Immunoelectron microscopic study of substance P-containing fibers in feline cerebral arteries

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Brain Research, 369 ( 1986~ 12-21! Elsevier

BRE 11530

Immunoelectron Microscopic Study of Substance P-Containing Fibers In Feline Cerebral Arteries LEE-YUAN LIU-CHEN, THEODORE M. LISZCZAK, JOAN C. KING and MICHAEL A, MOSKOWITZ Neurosurgery and Neurology Services, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114 and Department of Anatomy, Tufts University School of Medicine, Boston, MA 02110 (U.S.A.)

(Accepted July 23rd, 1985) Key words: substance P - - ultrastructure - - cerebral blood vessel - - immunohistochemistry - - sensory nerve fiber

The ultrastructure of substance P-containing fibers in feline cerebral arteries was examined by combining substance P immunohistochemistry and electron microscopy. At the light and electron microscopic level, positive fibers were observed in the adventitia and at the border between the adventitia and media, but not within the media or the endothelium. The substance P-containing fibers were unmyelinated with diameters consistent with C-fiber caliber. Positive axons were in close contact with Schwann cell processes. Positive axons contained 24 nm microtubutes, 10 nm neurofilaments, clear vesicles and scattered mitochondria. The number of mitochondria and organelles resembling vesicles appeared to increase in presumptive axon terminals. No synaptic membrane specializations were observed.

INTRODUCTION There is a paucity of information regarding the ultrastructural appearance of sensory fibers surrounding the cranial vasculature. At the light microscopic level, sensory fibers were recently found to project from small and large diameter neurons within the first division of the ipsilateral trigeminal ganglia in cats 33,34. Some of these trigeminovascular axons contain the putative neurotransmitter substance p33,34. This peptide resides in some small neurons in dorsal root ganglia and in unmyelinated C-fibers 16 and has been associated with the transmission of nociceptive information (see ref. 24 for review). Myelinated fibers have also been noted surrounding brain blood vessels 7,10.45,46. These were postulated to be of primary sensory origin because they failed to degenerate following superior cervical ganglionectomy and because they gave rise to a unique profile of unmyelinated axons containing few vesicles, but with numerous oval mitochondria3, 22,23. In two reports, elaborate Schwann cell-axonal complexes were also found in

the adventitial layer of cerebral vessels. These reportedly resembled specialized afferent terminations in skin 7,46. We determined previously that the majority of substance P-containing perivascular fibers originate from sensory neurons 32. Hence, major reductions in both the content of substance P (as measured by radioimmunoassay) and the number of immunohistochemically visualized fibers were found in the arteries of the circle of Willis following unilateral trigeminal ganglionectomy in cats and rats 33,47. Similarly, pretreating guinea pigs with capsaicin, a drug which decreased substance P in primary sensory neurons, abolished both the visualized fibers 13 and the content of this peptide within brain vessels 9. In addition, substance P immunoreactivity could be released from these fibers by capsaicin or high concentrations of potassium 41. Substance P is a potent cerebrovasodilatorn. All these observations suggest that substance P-containing fibers perform important regulatory functions in cerebral arteries. In the study reported herein, we describe the ultrastructural features of a

Correspondence: M.A. Moskowitz, Director, Stroke Research Lab., Massachuss~ts General Hospital, Boston, MA 02114, U.S.A.

0006-8993/86/$03.50 (~) 1986 Elsevier Science Publishers B.V. (Biomedical Division)

13 population of perivascular sensory fibers by combining substance P immunohistochemistry with electron microscopy. MATERIALS AND METHODS Five adult cats (3.5-6.0 kg) were anesthetized with ketamine hydrochloride (20 mg/kg, i.p.) and sodium pentobarbital (15 mg/kg, i.p.) and perfused through the aorta (after clamping the descending aorta) with approximately 400 ml of heparinized saline and then 500 ml of 5% acrolein (Polyscience, Warrington, PA) in 0.1 M phosphate buffer (pH 7.0, PB) containing 10 mM magnesium chloride25.26.53. After removing the brain, the organ was washed several times with 0.1 M PB at 4 °C. Large pial arteries were carefully removed using microscissors and an operating microscope and washed with 0.01 M phosphate-buffered saline (pH 7.0, PBS). Vessels were then treated with 0.01 M sodium meta-periodate in 0.05 M PB (15 min), washed in 0.05 M PB (3 × 5 min), treated with 1% sodium borohydride in 0.05 M PB (10 min), and incubated with 5% DMSO (10 min). Vessels were then processed for immunohistochemistry with the avidin-biotin-peroxidase complex (ABC) methodlS, 20 or the peroxidase-antiperoxidase (PAP) method 54. Tissues were incubated for 40-64 h at 4 °C with antisera raised in rabbits against substance P (Immunonuclear, Stillwater, MN, lot no. 27231) at a dilution of 1/3000. Following 3 rinses with 0.01 M PBS, tissues were incubated with either biotinylated goat anti-rabbit IgG (7.5 ~g/ml) (Vector Labs., Burlingame, CA) (for avidin-biotin-peroxidase method) or goat anti-rabbit IgG (Antibodies, Davis, CA) at a dilution of 1/80 (for peroxidase-anti-peroxidaxe method) on ice for 90-120 min. This was followed by rinsing 3 times with PBS before incubating with avidin-biotin-peroxidase complex (ABC, Vector Labs.) at 1/100 dilution or peroxidase-anti-peroxidase complex (SternbergerMeyer) at 0 °C or room temperature for 90-120 min. Tissues were then subjected to another 3 rinses with PB, reacted with a freshly prepared solution of 3,3'diaminobenzidine tetrahydrochloride (DAB) (0.006%) (Sigma) and hydrogen peroxide (0.0018%) for 5-10 min in 0.01 M PB. Brown reaction product identified structures containing substance P-like immunoreactivity (SPLI). Controls were treated as out-

lined above except that the primary antiserum was replaced with normal rabbit serum or antiserum pretreated with an excessive amount of SP (Beckman Instruments, Palo Alto, CA) at a concentration of 5-10 /~g/ml diluted antiserum for 16-24 h prior to use. The tissues were placed in 2% Dalton's osmium tetroxide in water at room temperature (1 h), rinsed with distilled water (10 min), reacted with uranyl acetate overnight, dehydrated through 70, 80, 95% ethanol (1 h each), 100% ethanol (two 30-min periods), and propylene oxide (two 30-min periods) at room temperature, incubated on a rotator plate at room temperature with propylene oxide/Epon 812 (50/50 and 25/75) (1 h each), 100% Epon (1 h x 2) and embedded in Epon 812 using a procedure described by King and Anthony zS. Tissues were placed on a sheet of Aclar (33c film, 7.8 mil, Allied Chemicals) in fresh Epon 812, coverslipped with glass slides previously coated with dimethyldichlorosilane (Sigma), and placed in an oven at 60 °C for 24-60 h. Tissues were then examined under a light microscope for the presence of SPLI. Ultrathin sections (60-80 nm) were cut with a microtome (LKB Ultratome 3) perpendicular to the long axis of blood vessels and placed on uncoated copper grids. Serial sections were cut parallel to the long axis of blood vessels and placed on formvar-coated grids. A Zeiss Em 9 electron microscope was used to view sections at 60 kV. Structures were considered immunopositive if two investigators (T.L., L.-Y. L.-C.) both agreed the axons possessed reaction product as shown by accumulations of electron-dense material over background. A micrometer was used to make morphological measurements from photographs of known magnification. Values represent the mean + standard deviation. RESULTS As reported in previous studies, SP immunoreactive fibers were observed in all cerebral arteries of cats examined at the light microscopic level, including the anterior, middle, and posterior cerebral arteries, both anterior and posterior communicating arteries, superior cerebeUar, anterior inferior cerebellar and posterior inferior cerebellar arteries as well as basilar arteries. There was no consistent variation in the density of SP immunoreactive fibers from vessel to vessel. Bundles of axons and numerous fine fibers

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Fig. 1. Wholemount preparation of cat middle cerebral artery. Note the large number of positive axons coursing in parallel and perpendicular directions to the long axis of the blood vessel. Beading is evident along individual fibers. Top ×334; bottom × 145.

(Fig. 1) were observed to course parallel and perpendicular to the long axis of the vessel, some in a 'barber pole configuration'. In many fibers, varicosities were seen at irregular intervals. Control vessels incubated with normal rabbit serum and SP antiserum pre-absorbed with an excess amount of synthetic SP (5 ktg/ml diluted antiserum) did not exhibit immunoreactivity. Under the electron microscope, numerous myelinated and unmyelinated nerve fibers were found in the periadventitia, adventitia and at the border between the vessel adventitia and the media. No fibers were observed in the tunica media. Reaction product was present only in unmyelinated fibers with diameters of 350 + 170 nm (95 axons measured from 2 cats). Within the periadventitia, a single Schwann cell ensheathed both SP-positive and negative fibers within fascicles (Fig. 2). The ratio of SP-positive-tonegative fibers in this fascicle was 2 to 8. Overall, the percentage of positive fibers in 5 fascicles containing 92 axons was approximately 9%. Positive axons are

associated with Schwann cells processes and contained 24 nm microtubules, 10 nm neurofilaments, scattered mitochondria, clear vesicles, and reaction product associated with all membranes (Fig. 2C). Some relatively large diameter immunonegative axons (419 + 209 rim, n -- 7) contained 24 nm tubules and electron lucent vesicles (with diameter of 50 + 4 rim, n = 176) and represent boutons en passant (Fig. 3a). Many of the vesicles were oval. Their smallest dimension was measured in all instances. Schwann cells and their processes surrounded fascicles and single axons. Within the tunica adventitia, SP-positive fibers associated with Schwann cell processes were seen coursing through the adventitia at distances as close as 196 nm from muscle cells (Fig. 3b). These axons presumably represented the most distal branches of SPLI-containing fibers and might be analogous to free nerve endings observed in other tissues. Because reaction product was not observed in the sparse population of myelinated fibers, it is assumed that free nerve endings probably derive from immunopositive unmyelinated axons. Terminal axons were often found adjacent to immunonegative boutons. The basal lamina was poorly distinguished owing to the employed fixation and histochemical conditions. Schwann cells surrounding fascicles appeared similar to those surrounding individual axons and contained relatively few mitochondria, some free ribosomes, and nuclei with marginated heterochromatin. Vesicle-containing positive terminal fibers represented a relatively small proportion of all immunopositive fibers visualized in the adventitia. Positively stained axons were oval in appearance. Axons contained as many as 5 somewhat oval and rounded appearing mitochondria with 3 or less cristae. An infrequent mitochondria was found in negative axons. Parallel arrays of filaments and tubules were not seen in these axon terminals. The immunoreaction product was associated with all cellular membranes and at times present in vesicle-like organelles. In most cases, the ubiquity and denseness of the reaction product obscured the ultrastructures of the subcellular organelles. In rare instances in which the reaction product did not obscure vesicles, a diameter of 110 --, 7 nm (n = 7) was measured which is similar to measurements of SP-positive vesicles within lamina I and II of the dorsal horn of cat spinal cord (108 + 10 nm, n = 6), and

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Fig. 2.a: Sclawann cell ensheaths two substance P-positive (arrows) and 8 immunonegative adventitial axons. Fibroblast (fibro), subarachnoid space (sas). x 11,450 (bar = 1 ~m). b: unmyelinated nerve fascicle contains 30 immunonegative and immunopositive axons. A myelinated axon lies adjacent, x 22,250 (bar = 1 am). c: two immunopositive axons contain mitochondria (m), 24 nm tubules, and vesicles (v). Collagen bundles and negative axons are present in the immediate vicinity. Schwann cell processes can be seen ensheathing these positive axons, x 14,150 (bar = 1 ~m).

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Fig. 3.a: a nerve fascicle contains unmyelinated axons, two enlarged unmyelinated axons possessing multiple clear vesicles (boutons en passant) (solid arrows) adjacent to one immunopositive axon (open arrow), x38,900 (bar - 1~m) b: substance P positive axon and an immunonegative axon in close proximity to a smooth muscle cell (Sm). Mitochondria (m) are abundant in the positive axon. Note the vesicles (v) within smooth muscle. The immunonegative axon contains large granular vesicles (g) (97 _+ 23 nm, see text). The positive axon is surrounded in part by a Schwann cell process. At no time were synaptic contacts or junctional complexes observed between fibers or terminal axons, x 19.650 (bar - 1Hm).

with published results 8.12.5°. Large varicosities present in spinal cord (Fig. 4) were not observed in blood vessels. Some positive perivascular axons did contain clear vesicles (Fig. 3), although the association of the D A B reaction product with all cellular m e m b r a n e s in our preparations may have obscured these organelles in other axons (Fig. 4). Some SP-positive terminal fibers in blood vessels were observed adjacent to immunonegative fibers containing large granular vesicles (Fig. 3b). Large granular vesicles (97 _ 23 nm) and small and agranular vesicles (diameter 50 + 4 nm; derived from 176 vesicles, 7 boutons, one cat) were present in abundance within negatively stained fibers. No synaptic m e m b r a n e specializations were observed for substance P-containing fibers and no complex differentiated endings were observed for immunopositive or negative axons.

DISCUSSION A t the light microscope level, substance P immunoreactive fibers were present in all cat cerebral arteries examined 33. T h e r e was no consistent variation in the density of immunopositive fibers in the examined vessels. This agrees with determinations m a d e by radioimmunoassay in individual arteries 47 but differs from the estimates made by immunohistochemistry suggesting lower fiber densities in posterior cerebral and basilar arteries 11. The ultrastructural a p p e a r a n c e of substance Ppositive axons described in cerebral arteries is compatible with the conclusion that immunopositive nerve fibers originate from primary sensory neurons. Thus immunoreactivity was observed within small unmyelinated perivascular axons associated with Schwann cell processes travelling in fascicles containing both positive and negative fibers. M o r e distal

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Fig. 4. Perivascular axon (a) within tunica adventitia showing reaction product concentrated in rounded-appearing structures with diameters similar to membrane bound vesicles found in substance P immunopositive bouton within dorsal horn of spinal cord. ×53,350 (b) (see text). The arrows point to presumptive vesicles containing reaction product, x56~250(bar = 1/~m).

arbors became single and possessed a thin Schwann cell envelope. Axon profiles possessed vesicles and numerous mitochondria reminiscent of sensory nerve terminals in other tissues. Munger and Halata summarized characteristics of free nerve terminals of primary sensory fibers44; these include the tendency for

each Schwann cell to invest one unmyelinated axon; the absence of an intervening Schwann cell envelope between axoplasm and basal lamina; the abundance of mitochondria, vesicles, occasional glycogen granules and electron opaque lipoidal inclusions. Some of these characteristics were present in substance P-

18 containing nerve fibers except for electron opaque inclusions and glycogen particles which would not be preserved using the employed methods. (The slow embedding procedure using uranyl acetate leaches out glycoprotein.) Basal lamina could not be distinguished. Differentiated nerve endings or synaptic contacts were not observed. Distal branches lacking Schwann cell coverings and lying adjacent to vascular smooth muscle probably were analogous to free nerve endings found in other tissues. The absence of an elaborate axon-Schwann cell complex in distal arbors distinguishes between the ultrastructural appearance of mechanical nociceptor endings in hairy skin and that surrounding cerebral arteries 27. Substance P-containing peripheral fibers exhibit two or more populations of vesicles as do substance P containing central terminals ~-2,~,49. We have observed reaction product in spheroid structures whose diameters resemble those of vesicles visualized in dorsal horn~,S0 and those characterized biochemically from brain t2. It has been reported that vesicles in peripheral terminal axons of sensory fibers tend to be scattered, irregular in shape and size 2~. Others note that peripheral sensory fibers may contain more than a single population of vesicles; for example, unmyelinated terminal axons were reported to possess clear, pleomorphic and/or dense-core vesicles 13,14,16-19,27,37. In other studies in which electron microscopy was performed after unilateral trigeminal ganglionectomy, some degenerating axon profiles exhibited both large granular and small clear vesicles (unpublished observations). Since substance P is present in 20% of some primary sensory neurons and found in trigeminal neurons 6, support is lent to the notion that segregation of vesicle populations does not occur in the central and peripheral axons of primary sensory fibers. Consistent with another report 48, some immunonegative axons contained clusters of small dense core vesicles (30-60 nm in diameter) characteristic of noradrenergic nerves, and some contained clusters of small clear vesicles (30-60 nm in diameter) characteristic of cholinergic nerves. Varicosities containing vesicles in clusters are more reminiscent of terminal axons belonging to autonomic fibers. Other immunonegative axons contained both large granular and small clear vesicles reminiscent of peptide-containing vesicles. Preliminary immunoelectron microscopic studies in rat cerebral

vessels show similar features to those we have described in cats (unpublished observations). Despite the fact that the vast majority of ipsilateral fibers containing reaction product disappeared under the light microscope following unilateral trigeminal lesions, other possible sources in addition to primary sensory neurons should be considered for those fibers we observed. As much as 30-50% of the radioimmunoassayable substance P remained in arteries of the circle of Willis 2-3 weeks after unilateral trigeminal surgery 47. The reason for this discrepancy between radioimmunoassay and immunohistochemistry remains obscure. The observation that the profiles of immunoreactive axons were remarkably homogeneous in appearance somewhat mitigates against the notion that multiple sources contribute to the population of substance P fibers surrounding vessels. Using horseradish peroxidase histochemistry, only cells in trigeminal, sympathetic or parasympathetic ganglia contained transported label after tracer was introduced around the middle cerebral artery 39,40. Ganglia of the vagus nerve, glossopharyngeal and geniculate were without label. Since unilateral or bilateral sympathectomy or treatment with 6-hydroxydopamine failed to decrease substance P levels in cerebral vessels (Liu-Chen and Moskowitz, unpublished observations) the sympathetic nervous system seems an unlikely source of substance P-containing fibers in cerebral arteries. Nevertheless, other candidates to consider should include both intrinsic brain neurons and otic ganglia. The latter, a parasympathetic ganglia known to contain substance P in perikarya 52 was also found to project to cerebral arteries recentlyS5. The presence of vesicles in substance P-containing perivascular axons suggests that this peptide may be released from primary sensory fibers. This has been shown in vitro by high levels of extracellular potassium (20 raM) or capsaicin ( > 0.01 I,M) by calcium-dependent mechanisms in arteries of the circle <. Release of substance P from peripheral nerves has been associated with the development of neurogenic inflammation as characterized by vasodilation and increased vascular permeability to Evans blue-albumin complex30.31. This has been observed in the nasal mucosa, eye38 and skin of the face following stimulation of the trigeminal nerve. Stimulating the saphenous nerve 30 or vagus nerve 35 has also been associated with the development of similar responses.

19 The effects of saphenous nerve stimulation could also

cerebrovasculature, some of the substance P-con-

be mimicked by intraarterial substance P administra-

taining C-fibers identified in this study may transmit

tion 30. The effects of both nerve stimulation and pep-

information of a nociceptive nature from the blood

tide administration became attenuated by pretreatment with substance P receptor-blocking drugs. The

vessel to the central nervous system. By so doing, these fibers may be of importance to mechanisms of vascular head pain 42.43.

effects of depolarization-induced release of substance P from sensory axons surrounding cerebral arteries has not yet been determined, but no change in the transport of Evans blue has been demonstrated as yet following chemical depolarization or substance P

ACKNOWLEDGEMENTS

administration51. The increases in cerebral blood

Supported by G r a n t 5-PO NS 10828, NSF PCM-

flow reported to follow trigeminal nerve stimulation in two reports 28.29 might be mediated via a substance

8402540 and a grant-in-aid from the AmeriCan Heart Association. M . A . M . is an Established Investigator

P mechanism. Important to the functioning of the

of the A m e r i c a n Heart Association.

REFERENCES

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