Innervation of the Cerebral Superficial Arteries and the Parenchymal Small Vessels in Elaphe quadrivirgata(Endocrinology)

8 Pages·1991·13.2 MB·English

by  Masahiko Kuroda

ZOOLOGICAL SCIENCE 8: 751-758 (1991) g 199] ZoologicalSocietyofJapai
Innervation of the Cerebral Superficial Arteries and the
Parenchymal Small Vessels in Elaphe quadrivirgata
Masahiko Kuroda, Takasuke Tagawa, Yumiko Enomoto
and Chiho Hirota
Department ofAnatomy, School ofMedicine, Fukuoka University,
Fitkuoka 814-01, Japan
—
ABSTRACT The innervation ofthe eerebral arteries in the striped snake. Elapha quadrivirgata, was
studied b) catecholamine fluorescence, acetylcholinesterase (AChE)-activation staining, and immuno-
histochemistr\. Aminergie nerve fibers (Amn) showed dense meshworks of fine fibers not only in the
eerebral carotid and basilar arteries but also in the cerebral parenchymal small vessels. Cholinergic
nerve fibers (Chn) were distributed more densely than Amn in the eerebral carotid and basilararteries,
but were sparse in thecerebralparenchymalsmallvessels. Thevasoactiveintestinalpolypeptide (VIP)-,
substance P(SP)-.neurokininA(NKA)-andcalcitoningene-relatedpeptide (CGRP)-like immunoreac-
tive (LI) nerve fibers were very densely distributed in the cerebral carotid and basilar arteries, and
moderately distributed in the cerebral parenchymal small vessels. Neuropeptide Y (NPY)-LI nerve
fibers were not detected in the superficial cerebral arteries, but they were distributed in the cerebral
parenchymalsmallvessels. NodifferencesofthedistributionsofAmn. Chn andpeptide-LI nerve fibers
were observed in the normal halfsideorin the undeveloped halfofthecerebralcarotid arteries. When
examined by double staining. SP-. NKA- and CGRP-LI nerve fibers showed almost identical
distribution patterns. Three different patterns existed concerning the relationship between Chn and
YIP-LI nerve fibers: 1) Chn and VIP-LI nerve fibers coexisting, 2) Chn living alone, and 3) VIP-LI
nerve fibers livine alone.
studies were performed only for the superficial
INTRODUCTION
cerebral arteries in mammals and the examination
It has been thought that the cerebral blood of superficial cerebral arteries or parenchymal
vessels consist of the cerebral carotid and basilar small vessels in submammalian animal species has
systems which are controlled by aminergie nerve rarely been performed.
fibers (Amn) and cholinergic nerve fibers (Chn) Using immunohistochemical techniques, distri-
whose neurotransmitters are noradrenaline and butional patterns and the connection ofAmn. Chn
acetylcholine, respectively, with these neurotrans- and peptide-LI nerve fibers, including substance P
mitters playing a main role in vasomotor modula- (SP)-, neurokinin A (NKA)-, calcitonin gene-
tion [1-9]. related peptide (CGRP)-, vasoactive intestinal
Recently, immunohistochemical developments polypeptide (VIP)- and neuropeptide Y (NPY)-LI
have enabled the discovery of a number of pep- nerve fibers were examined in the superficial cere-
tide-like immunoreactive (LI) nerve fibers in the bral arteries and parenchymal small vessels oi
walls of cerebral blood vessels and. as a result, it Elaphe quadrivirgata, which have a specific vascu-
appears that vasomotor modulation involves a lar system consistingof an undeveloped hall side ol
complicated neuro-control mechanism [10-20]. the cerebral carotid artery.
However, Since these findings have all been re-
ported separately, a connection among the various MATERIALS \M) METHODS
nerves has not been elucidated. Moreover, these
\ group ol 30 striped snakes. /;. quadrivirgata,
Accepted April 1. 1991 were used in this study. To detect Amn and Chn.
Received September 3, 1990 the catecholamine fluorescence technique by
752 M. Kuroda, T. Tagawa, Y. Enomoto and C. Hirota
Falck-Hillarp [21] and the acetylcholinesterase que and in frozen slices using an AChE-activation
(AChE)-activation staining technique by staining technique, respectively.
Karnovsky-Roots [22] were used. The detection of After fixation by perfusion with cold Zamboni's
these nerveshas been already reported in detail by solution, the whole-mount preparation of the su-
Tagawa et al. [23]. perficial cerebral arteries, was re-fixed with the
In the immunohistochemical studies, each pep- same solution at4°Cfor48hr, andreactedfor 1 hr
M
tide-LI nerve fiber was detected using the perox- with 0.1 phosphate buffer saline (PBS) contain-
idase-antiperoxidase (PAP) technique [24] or the ing 1% H2 2 to abolish the non-specific perox-
avidin-biotin peroxidase complex (ABC) techni- idase reaction. ThePAPtechniquewasperformed
M
que [25], in the superficial cerebral arteries or the while washing with 0.1 PBS containing 0.3%
parenchymal small vessels, respectively. Triton-X (PBST). After treatment with anti-SP,
After fixation by perfusion with cold Zamboni's anti-NKA, anti-CGRP, anti-VIP and anti-NPY
solution [26], the cerebral parenchym was post- antibodies (at 4°C for 2hr each) followed by
fixed in block form at 4°C for 24hr with the same washing with PBST, the slices were reacted with
M
solution. The block was incubated in 0.1 phos- goatanti-rabbitIgG (CappelPA, 1 :200) at4°Cfor
phate buffer (PB) containing 10% and 20% su- 24hr and again washed with PBST. They were
crose at 4°Cfor48 hreach andmounted in gelatin. thenreactedwith anti-rabbitPAP(Zymed, 1 :200)
After re-fixation with Zamboni's solution at 4°C at 4°C for 2hr and stained with a solution of
M
for 24hr and incubating in 0.1 PB containing 3,3'-diaminobenzidine (DAB, Sigma). Lastly, the
10% and 20% sucrose at 4°C for 48 hr each, the slices were dehydrated, mounted and observed
block was frozen with dry-ice isopentane and 15- under a microscope.
20jum slices were reacted with the ABC reagent Thecoexistence ofan autonomicneurotransmit-
(Vector Inc., ABC KIT). The slices were treated ter and a peptide, or a peptide and a peptide was
with the first antibody (anti-SP, anti-NKA, anti- examined using the double staining technique. In
CGRP, anti-VIP and anti-NPY antibody) and the the double staining of VIP-LI nerve fibers and
biotin conjugated second antibody at room Chn, VIP-LI nerve fibers were stainedbythe PAP
temperature for 30min each and the ABC reagent techniqueusing4-chloro-l-naphtholandnext, Chn
at room temperature for 60min. They were then was identified with the AChE-activation staining
stained with peroxidase-substrate solution for 8 technique. Double staining with SP-NKA or SP-
min, counter-stained with methyl-green. Then the CGRP was examined using a fluorescein labeled
parenchymal small vessels were observed under antibody technique [27]. The slices were treated
the microscope. Amn and Chn in the cerebral with anti-rabbit NKA or anti-rabbit CGRP anti-
parenchym were observed in paraffin embedded body at 4°C for 48hr, followed by washing, and
slices using a catecholamine fluorescence techni- reacted with tetramethylrhodamine isothiocyanate
Table 1. Details of antibodies used
First antibody Antibody code Host Dilution Sourse Reference
SP FP03 Rabbit 1:5000 Dr. H. Kamiya Takano et al.
Fukuoka Univ. [33]
NKA FK03 Rabbit 1 :5000 Dr. H. Kamiya Takano et al.
Fukuoka Univ. [33]
CGRP R.842701 Rabbit 1 :1200 MMiallambo, Sweden U[3d4]dman et al.
VIP R.785202 Rabbit 1 :1200 Milab Matsuyama et al.
Malmo, Sweden [13]
NPY R.840403 Rabbit 1 :1200 Milab Itakura et al.
Malmo, Sweden [29]
Mo-SP B7A35 Rat 1:500 sera-lab Cuello et al.
England [35]
a
Cerebral Vascular Innervation in Snake 753
(TRITC)-labeled anti-rabbit [gG (SEIKAGAKU
RESULTS
KOGYO Co.. 1 :200) at 38°C for 1 hr. Then, the)
were reacted with anti-rat monoclonal SP antibody A schematic illustration of the angio-
at 4 C tor 48 hr. washed and again reacted with architecture of the cerebral arteries in E.
fluorescein isothiocyanate (FITC)-labeled anti-rat quadrivirgata is shown in Figure 1 and the distribu-
IgG (ICN. 1 :200) at 38°C for 2 hr. The sliceswere tional densities ofAmn, Chn and pcptidc-LI nerve
washed, mounted with glycerin-PBS (1:1) and libers in the superficial cerebral arteries and the
observed under a fluorescence microscope. parenchymal small vessels is shown in Tabic 2.
The names of the companies at which the first Both the cerebral carotid and basilar arteries were
antibodies were prepared, and their dilution rates found to be well innervated by Amn. Amn in the
are shown in Table 1. In order to examine the cerebral carotid and basilar arteries showed a
specificities of the antibodies, absorption tests dense distribution consisting of meshworks of fine
were performed. fibers (Figs. 2, 3). These fine Amn were also
evident in the cerebral parenchymal small vessels
as well as in the superficial cerebral arteries (Fig.
4). In addition, the cerebral carotid and basilar
1 arteries were well innervated by Chn with a very
dense distribution of meshworks (Figs. 5, 6),
although the distribution wassparse in the cerebral
parenchymal small vessels (Fig. 7).
The distribution of VIP-LI nerve fibers was
markedly dense, covering the cerebral carotid and
basilararteriesand the cerebral parenchymal small
vessels (Figs. 8-10). Although both SP- and
NKA-LI nerve fibers were distributed rather de-
nsely in both the cerebral carotid and basilar
arteries (Figs. 11, 12, 14, 15), their distributions in
the cerebral parenchymal small vessels were far
more dense as compared with the cerebral carotid
and basilar arteries (Figs. 13, 16). CGRP-LI nerve
fibers revealed a very dense distribution over the
cerebral carotid arteries (Fig. 18), but their dis-
tributions in the basilar arteries and the paren-
chymal small vessels were only moderately dense
(Figs. 17, 19). Although NPY-LI nerve fibers
could not be observed in eitherthe cerebral carotid
or basilar system, they were found with NPY-
positive nerve cells in the cerebral parenchyma.
No differences were found among the distribution-
al densities of Amn. Chn or pcptidc-LI nerve
Elaphe quadriviryata atra fibers in the cerebral carotid arteries in the normal
Fig. 1. Diagram of the arterial supply to the ventral half side or in the undeveloped half.
surface of the brain in the Elaphe quadrivirgata. A similar distributional pattern was shown
me.a. ethmoidal artery: a.r.a. anterior cerebral among SP- and NKA-I.I or SP- and CGRP-LI
artery: c.e.a. cerebro-cthmoidal artery: m.c.a. mid- nerve fibers when examined by double Staining
dblrealcecraerbortaildaratretreyr:y:r.ar..pa.nteproisotreriroarmusr.amcu.sC.:a.p.cerr.ea.- (1 igS. 20. 21). However. VIP-I.I nerve fibers and
posterior cerebral artery; b.a. basilar artery: v. ( hn were found in three patterns: 1 ) VIP I I n
vertebra] aretr\ fibers and Chn existing together, 2) VIP-I.I fibers
.
754 M. Kuroda, T. Tagawa, Y. Enomoto and C. Hirota
Fig. 2. Aminergic innervation ofthe cerebral artery. Arrow indicates Amn. X100.
Fig. 3. Amn (arrow) ofthe cerebral carotid artery and its branch, a. cerebralcarotid artery; b. posteriorramus; c.
anterior ramus X100.
Fig. 4. Amn (arrow) which distributed in the small vessel ofcerebral parenchym. X250.
Fig. 5. Chn (arrow) ofthe basilar artery. XlOO.
Fig. 6. Chn (arrow) ofthe cerebral carotid artery and its branch, a. cerebral carotid artery; b. posteriorramus; c.
anterior ramus X100.
Fig. 7. Chn (arrow) in the cerebral parenchymal small vessel. XlOO.
Fig. 8. VIP-LI nerve fiber (arrow) ofthe basilar artery. X100.
Fig. 9. VIP-LI nerve fiber (arrow) of the cerebral carotid artery and its branch, a. cerebral carotid artery; b.
posterior ramus; c. anterior ramus XlOO.
Fig. 10. VIP-LI nerve fiber (arrow) which distributed in the cerebral parenchym. X160.
Table2. Density of Amn, Chn and peptide-like immunoreactive (LI) fibers of cerebral arteries and
intracerebral parenchymal small vessels in Elaphe quadrivirgata
SP-LI NKA-LI CGRP-LI VIP-LI NPY-LI
Amn Chn fiber fiber fiber fiber fiber
cerebral carotid arteries 4f tt # 4f # Iff -
-
basilar arteries # 1 H- 4f 4f ttr
intracerebral parenchymal M- ± ttf ttf -H- +ft +f
small vessels
The relative number of nerves was graded arbitrarily; — no nerve was observed
±. few nerves
+ moderate number of nerves
4f rich supply of nerves
-H+ very rich supply of nerves
Cerebral Vaseular Innervation in Snake 755
14 IS >s 16
I
/
@
a
.-A
\
\
B
Fig. 11. SP-LI nerve fiber (arrow) of the basilar artery. X100.
Fig. 12. SP-LI nerve fiber (arrow) of the cerebral carotid artery and its branch, a. cerebral carotid artery; b.
posterior ramus; c. anterior ramus X100.
Fig. 13. SP-LI nerve fiber (arrow) which distributed in the cerebral parenchym. x 160.
Fig. 14. NKA-LI nerve fiber (arrow) of the basilar artery. X100.
Fig. 15. NKA-LI nerve fiber (arrow) of the cerebral carotid artery and its branch, a. cerebral carotid artery; b.
posterior ramus; r. anterior ramus X100.
Fig. 16. NKA-LI nerve fiber (arrow) which distributed in the cerebral parenchym. x 160.
Fig. 17. CGRP-LI nerve fiber (arrow) of the basilar artery, x100.
Fig. 18. CGRP-LI nerve fiber (arrow) of the cerebral carotid artery and its branch, a. cerebral carotid artery; b.
posterior ramus; c. anterior ramus x 100.
Fig 19. CGRP-LI nerve fiber (arrow) which distributed in the cerebral parenchym x 100
} !'. 20. SP-LI (A) and NKA-LI (B) nerve fibers of the posterior cerebral artery. K KM).
1 i'. 21. CGRP-LI (C) and SP-LI (D) nerve fibers of the posterior cerebral artery. - 100.
Fir, 22 Chn and VIP-LI nerve fiber ofthe posterior cerebral artery. Large arrow indicates YIP I I nerve fiber and
small arrow Chn. x100.
observed alone and 3) Chn observed alone (Fig. DISCUSSION
Nearly comparable amounts of Ami) and ( hn in
the superficial cerebral arteries were reported in
756 M. Kuroda, T. Tagawa, Y. Enomoto and C. Hirota
rats [8] and larger amounts of Amn as compared longation of superficial and deep vessels was not
with Chn were reported in domestic fowls [23] and elucidated. Nevertheless, it seems that the exist-
tortoises [6], while, in bullfrog [7] or lamprey [28], ence of NPY-positive nerve cells and NPY-LI
Amn or serotonergic nerves alone were found. nerve fibers in the cerebral parenchyma suggests
The distributional densities of both Amn and Chn an objective for the study ofthe origin ofpeptide-
were higher in the cerebral carotid system than in LI nerve fibers in the future.
the basilar system in rats [8], domestic fowls [23] In mammalians the coexistence of Amn, Chn
and tortoises [6], whereas, in bullfrogs [7], Amn and peptide-LI nerve fibres or that of differing
alone was found, with larger amounts in the cere- peptidergic nerve fibers was reported. In the
bral carotid system than in the basilar system. cerebral arteriesofE. quadrivirgata, themodeand
However, in E. quadrivirgata, the distributional the density of distribution were mutually equal
density of Amn was nearly equal in both the between SP- and NKA-LI nerve fibers and be-
cerebral carotid and basilar systems and a dense tween SP- and CGRP-LI nerve fibers and accor-
meshworks offine Amn was found in all the areas dingly, the coexistence ofSP, NKA and CGRP in
of the cerebral carotid and basilar systems. Chn the same fiber was suggested. The coexistence of
was nearly equally and densely distributed in both SPandCGRPinthecerebralarteriesortrigeminal
arterial systems. Chn density was higherthan that ganglion was reported by Hanko etal. [18] in cats
of Amn in both systems. These findings suggest and Matsuyama etal. [17] in rats and guinea pigs.
that Amn and Chn possessed different densities at AlthoughLundbergetal. [31] reportedthecoexist-
each site depending upon the species. ence of VIP-LI nerve fibers and Chn in cat sub-
Amongthe peptide-LI nerve fibers, SP-, NKA-, maxillary glands and Kobayashi et al. [11] as well
CGRP- and VIP-LI nerve fibers were each found as Hara et al. [20] reported this in rat cerebral
to possess a similar density between the cerebral arteries, the distributional mode of VIP-LI nerve
carotid and basilar systems. The distributions of fibersandChninthesuperficialcerebralarteriesof
CGRP- and VIP-LI nerve fibers were more dense E. quadrivirgata was mutually different in spite of
compared with those of SP- and NKA-LI nerve theirsimilar distributional density. Three patterns
fibers in both the system. These findings are not were observed: 1) the coexistenceofVIP-LInerve
coincidental with the results previously presented fibersandChn, 2)VIP-LInervefibersaloneand3)
[11, 17, 29, 30], in which the distributions of Chn alone. This is coincidental with a connection
peptide-LI nerve fiberswere higherinthe cerebral between VIP-LI nerve fibers and Chn in the cere-
carotid system than in the basilar system. Howev- bral arteries of hamsters [unpublished] and
er, according to a study in our laboratory on Leiothrix lutea [unpublished] examined in our
hamsters, a difference in the distributions of SP- laboratory.
and CGRP-LI nerve fibers was not observed be- Amn [2, 3] and NPY [14, 32] have a vasocon-
tween these two arterial systems, although VIP-LI strictive action and Chn [4, 9] and VIP [10, 14]
nerve fibers alone possessedahigherdensityin the have a vasodilative action. Although the direct
cerebral carotid system. These findings suggest action of SP, NKA and CGRP for the vascular
that and the distributional mode and density of smooth muscle is vasoconstrictive, it is said to be
peptide-LI nerve fibers in the cerebral arteries vasodilative when vascular endothelia exist
differ considerably with each order or species. together. Eitherway, elucidationoftheoriginand
The distribution in the cerebral parenchymal the fibrous prolongation as well as the function of
smallvesselsofeach kindofnervevariedconsider- each kind of nerve was not possible because an
ably from those in the cerebral carotid and basilar anesthetic technique for E. quadrivirgata has not
systems; the SP-, NKA- and VIP-LI nerve fibers been established and their survival after biopsy is
were distributed very densely while the Chn alone impossible. In the future, an anesthetic technique
were very sparse. Survival ofthe reptiles was rare should be developed and various morphologic,
and like in the case of birds after anesthesia for pharmacologic and physiologic studies of these
experimental purposes, the origin and fibrous pro- remaining questions should be undertaken.
Cerebral Vascular Innervation in Snake 757
tribution of vasoactive intestinal polypeptide-
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