Trigeminal Nerve Pathways to the Cerebral Arteries in Monkeys

Home , Maxillary nerve, Ophthalmic nerve, Trigeminal ganglion

J. Anat. (1987), 155, pp. 23-37 23 With 11 figures Printed in Great Britain Trigeminal nerve pathways to the cerebral arteries in monkeys

G. L. RUSKELL AND TERESA SIMONS Department of Optometry and Visual Science, The City University, London EC1 V OHB (Accepted 27 January 1987)

INTRODUCTION Nerve fibres are plentiful within the adventitia of the cerebral arteries of the circle of Willis. Most of them are autonomic motor but some are thought to be fibres of primary afferent neurons. Suggestive evidence for a sensory innervation in man was obtained from the responses of patients to stimulation of the cerebral vessels. For example, pain was elicited when the pial or dural arteries were stimulated electrically in patients under local anaesthesia (Fay, 1932; Penfield & McNaughton, 1940; Ray & Wolf, 1940). Sensitivity was greatest when the large pial arteries were stimulated proximally. Provision ofsensory nerve branches to the internal carotid artery probably occurs in the cavernous sinus where the ophthalmic and maxillary branches of the trigeminal nerve pass close to the artery but they have not been demonstrated satis- factorily. Several fine nerves located betweeen the trigeminal ganglion and internal carotid artery were noted by Hovelacque (1927) in dissected human material but he was uncertain whether they were branches of the trigeminal ganglion or sympathetic twigs passing through or to the ganglion. McNaughton (1938) and Fang (1961) assumed that trigeminal fibres joined the nerves of the sympathetic internal carotid plexus within the cavernous sinus and were distributed together to the intracranial branches of the internal carotid artery. They demonstrated the recurrent branches of the trigeminal ganglion to the tentorium in man but they were unsuccessful in their endeavours to identify and follow others passing forward from the ganglion or from the ophthalmic or maxillary nerves in the cavernous sinus. Numerous workers have observed substance P-like immunoreactivity in intracranial pial arteries including those of primates (Chan-Palay, 1977; Uddman, Edvinsson, Owman & Sundler, 1981; Edvinsson, Rosendal-Helgesen & Uddman, 1983) and there is evidence that such reactivity is associated especially with sensory nerves (Hokfelt, Kellerth, Nillson & Pernow, 1975; Jessel & Iversen, 1977; Duckles & Buck, 1982). Some reduction was found in the density and intensity of substance P (SP)-containing fibres in pial arteries of cats (Liu-Chen, Han & Moskowitz, 1983), and the quantity of SP measured by assay was approximately halved (Norregard & Moskowitz, 1985), after unilateral trigeminal ganglionectomy. The same manoeuvre in gerbils and guinea-pigs reduced SP fibres in the carotid system of arteries but had limited or no effect on their number in the walls of vertebral and basilar arteries (Yamamoto et al. 1983; Matsuyama et al. 1984) indicating that the trigeminal ganglion shares responsibility for provision of sensory fibres to the cerebral arteries; there is good evidence that the superior vagal ganglion is a source (Keller, Beduk & Saunders, 1985). These recent studies, employing mainly chemical markers, amply confirm earlier evidence for a sensory innervation of the cerebral vessels without providing details of nerve access to the vessels. We have traced three nerve pathways from the trigeminal 24 G. L. RUSKELL AND TERESA SIMONS ganglion to the cerebral arteries in cynomolgus monkeys and they are the subject of this report.

MATERIALS AND METHODS Nine young adult cynomolgus (Macaca fascicularis) monkeys of both sexes were sedated parenterally with 2-3 mg/kg ketamine and anaesthetised with 15-25 mg/kg Sagatal (sodium pentobarbitone) given via the saphenous vein or intraperitoneally. The calvaria was removed and the left lateral lobe of the brain elevated to expose the trigeminal nerve. To minimise the risk ofdamage to adjacent nerves and the cavernous sinus, the ophthalmic nerve sheath was slit parallel to the nerve axis close to the trigeminal ganglion. The nerve was freed from sheath attachments, separated from adjacent structures with a hook, and divided and reflected in six animals. In addition, a short length of the maxillary nerve was removed close to its entry into the foramen rotundum in three of the animals. In three other monkeys only the maxillary nerve lesion was made. The operations were designed to induce Wallerian degeneration so that fibres from the divided nerves to the internal carotid artery could be traced. Maxillary neurectomy was included because earlier work on monkeys had shown that an extracranial branch of the maxillary nerve, the orbitociliary nerve, re-enters the cranium from the pterygopalatine fossa and in part joins branches from the internal carotid plexus (Ruskell, 1974). Consequently, the orbitociliary nerve is a potential source of cerebral artery sensory nerve fibres. From three to seven days after operation animals were sedated, anaesthetised and given an injection of the anticoagulant heparin sodium (1500 units). The external jugular veins and the inferior vena cava were cut and a 2% glutaraldehyde, 3 % paraformaldehyde cacodylate-buffered solution was perfused through the heart. The heads were stored in the fixative at approximately 4 °C and dissected while immersed in a buffered sucrose solution using a dissection microscope. The cavernous sinus with its contents was removed in one piece from both sides. The internal carotid artery was cut posterior to the trigeminal ganglion and again a few millimetres beyond the position where it emerges dorsally from the cavqrnous sinus and all the soft tissue between these positions was freed from the cranial floor. The dissected tissues included the trochlear, ophthalmic and abducent nerves with a short length of the maxillary nerve, and the piece was divided vertically into anterior and posterior halves. Only the part ofthe trigeminal ganglion opposite the ophthalmic and maxillary nerves was retained; the hypophysis and the oculomotor nerve, where it lay external to the cavernous sinus, were discarded. The contents of the upper part of the pterygopalatine fossa, including the orbitociliary nerve and the rami orbitales, were left attached to the sinus tissues. Finally, in some preparations an additional length of the internal carotid artery was removed from the carotid canal together with the thick sleeve of connective tissue (including periosteum) enclosing the artery. Care had to be taken to ensure that the tissues were free of bone fragments. The pia-arachnoid canopy of the pons from the level of the superficial origins of the oculomotor nerves to the medulla was separated from the underlying nervous tissue. Incisions were made through the canopy just lateral to the line of cranial nerve origins on each side and the canopy was then grasped with forceps at its cut medullary end and slowly peeled free. The caudal cerebral vessels and the stumps of the abducent nerves were naturally retained within the thin sheet of tissue. Tissues of the cavernous sinus were postfixed in 1 % unbuffered osmium tetroxide, Trigeminal nerve pathways to cerebral arteries 25 dehydrated, embedded in Araldite and transversely sectioned using glass knives. Sections were cut 1 ,sm thick at intervals of 120-240 um in areas of least interest and at 60,um intervals or every fifth section elsewhere and stained with 1 % toluidine blue in an equal volume of 2-5 % sodium carbonate. Special attention was given to tissue areas that included the site of the lesion. The face of each large block of tissue was trimmed at appropriate points to present a small area for electron microscopical examination. Sample sections of the separate lengths of internal carotid artery were treated for light and electron microscopy. Sections for electron microscopy were mounted on unfilmed copper grids, immersed in a saturated solution of uranyl acetate in 30-70% ethanol for about 20 minutes, washed and immersed in 0-4 % lead citrate in 01 N sodium hydroxide for about 10 minutes. The pia-arachnoid canopy was similarly treated and embedded whole in an Araldite bath. Pieces pertinent to the present study were identified, cut out and prepared in the same manner as the sinus tissues. Histological evaluation of the ophthalmic lesions revealed some damage to the trochlear nerve in two monkeys but otherwise lesions were confined to the ophthalmic nerve. Retention of full integreity of autonomic filaments of the carotid plexus, which was crucial for the study, was verified in each animal.

RESULTS Figure 1 includes most of the structures examined in the present study together with some neighbouring structures. The trigeminal ganglion was included in the first sections of each series and in all preparations ganglion cells infiltrated the ophthalmic nerve in large numbers. They made up the bulk of the nerve in the first millimetre reducing gradually to a few, 2 5 mm from the ganglion on average. Further forward, isolated or small groups of cell bodies were present. Animals displayed little variation in the length of nerve infiltrated. As a consequence large numbers of intact trigeminal cells lay distal to the damaged area following ophthalmic neurotomy. In contrast, the maxillary nerve was entirely free of cell bodies. Fine branches from the ophthalmic and maxillary nerves joined the plexus of autonomic nerves in the cavernous sinus before passing to the walls ofcerebral arteries or to other structures (Fig. 2). Autonomic nerves were derived from two sources, one from the sympathetic superior cervical ganglion via the internal carotid nerves and the others from the parasympathetic pterygopalatine ganglion via the rami orbitales. Identification of internal carotid nerves at the level of the carotid canal and the rami orbitales in the pterygopalatine fossa, and tracing of these nerves to the plexus was regarded as satisfactory evidence of the duality of the autonomic contribution. The cavernous plexus consisted ofa complex mesh offine nerves extending through- out the length of the sinus from close to the trigeminal ganglion posteriorly to just beyond the internal carotid artery anteriorly, where it continued as the retro-orbital plexus. The nerves lay in the walls of the trabeculae of the sinus or in the connective tissue surrounding the cranial nerves. Ophthalmic and maxillary nerve contributions to the plexus will be described separately. Ophthalmic contribution to the cavernous plexus Two or three small divisions of the ophthalmic nerve joined the plexus in most preparations but more were found in others, up to a maximum of six. Commencing at about 15 mm from the trigeminal ganglion, fine branches issued from the intracavernous part of the ophthalmic nerve. The branches contained unmyelinated fibre

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Fig. 1. Drawing of the structures associated with the cavernous sinus in the cynomolgus monkey (right side). The cavernous sinus is indicated by a mesh; most of the length of the internal carotid artery is dotted and the trigeminal ganglion is mottled. The oculomotor (0), trochlear (1), abducent (A) and ophthalmic nerves leave the cranial cavity through the superior orbital fissure (F). The maxillary (Mx) nerve is obscured after entering the foramen rotundum (R) and appears again to the

right in the pterygopalatine fossa. The positions of the ophthalmic and maxillary nerve lesions are indicated by asterisks. Md, mandibular nerve, On, optic nerve; Oc, orbitociliary nerve; P, pterygopalatine ganglion; Ro, rami orbitales.

Fig. 2. Drawing of a section through the cavernous sinus region at the level of the arrows shown in Figure 1. The ophthalmic nerve, which has two divisions in this position, is shown in solid black. All the branches of the cavernous plexus are shown in outline except for their ophthalmic or maxillary fractions which are also shown in solid black. The 21 branches are relatively minute and are located close to the ophthalmic and abducent (large cross hatched profile) nerves and in the adventitia of the internal carotid artery (IC). The largest colony of sensory fibres, apart from the ophthalmic nerve, was found at the surface of the abducent nerve in this specimen. The unfilled area surrounding the internal carotid artery and near the trochlear nerve (small cross-hatched) are the venous spaces of the cavernous sinus. The trigeminal fractions in the cavernous plexus and abducent nerve were identified by degeneration following ophthalmic and maxillary neurotomy. L, lateral surface. Trigeminal nerve pathways to cerebral arteries 27 bundles and numerous myelinated fibres of various diameters which permitted them to be distinguished from the neighbouring autonomic branches of the plexus whose myelinated fibres, by comparison, were infrequent and of nearly uniformly small diameter. Few branches had a length greater than 1 mm before joining the plexus and some ophthalmic fibres were picked up directly by autonomic plexus branches entering the ophthalmic nerve, staying adjacent to the perineurium and leaving the nerve one or two millimetres further on with an additional complement of fibres. The smallest of the ophthalmic branches contained two dozen or so fibres but most of them were considerably more substantial, frequently containing one or two hundred myelinated fibres with a similar number of unmyelinated bundles (Figs. 3, 4). Branching from the nerve and from the plexus appeared to follow no regular pattern but the most dense part of the plexus usually lay between the internal carotid artery, ophthalmic and abducent nerves close to where the artery turned upwards to leave the sinus. Ophthalmic nerve lesions were complete in four animals and partial in two others and in each case degenerated fibres were observed in the ophthalmic branches to the cavernous plexus. Few branches displayed degeneration of all nerve fibres which was to be expected in the preparations with incomplete lesions. Yet complete lesions gave a similar result, except that fully degenerated branches were more common, and in all six animals there were many fibres of normal appearance in the parent nerve distal to the lesion, increasing in number with distance from the lesion. No doubt these were the fibres of cell bodies lodged in the nerve where they were unaffected morpho- logically by the proximally placed lesion. The significant point for attention here is that the lesions provided an effective qualitative marker for ophthalmic fibre tracing. This was of considerable help in following ophthalmic fibres through and from the cavernous plexus (Fig. 5). Maxillary contribution to the cavernous plexus The serial sections revealed no branches passing to the cavernous plexus from the intracranial portion of the maxillary nerve but the orbitociliary nerve, observed in the tissues of the pterygopalatine fossa, sent branches to the cavernous plexus. The orbitociliary nerve branches dorsally from the maxillary in the fossa, enters the orbit and joins the ciliary ganglion (Ruskell, 1974). One or two small filaments branched from the nerve, re-entered the cranial cavity through the medial infraorbital fissure and joined the plexus. The filaments took a similar course to the rami orbitales, which issue dorsally from the pterygopalatine ganglion in the fossa, but they were usually readily distinguished from them by their typical trigeminal fibre content. Moreover they were degenerated following maxillary neurotomy. But degeneration was not always confined to the filaments as small groups of degenerated fibres were also seen in rami orbitales in some animals, indicating that filaments had joined rami in the fossa or that maxillary fibres had passed directly into the rami at the ganglion. The maxillary filaments passed backwards in or close to the inferior wall of the cavernous sinus, beneath the internal carotid artery and usually joined the plexus at its most dense part. Microganglia Microganglia were located at several points in the plexus especially anteriorly, close to the upturn of the internal carotid artery and also inferiorly, where rami orbitales joined the plexus. The ganglia were of various sizes, their content ranging from less than a score to several hundred cells. Ganglia tended to be elongated, conforming to

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Fig. 3. Cavernous plexus nerve junction consisting of a substantial division (S) of the ophthalmic nerve (0) with several autonomic branches (arrows) of different orientations. A, abducent nerve. x 253. Fig. 4. Small division (S) of the ophthalmic nerve (0) about to separate before joining the cavernous plexus. A, abducent nerve. x 150. Fig. 5. Electron micrograph of a plexus branch containing degenerated myelinated nerve fibres. A low density flocculent material with small depositions of greater density, and membrane whorls have replaced the normal axoplasmic organelles. Myelin shows few signs ofdisruption except in two cases. Most of the unmyelinated nerve fibre bundles have a normal appearance but examples of Schwann cell processes without enclosed axons or displaying hyperplasia and abnormal configurations are present (arrows). x 4000, fixed four days after ophthalmic neurotomy. Fig. 6. Part of a microganglion of the cavernous plexus showing extreme variation in nerve cell diameter. A group of nerve fibres above the larger neuron contains myelinated axons with a variety of diameters typical of trigeminal nerve plexus branches. x 158. Trigeminal nerve pathways to cerebral arteries 29 Table 1. Proportion of degenerated nerve fibres in the adventitial nerves of the internal carotid artery above the cavernous sinus

Degeneration Myelinated fibres Unmyelinated fibres Operation Reference time (days) normal/degenerated normal/degenerated Ophthalmic M49 7 27/20 100/35 neurotomy M50 6 29/24 201/82 M86 7 26/56 455/134 Maxillary M46 4 63/37 536/33 neurotomy M80 4 55/5 357/15 M82 4 67/12 381/25 Ophthalmic and M43 6 3/38 90/45 maxillary M83 3 32/7* 26/48 neurotomies M84 4 21/24* 90/45 * Incomplete ophthalmic neurotomy. Retention of adventitial nerves was incomplete in some animals. Degenerated fibres were absent from the control sides.

the orientation of neighbouring structures, so that a true impression of their size was seldom conveyed in single cross sections where even the largest ganglia may reveal a mere twenty cells or so. Serial sections were cut through one of the largest ganglia and by counting nucleoli in every fifth section a total of 509 cells was estimated to be present (nucleoli were counted and the total multiplied by a factor of 3/2 - section thickness 0 75 ,um, nucleolus diameter < 2 5 ,m). A large majority of the microganglion cells ranged in diameter from 9 to 33 tum (mean, 21 ,um, n = 556) and had the appearance ofsuperior cervical or pterygopalatine ganglion cells but were similar enough to frustrate attempts to distinguish the two. However, small groups of cells within the microganglia and others occasionally observed along ophthalmic branches before they had reached the plexus were quite distinctive and characteristic of trigeminal ganglion cells (Fig. 6). They were larger than the others (mean 31 Jsm, n = 42) and included several cells with diameters greater than 40 /sm. Their cytoplasm was lighter, Nissl granules were more contrasty and nuclei of the capsular cells more obviously indented the cytoplasm of the neurons. Cells of the trigeminal type were not found in the microganglia of all animals. Trigeminal branches innervating the internal carotid artery Cavernous plexus branches advanced to the adventitia of the artery through trabeculae ofthe sinus. Some, without a trigeminal fraction, entered the adventitia in a proximal position, but the majority delayed their passage to the artery until shortly before, or as it turned upwards to leave the sinus. Trigeminal nerve fibres formed part of the content of some of the distal branches. Confirmation of the trigeminal source of fibres within adventitial nerves of the artery was provided by the changes induced by the nerve lesions. Most ofthe degenerated fibres advanced in the adventitia towards the dorsal exit of the internal carotid artery from the sinus. Others passed caudally, reducing in number until none were observed opposite the trigeminal ganglion. Several nerves contained numerous degenerated fibres at the dorsal exit ofthe artery where the mean proportion of degenerated myelinated fibres in all adventitial nerves was 55 % following combined ophthalmic and maxillary neurotomy and 51 % and 20% following ophthalmic and maxillary neurotomies respectively. The corresponding propor- tions ofunmyelinated fibres showing induced changes were 32%, 26% and 5 % (Table 30 G. L. RUSKELL AND TERESA SIMONS

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410~~~~~~~~~~~~4S' 79 eh a v Trigeminal nerve pathways to cerebral arteries 31 1). Few of the larger myelinated fibres were without induced changes following ophthalmic neurotomy except in one case of incomplete neurotomy. Degenerated nerves were not seen in the adventitia of the internal carotid artery before it entered the cavernous sinus either in its course within the carotid canal or in the foramen lacerum. Trigeminal branches innervating the basilar artery A substantial branch of the cavernous plexus joined the abducent nerve and passed back towards the brainstem: this plexus branch will be referred to as the recurrent nerve of the plexus. It was present in all animals, bilaterally in seven and unilaterally in two. The mean content was 71 myelinated and 92 unmyelinated fibres, the largest with 243 and 226 fibres and the smallest with 23 and 17. The content of pairs of recurrent nerve was often numerically dissimilar and when present unilaterally the nerves were among the largest found. The large fraction of myelinated fibres of varied diameters in the recurrent nerves was adequate evidence of their trigeminal source but aggregations of unmyelinated fibres in some of them suggested that autonomic fibres might also be present. Tracing the nerve within the abducent nerve presented little problem because it usually persisted as a single colony of fibres with an appearance contrasting with the content of the host nerve and it often remained at or close to the surface (Fig. 7). In some instances it split up into two or three groups and occasionally it passed deep within the abducent nerve. Further back, behind the cavernous sinus the recurrent nerve branched from the abducent, looped medially across the face of the pons and entered the adventitia of the basilar artery. Sometimes it issued from the abducent as two or three branches, the last of these leaving the nerve close to its superficial origin from the brainstem. The recurrent nerve divided further on reaching the basilar artery and most ofthe divisions passed upwards towards the circle ofWillis, distributing filaments to pontine branches of the basilar, anterior cerebellar and posterior cerebral arteries. This distribution was shared with the recurrent nerve from the opposite side with which it anastomosed. Inspection of osmium-stained preparations, such as those illustrated in Figures 9 and 10, revealed fine branches terminating in the basilar and other recipient arteries. Degeneration experiments fulfilled their purpose in confirming the trigeminal origin ofmany ofthe fibres in the recurrent nerve (Figs. 7, 8) but by chance the operated sides of two of the animals were those that lacked a recurrent nerve. There was little degeneration in two preparations with substantial recurrent nerves following maxillary neurotomy and none in a third. Many of the unmyelinated fibres were unchanged by any of the experiments, indicating an autonomic fraction.

DISCUSSION Serial section reconstruction adequately revealed the pathways followed by trigeminal afferents to the cavernous plexus but because of the complex relationship between trigeminal branches and autonomic nerves in the cavernous sinus their pas- Fig. 7. Part of the abducent nerve (A) containing the recurrent nerve of the cavernous plexus (R) at its surface. The recurrent nerve is made up of thinly myelinated fibres, smaller than the main group of somatic motor fibres but varying in diameter; unmyelinated fibres are also present. Several fine nerves of the cavernous plexus (P) lie close to the abducent. x 617. Fig. 8. Electron micrograph ofpart ofthe recurrent nerve shown in Figure 7. Many ofthe myelinated fibres and a few of the unmyelinated are degenerated. Some without normal axoplasm and others more severely disrupted are indicated (arrows). Adjacent large somatic motor fibres (A) contain normal axoplasm. x 3400. 32 G. L. RUSKELL AND TERESA SIMONS

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...... Trigeminal nerve pathways to cerebral arteries 33 sage through the plexus and beyond was often difficult and sometimes impossible to follow. The degeneration experiments provided a solution to the problem. The initial expectation that the degeneration technique would permit a total count of the trigeminal fraction innervating the cerebral arteries was frustrated for several reasons. Lesions had to be made close to the trigeminal ganglion to avoid excessive damage to the cavernous sinus with the risk of intractable bleeding. This constraint meant that the large numbers of somata housed in the ophthalmic nerve, well forward of the ganglion, together with their distal processes survived the lesions. Microganglia were distributed widely in the cavernous sinus region and although there is little doubt that they were composed mostly and sometimes entirely of autonomic ganglion cells, some contained cells resembling those of the trigeminal ganglion; these too were spared by the lesions. Endeavours to avoid damage to underlying autonomic nerves led to incomplete lesions of the ophthalmic nerve in two animals. The cavernous sinus region harbours numerous autonomic filaments as the comple- mentary observations of this study bear witness and consequently manoeuvres to section trigeminal branches within or near the sinus placed them at risk. This problem was avoided for the maxillary nerve by making the lesion at the foramen rotundum after the nerve emerges from the sinus and where it has no neighbouring autonomic nerves. However autonomic nerves are present along most of the intracranial length of the ophthalmic nerve and some enter it transiently, but preliminary observations showed that the lateral face of the initial portion of the nerve is free of autonomic intrusion. Nerve sections were confined to this region. Autonomic nerves lying deep to the ophthalmic nerve were damaged in one animal and the results were not used for the purposes of this study. Lesions were successfully confined to the ophthalmic nerve in all other cases and the normal appearance of autonomic nerves was verified proximal, opposite and distal to the lesions. Nerve fibre tracing by induced degeneration therefore provided a reliable qualitative assessment of the ophthalmic nerve distribution to cerebral arteries. The quantitative accuracy of the maxillary nerve results is subject only to the remote possibility of associated microganglion cells. The cavernous plexus If the habit of regarding the cavernous plexus as a sympathetic plexus remains, as originally proposed by Arnold (1831), then the present results should be discouraging. That a parasympathetic element joins the plexus from the pterygopalatine ganglion was demonstrated long ago (Ruskell, 1970) and it may have as a target, among others, the walls of cerebral vessels (Hara, Hamill & Jacobowitz, 1985). To this we now add a sensory element and identify the plexus as a confluence ofthree anatomically distinct neural subdivisions preparatory for issue to numerous structures (Fig. 11). The internal carotid artery is one, the basilar artery is another and various orbital structures also receive branches from the plexus (Ruskell, 1985). We have evidence, to be reported

Fig. 9. The basilar artery and the stumps of the abducent nerves in the pia-arachnoid canopy after removal from the brainstem. The abducent nerves became detached from the brainstem at their superficial origin where the broken ends of the rootlets (S) can be seen, and were cut at the other end close to the position of entry into the dura mater. Fine divisions (R) of the recurrent nerve of the cavernous plexus pass from each abducent nerve to the wall of the basilar artery (B). The nerve passing along the anterior inferior cerebellar artery (C) on the right appeared to issue from the vagus nerve. L, labyrinthine artery; V, vertebral artery. Osmium stain, transilluminated. x 15. Fig. 10. Another example of the pia-arachnoid canopy with a unilateral recurrent nerve to the basilar artery. Incident illumination. x 12. 34 G. L. RUSKELL AND TERESA SIMONS

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1 1 Fig. 11. Summary diagram of a medial view of the right cavernous plexus. Sensory elements of the plexus are shown as dotted lines and autonomic motor as solid lines. Sensory branches to the plexus issue from the ophthalmic nerve (alternate thick and thin stripes) at the arrows and from the orbitociliary branch (arrow) ofthe maxillary nerve (Mx). Plexus branches containing both fibre types pass (1) to the adventitia ofthe internal carotid artery (IC) at the distal but not the proximal end (only autonomic fibres are present proximally), (2) to the basilar artery (B) via the abducent nerve. The three cross hatched structures are respectively from the top of the figure, the oculomotor, trochlear and abducent nerves. The trigeminal ganglion is marked by a grid. Innervation of the internal carotid artery by pterygopalatine ganglion neurons indicated in the figure was assumed, not demonstrated. C, crus cerebri; Md, mandibular nerve; 0, ophthalmic artery; P, pterygopalatine ganglion; Po, pons; Ro, rami orbitales; S, superior cervical ganglion. later, that plexus branches are partly responsible for the innervation of the dura mater and there is little reason to suppose that this exhausts the list of recipient structures. Although the ophthalmic nerve commonly provided only two or three primary branches for the plexus, by dividing and mixing, ophthalmic fibres became broadcast within the meshes of the plexus as indicated most clearly by the distribution of degenerated fibres following ophthalmic neurotomy. The maxillary contribution was similarly distributed in the plexus stemming sometimes from only one division of the orbitociliary nerve. Maxillary and ophthalmic fibres mingled in the cavernous plexus and only induction of degeneration in one or the other nerve permitted identification of their separate origins. So the plexus is not only a locus for convergence of afferent and autonomic fibres but also of the two trigeminal sources of afferents. Cavernous plexus branches to the internal carotid artery Trigeminal afferent fibres were conducted to the adventitia of the internal carotid artery in the numerous trabeculae traversing the venous space of the cavernous sinus. A large majority ofunmyelinated fibres in the adventitial nerves were unaffected by the Trigeminal nerve pathways to cerebral arteries 35 lesions and although a few of these may have been afferents that had escaped damage, the bulk of them were autonomic. They probably consisted of both sympathetic and parasympathetic fibres, branches of internal carotid nerves and rami orbitales having been traced to the cavernous plexus, but a slight doubt remains. Fibres of the two autonomic types could not be distinguished within the plexus and whether or not both were finally presented to the wall of the artery was uncertain. The possibility that one or the other type was selectively filtered to serve another target is worthy ofconsidera- tion but previous literature is ambivalent on this matter. Evidence for a sympathetic innervation in mammalian cerebral arteries from the superior cervical ganglion is secure (Purves, 1972) and although a parasympathetic innervation appears likely, the question of its source is unsettled. Vasoactive intestinal polypeptide(VIP)-immuno- reactive cells are present in the pterygopalatine ganglion (Uddman et al. 1980) and VIP-positive nerves have been found in the walls of cerebral arteries in numerous mammalian species (Larsson, 1977) including a monkey (Edvinsson et al. 1980). Add to this the proximity of ganglion efferents and the internal carotid artery in the cavernous sinus and the conclusion that the ganglion is a source becomes compelling. Yet disparate results are obtained when the ganglion is damaged; the density of VIP nerves was reduced in rats (Hara et al. 1985) but not in cats (Edvinsson et al. 1980). Many of the myelinated fibres in the adventitial nerves were degenerated following combined ophthalmic and maxillary lesions and few were without induced changes in one preparation with complete ophthalmic and maxillary lesions. In particular the larger myelinated fibres were affected by the lesions, consistent with the results of serial section reconstruction of control material identifying a trigeminal source for such fibres. Substantial numbers were again degenerated when only one of the trigeminal branches was cut but more were degenerated following ophthalmic nerve lesions; the same applied to the unmyelinated nerve fibres. A substantial proportion of the myelinated fibres around the internal carotid artery are therefore afferent, most of them issuing from the ophthalmic nerve. Predominance of ophthalmic nerve provision of afferents to the carotid cerebral branches may also apply to cats. Horseradish peroxidase applied to the proximal middle cerebral artery was retrogradely transported to cell bodies occupying the portion of the ipsilateral trigeminal ganglion corresponding mainly to the ophthalmic division (Mayberg, Zervas & Moskowitz, 1984). These comments cannot be extrapolated to include the whole cerebral vasculature and the existence of other sources of afferents to the caudal vessels is a strong possibility (Matsuyama et al. 1984; Keller et al. 1985). Most of the vascular nerves passed dorsally and emerged from the sinus with the artery and they were presumably destined to terminate in the wall of the artery and its branches on the surface of the brain. Other fine nerves passed back along the artery. Both groups contained degenerated fibres but they were few in the nerves passing caudally and at a position approximately opposite the trigeminal ganglion none were left. Moreover, degenerated fibres were not found in the carotid canal. It therefore appears that the internal carotid artery lacks an afferent innervation until it reaches the cavernous sinus. None of the adventitial nerves within the canal contained fibre groups with the morphological variety of afferent fibres so the possibility that this part of the internal carotid artery is served by another afferent source is unlikely.

Cavernous plexus branches to the basilar artery A single access route for trigeminal afferents to the cerebral vasculature via the internal carotid artery had been anticipated at the outset of this study for nothing in 36 G. L. RUSKELL AND TERESA SIMONS the literature led us to expect the abducent nerve to act as a conduit for recurrent trigeminal fibres, or for autonomic fibres for that matter. Our results indicate that the recurrent nerves may regularly have major responsibility for the afferent supply to the caudal circle of Willis and the basilar artery because most of the vascular nerves stained in the pontine canopy preparations were traced to the abducent nerve. Maxillary neurotomy was relatively ineffective in pro- ducing degeneration in the recurrent nerve compared with ophthalmic neurotomy and since maxillary lesions were routinely complete one may safely conclude that its trigeminal content is largely derived from the ophthalmic branch. The occasional contralateral inequality of the nerves or unilateral absence is not surprising as they supply a midline structure and a reciprocal relationship between pairs of nerves is a reasonable expectation. Division of the nerves and attenuation of the myelin stain in the wall of the basilar artery, its branches and posterior branches of the circle of Willis is satisfactory evidence of their termination in the vessel walls. A detailed evaluation of the terminals will be reported later. Whether or not the basilar branch of the cavernous sinus is regularly present in mammals possessing a comparable pattern of arteries at the base of the brain remains to be seen. Our effort to answer this question is so far represented by a single rhesus monkey preparation which displayed a basilar branch from the abducent nerve on each side. Does the basilar branch have full responsibility for afferent innervation of the basilar artery in monkeys? The pattern of myelinated fibres in the walls of the basilar and vertebral arteries suggests that it does not and that trigeminal sources are augmented by at least one other as in guinea-pigs and cats (Matsuyama et al. 1984; Keller et al. 1985).

SUMMARY Two or three or sometimes more fine intracavernous branches were traced from the ophthalmic nerve using serial section reconstruction and induced nerve degeneration. They joined the cavernous plexus and were distributed forward with autonomic nerves to the adventitia of the internal carotid artery, emerging from the sinus with the artery. A strong recurrent branch from the plexus joined the abducent nerve, passed back and left the nerve at pontine level to innervate the basilar artery and the caudal circle of Willis. The recurrent nerve was absent from one side of two animals and showed asymmetry in others. No branch issued intracranially to the plexus from the maxillary nerve, but in the pterygopalatine fossa the orbitociliary branch of the maxillary nerve gave off one or two filaments that re-entered the cranial cavity through the medial infraorbital fissure andjoined the cavernous plexus. Their content augmented the ophthalmic afferent distribution. All plexus branches with trigeminal fibres also contained autonomic fibres. The results show, firstly, that the cavernous plexus consists of a mixture of sensory and autonomic nerves (sympathetic and parasympathetic) and, secondly, that afferents of the internal carotid artery and rostral circle of Willis and those to the basilar artery and caudal circle of Willis are distributed separately.

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