.
Many proteins and dyes are partially or
entirely excluded from the endoneurium of
peripheral nerve after they are injected
into the blood or into the space around
the nerve (Waksman, '61; Waggener et al.,
'65; Mellick and Cavanagh, '66; Olsson,
'66; Olsson and Reese, '71; Aker, '72). It
has been suggested that a malfunction of
this barrier system may be involved in
peripheral nerve lesions such as argyria
and diphtheritic polyneuritis (Waksman,
'61), nutritional disorders (Olsson, '68),
and diabetic nerve lesions (Seneviratne,
'72).
Protein tracers injected into the space
around the sciatic nerve are prevented
from entering the endoneurium by the
perineurial epithelium of the rat (Waggener et al., '65) and the mouse (Olsson
and Reese, '71 ). Since horseradish peroxidase (mol wt 40,000) penetrates into the
deeper layers of the perineurium more
rapidly than ferritin (mol wt 500,000)
(Waggener et al., '65; Olsson and Reese,
'71), the perineurium may be more permeable to smaller proteins than larger proteins. Olsson ('67; '71) reported that of
nine animal species examined, only the
nerves of rats and mice were completely
impermeable to exogenous albumin and
globulin. Since previous investigations have
ANAT. REC., 181: 755-766.
used proteins with molecular weights of
40,000 and larger (Waggener et al., '65;
Olsson, '71; Olsson and Reese, '71), the
use of a smaller protein such as cytochrome-c (mol wt 12,000) may prove to
be a useful histochemical probe to further
characterize macromolecular permeability
barriers in rat peripheral nerve.
MATERIALS AND METHODS
The permeability of the perineurial epithelium to cytochrome-c was examined in
nerve branches of various sizes in the
tongues of 30 male Sprague-Dawley rats.
Two-month old (281-428 g), 1-month old
(112-152 g), and 14-day old (23-37 g)
rats were injected in the tongue with a solution of cytochrome-c (type 111, from horse
heart, Sigma Chemical Company, St. Louis,
Mo.) at a concentration of 10 or 100 mg/
ml in saline and a dose of 1.5-15 mg/kg
body weight. Controls were either uninjected or injected with an equal volume of
saline. One hour after injection, the rats
were anesthetized and perfused through
the heart (adult at 100 cm pressure; 14day at 30 cm pressure) with 4% formaldehyde (from paraformaldehyde) in pH 7.3
Received Ma. 29, '74. Accepted Sept. 16, '74.
1 This work was submitted i
n partial fulfillment for
the requirements of the Ph.D. degree.
755
756
LESLIE T. MALMGREN AND JOHN J. BRINK
(0.1 M ) phosphate buffer. Slices of tongue
were immersed in the same fixative for 45
minutes at 4°C and then run through several changes of 5% sucrose in 0.1 M phosphate buffer (pH 7.3) at 4°C for 1-2 hours.
Cryostat sections (16
thick) were cut
and incubated in 10 ml of freshly prepared
50 mM citrate buffer (pH 3.9) containing
35 IJ. of 30% HzOzand 20 mg 3,3'-diaminobenzidine-tetra-HC1 for 30-60 minutes at
room temperature (Karnovsky and Rice,
'69). Unstained sections were examined by
brightfield and phase contrast microscopy.
DISCUSSION
After sections were incubated as indicated, the endogenous peroxidase activity
of mitochondrial cytochrome-c (Anderson,
'72) was not detectable with the light microscope in nerves of control animals
(fig. 1).
The results indicate that the perineurial
epithelium of 1-month old and adult rats
impedes the penetration of exogenous cytochrome-c into the endoneurium from the
tissue space surrounding the nerve (figs. 2,
4). Although cytochrome-c is smaller than
proteins previously used in investigations
of this type, our results do not differ from
RESULTS
those obtained in similar studies of perNerves in tongue sections from uninmeability of the perineurium of larger
jected and saline injected control rats nerves in the mouse, rat or human (Wagwere free of endogenous peroxidase activ- gener et al., '65; Kristensson and Olsson,
ity (fig. 1).
'71; Olsson and Reese, '71; Soderfelt et al.,
One hour after injection of cytochrome-c '73).
into the tongue, the distribution of reacIt has previously been recognized that
tion product in the nerves of 1-month old the perineurial epithelium is necessarily
rats was the same as in those of adult discontinuous where blood vessels pierce
rats. Reaction product was concentrated the perineurium and that histological spein the epineurium and perineurium but did cializations at these sites may partially denot extend into the endoneurium of the termine the permeability characteristics
larger nerve branches that were not in the of the nerve (Olsson, '72). Little is known,
vicinity of the needle track (figs. 2, 5). In however, about the permeability of these
many of the larger branches however, re- sites to proteins that are normally lost from
action product was found within or sur- the blood into the tissue space surrounding
rounding axons bordering blood vessels the nerve (Olsson, '66; Olsson and Reese,
(figs. 4, 6) but not in the myelin of these '71). The presence of reaction product in
fibers. In some of the smaller nerve association with endoneurial blood vesbranches, thin linear strands of reaction sels after injection of cytochrome-c into
product fdled the interstices between nerve the tissue space surrounding the nerve
fibers (figs. 7, 8 ) .
branches of the tongue (figs. 2, 4, 5, 6)
When adult rats were injected with only may indicate a pathway into the endoneu1.5 mg cytochrome-c/kg body weight, the rium that is associated with the penetraarea of detectable reaction product did not tion of blood vessels through the perineuinclude the entire tongue. In these animals, rium. This type of channel has been demreaction product was associated with endo- onstrated by Burke1 ('67) who found that
neurial blood vessels within the limits of blood vessels that enter the endoneurium
tracer spread (fig. 2), but similar accumu- are surrounded by sleeves of perineurial
lations were not found in nerves in regions epithelium which are open-ended. Exogeof the tongue that were not included within nous proteins could move along the chanthe spread of reaction product (fig, 3 ) .
nel formed between this sleeve and the
After injection of cytochrome-c into the blood vessel and pass into the endoneurium
tongues of 14-day old rats, reaction prod- either through the single layer of sleeve
uct was diffusely distributed throughout or out of the open end of the channel.
the endoneurium (fig. 9). The density of
An alternative mechanism for the dereaction product was somewhat greater in position of cytochrome-c in association
the perineurium and epineurium and in with endoneurial blood vessels could inlinear strands within the endoneurium volve the uptake of injected cytochrome-c
into the blood followed by a loss of cyto(fig. 9).
NERVE BARRIERS TO CYTOCHROME-C
chrome-c from endoneurial blood vessels.
Since reaction product is not found near
blood vessels in nerves of the tongue that
are not within the limits of tracer spread
(fig. 3 ) , this second possible route is unlikely, Although this is indirect evidence
for the movement of cytochrome-c along
the channel formed between the blood
vessel and perineusial sleeve, the resolution of the light microscope does not permit localization of reaction product in this
channel. Ultrastructural studies are in
progress to examine the distribution of
cytochrome-c reaction product associated
with endoneurial blood vessels at the resolution of the electron microscope.
Reaction product associated with blood
vessels has been observed in several apparent stages of progressive penetration into
the endoneurium (figs. 2, 4, 5, 6). In some
cases reaction product appears to be localized within axons bordering blood vessels
but excluded from the myelin (fig. 6).
Krishnan and Singer ('73) have reported a
similar uptake of horseradish peroxidase
into the axons of newt peripheral nerve at
the node of Ranvier and to a lesser extent along internodal regions of the axon.
Since exogenous proteins injected into the
tongue undergo retrograde axonal transport (Kristensson et al., '71 ), the localization of reaction product at the time of
sacrifice might not reflect the site of initial
uptake of cytochrome-c into the axon but
rather material transported from remote
loci.
The small nerves in the tongue are probably more permeable to exogenous proteins
than larger nerves in the rat. Previous investigations have indicated that the sciatic nerve of the adult rat is impermeable
to albumin and horseradish peroxidase
(Kristensson and Olsson, '71; Olsson and
Reese, '71). On the other hand, Kristensson
et al. ('71) have found that when either
of these proteins are injected into the
tongue of the rat, they later appear in the
cell bodies of the hypoglossal nucleus after
retrograde axonal transport. This was interpreted as evidence that the branches of
the hypoglossal system in the tongue must
be permeable to proteins, but they did not
determine the sites of penetration. Waggener et al. ('65) have also reported that
smaller nerves are more permeable to exog-
757
enous proteins. They found that while the
rat sciatic nerve was impermeable to ferritin injected into the space around the
nerve, the small epineurial branches exhibited ferritin within Schwann cell cytoplasmic vacuoles. Furthermore, Burkel's
description of open-ended channels formed
in association with the penetration of blood
vessels into the endoneurium is based entirely on a study of the small termind
branches of nerves that supply rat extrinsic
eye muscles. Since there may be differences between smaller and larger nerves
with regard to structural organization and
permeability characteristics, it should not
be assumed that the permeability that we
have reported in association with the penetration of blood vessels into the endoneuriurn would also exist in larger nerves.
It has been found that the tissue space
of the endoneurium is continuous with
the tissue space surrounding the nerve at
the neuromuscular junction (Burkel, '67;
Saito and Zacks, '69). It is possible that
the reaction product that sometimes fills
the interstices between nerve fibers of very
small branches (figs. 7, 8) is due to the
penetration of cytochrome-c into the endoneurium at the neuromuscular junction.
Alternatively, Shantha and Bourne ('62)
have suggested that the greater permeability seen in terminal nerve branches may
be related to the fact that smaller nerve
branches have fewer layers of perineurial
epithelium ensheathing them,
Kristensson and Olsson ('71) have found
that albumin injected into the space around
the sciatic nerve penetrated into the endoneurium of immature rats but not into the
endoneurium of adult rats. Our results indicate similar age differences in the permeability of the small nerves of the tongue
to cytochromec (fig. 9). Cytochrome-c reaction product is diffusely distributed
throughout the endoneurium of 14-day old
animals (fig. 9 ) rather than limited to distinct linear accumulations by exclusion
from the myelin as in older rats (figs. 5-8).
The fact that formation and compaction
of myelin is still in progress in rat peripheral nerve 7 to 16 days after birth (Webster,
'71) may explain some of these age related
differences.
Waksman ('61) has pointed out that
rabbit nerve is impermeable to negatively
758
LESLIE T. MALMGREN AND JOHN J. BRINK
charged proteins such as diphtheria toxin
or serum albumin but partially permeable
to uncharged proteins such as serum
gamma globulin. Cytochrome-c (mol wt
12,000) is not only smaller than other proteins that have been used to investigate
the permeability of peripheral nerve, but
it is also a strongly cationic protein (PI
10.5). Schumaker ('58) demonstrated that
the binding of positively charged cytochrome-c to the cell surface of Amoeba
proteus induces extensive pinocytosis. Bock
('73) has reported that the accumulation of
horseradish peroxidase on the cell membranes of Schwann cells and axons may
be a result of their content of negatively
charged sites. The positive charge carried
by cytochrome-c could thus contribute to
its distinctive permeability characteristics.
ACKNOWLEDGMENT
The authors wish to thank Prof. Morris
J. Karnovsky of the Department of Pathology at Harvard Medical School for several
helpful suggestions in the preparation of
this manuscript,
LITERATURE CITED
Aker, F. D. 1972 A study of hematic barriers
in peripheral nerves of rabbits. Anat. Rec.,
174: 21-38.
1972 The use of exogenous
Anderson, W. A.
myoglobin as a n ultrastructural tracer. Reabsorption and translocation of protein by the
renal tubule. J. Histochem. and Cytochem.,
20: 672-684.
Bock, P. 1973 Adsorption von Meerettishperoxidase a n rutheniumot-positive Shuckturen
in markfreien Nerven. Acta Histochem., 46:
146-149.
Burkel, W. E. 1967 The histological fine structure of perincurium. Anat. Rec., 158: 177-190.
Karnovsky, M. J., and D. F. Rice 1969 Exogenous cyiochrome-c as a n ultrastructural
tracer. J. Histochem. and Cytochem., 17:
751-753.
Krishnan, N., and M. Singer 1973 Penetration
of peroxidase into peripheral nerve fibers. Am.
J. Anat., 136: 1--14.
Kristensson, K., and Y. Olsson 1971 The perineurium as a diffusion barrier to protein
tracers. Differences between mature and immature animals. Acta neuropath. (Berlin), 17:
127-138.
Kristensson. K.. Y. Olsson and J. Siostrand 1971
Axonal uptake and retrograde trhnsport of exogenous proteins i n hypoglossal nerve. Brain
Res., 32: 399-406.
Mellick, R., and J. B. Cavanagh 1966 The function of the perineurium and its relation to
the flow phenomenon within the endoneurial
spaces. Proc. Aust. Assoc. Neurol., 5: 521-525.
Olsson, Y. 1966 Studies on vascular permeahility in peripheral nerves. I. Distribution of circulating fluorescent serum albumin in normal,
crushed and sectioned rat sciatic nerve. Acta
Neuropath., 7: 1-15.
1967 Phylogenetic variations in the
vascular permeability of peripheral nerves to
serum albumin. Acta Path. et Microbiol. Scandinav., 69: 621-623.
1968 Studies on vascular permeability
in peripheral nerves. 3. Permeability changes of
vasa nervorum and exudation of serum albumin
i n INH-induced neuropathy of the rat. Acta
Neuropath., 1 1 : 103-112.
1971 Studies on vascular permeability
in peripheral nerves. IV. Distribution of intravenously injected protein tracers in the peripheral nervous system of various species. Acta
Neuropath. (Berlin), 17: 114-126.
1972 The involvement of the vasa
nervorum i n diseases of peripheral nerves. In:
Handbook of Clinical Neurology. Vol. 12, Chap.
24. P. J. Vinken and G . W. Bruyn, eds. NorthHolland Pub. Co., Amsterdam, pp. 644-664.
Olsson, Y., and T. S. Reese 1971 Permeability of
the vasa nervorum and perineurium in mouse
sciatic nerve studied by fluorescence and electron microscopy. J. Neuropath. and Exp.
Neurol., 30: 105-119.
Saito, A., and S. I. Zacks 1969 Ultrastructure
of Schwann and perineural sheaths a t the
mouse neuromuscular junction. Anat. Rec., 164:
379-3 90.
Schumaker, V. N. 1958 Uptake of protein from
solution by Amoeba proteus. Exp. Cell Res.,
15: 314-331.
Seneviratne, K. N. 1972 Permeability of Blood
nerve barriers in the diabetic rat. J. Neurol.
Neurosurgery and Psychiatry, 35: 156-162.
SGderfelt, B., Y. Olsson and K. Kristenssen 1973
The perineurium as a diffusion barrier to protein tracers i n human peripheral nerve. Acta
Neuropath. (Berlin), 25: 120-126.
Shantha, T. R., and G. H. Bourne 1962 The
perineural epithelium - A new concept. In:
The Structure and Function of Nervous Tissues.
Chap. 10. G. H. Bourne, ed. Academic Press,
New York, pp. 379-459.
Waggener, J. D., S. M. Bunn and J. Beggs 1965
The diffusion of ferritin within the peripheral
nerve sheath. A n electron microscopic study.
J. Neuropath. and Exp. Neurol., 24: 430443.
Waksman, B. H. 1961 Experimental study of
diphtheritic polyneuritis in the rabbit and
guinea pig. 111. The blood-nerve barrier in the
rabbit.
-- -- J. Neuropath. and Exp. Neurol., 20:
Bb--'l'l.
Webster, H. de F. 1971 The geometry of peripheral myelin sheaths during their formation
and growth in rat sciatic nerves. .T. Cell Biol..
48: s48-367.
PLATES
PLATE I
EXPLANATION OF FIGURES
760
la, b
Figure l a (phase contrast) shows a branch of the hypoglossal nerve
( N ) in the tongue of a 2 month old uninjected control. Figure l b
shows the same field with brightfield illumination. No endogenous
activity can be detected. x 390.
2
Cross section through a nerve in the tongue of a 2 month old r a t
1 hour after injection of 0.5 mg of cytochrome-c into the tongue.
Reaction product (broad arrows) does not extend past the perineurium and epineurium into the endoneurium ( E ) . Cytochrome-c
reaction product (thin arrows) is associated with endoneurial
blood vessels. x 390.
3a, b
Cross section (phase contrast and brightfield) through a nerve in
the tongue of the same animal as in figure 2 (0.5 mg cytochrome-c
at 1 hour. This nerve was not within the limits of tracer spread.
No reaction product comparable to that i n association with the
endoneurial blood vessels seen in figure 2 can be detected in the
nerves of this region (arrow). X 260.
NERVE BARRIERS TO CYTOCHROME-C
Leslie T. Malmgren and John J. Brink
PLATE 1
761
PLATE 2
EXPLANATION OF FIGURES
762
4
Cross section through a nerve in the tongue of a 2-month old rat
one hour after injection of 5 mg of cytochrome-c into the tongue.
Reaction product (arrows) surrounds axons near blood vessel (BV)
but has not penetrated into the axon in this case. x 1,320.
5
Longitudinal section through a nerve in the tongue of a 2-month
old r a t one hour after injection of 5 mg of cytochrome-c into the
tongue. Reaction product (broad arrows) does not extend past
the perineurium and epineurium into the endoneurium ( E ) . Cytochrome-c reaction product (thin arrows) is associated with an
endoneurial blood vessel. x 325.
6a, b
Cross section (phase contrast and brightfield) through a nerve in
the tongue of a Zmonth old rat one hour after injection of 5 mg of
cytochrome-c into the tongue. Reaction product can not be detected
in the myelin but appears to be present within some of the axons
adjacent to the blood vessel (arrows). x 1,350.
NERVE BARRIERS TO CYTOCHROME-C
Leslie T. Malmgren and John J. Brink
PLATE 2
763
PLATE 3
EXPLANATION OF FIGURES
764
7a, b
Cross section (phase and brightfield) through a small nerve branch
in the tongue of a 2-month old rat one hour after injection of 0.5
m g of cytochrome-c into the tongue. Cytochrome-c reaction product
(arrows) is present in the interstices between the nerve fibers.
X 1,160.
8
Longitudinal section through small nerve branch in the tongue of a
2-month old rat one hour after injection of 5 mg of cytochrome-c
into the tongue. Note thin linear strands of reaction product
(arrows) in the endoneurium. X 1,100.
9
Longitudinal section of a nerve in the tongue of a 14-day old rat
one hour after injection of 1 mg of cytochrome-c into the tongue.
Reaction product is diffusely distributed throughout the endoneurium ( E ) having the greatest density in the perineurial epithlium and epineurium (broad arrows) and i n the interstices between
the nerve fibers (thin arrows). x 1,980.
NERVE BARRIERS TO CYTOCHROME-C
Leslie T. Malmgren and John J. Brink
PLATE 3
765
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