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1From the Department of Ophthalmology, Tohoku University School of Medicine, Miyagi, Japan; the 2Department of Neuroanatomy, Field of Neurology, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan; and the 3Department of Oral Physiology, School of Dentistry, Health Sciences University of Hokkaido, Hokkaido, Japan.
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Abstract |
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METHODS. Cats anesthetized with a mixture of 
-chloralose (50 mg/kg) and urethane (100 mg/kg) were intubated and paralyzed by intravenous injection of pancuronium bromide. The central cut end of the lingual nerve (LN) was stimulated electrically to simulate somatic stimulation, and 1 µL of lidocaine (2%) was microinjected into the Vsp or the EW nucleus to determine its effect on the pupillary dilation induced by LN stimulation. The effect of electrically stimulating the Vsp or sectioning the superior cervical sympathetic nerve (CSN) on the pupillary response was also examined.
RESULTS. Stimulation of the LN or the trigeminal spinal nucleus (Vsp) evoked pupillary dilation in a frequency- and intensity-dependent manner. These responses were not affected by sectioning the ipsilateral or both CSNs. The pupillary responses were markedly suppressed by microinjecting lidocaine into the ipsilateral Vsp or the Edinger-Westphal (EW) nucleus, but not by injection into the contralateral Vsp.
CONCLUSIONS. These results indicate that the Vsp and EW nucleus act as bulbar relay centers for pupillary dilation elicited by LN stimulation and suggest that the efferent arc of the response is a parasympathetic pathway. The contralateral pupillary dilation appears to be mediated, at least in part, by fibers projecting from the Vsp to the contralateral EW nucleus.
During an experimental study on the effects of somatic stimulation on pupil size in urethane-anesthetized cats, we found that centrally directed LN stimulation consistently dilated the pupil that depended on both stimulus frequency and intensity. This response occurred even though the anesthesia was deep enough to prevent a reflex elevation of systemic arterial blood pressure (SABP) by a noxious stimulus (e.g., pinching the upper lip for approximately 2 seconds).
Somatic stimulation has long been known to induce a reflex pupillary dilation via the efferent autonomic nerve.12 13 14 However, the sympathetic and parasympathetic efferent nerve pathways for reflex pupillary dilation induced by somatic stimulation have still not been completely determined, and systematic studies have not been performed to analyze the central or peripheral neural pathways involved in the reflex pupil dilation.
In this study, we used the changes in pupil size and lip blood flow (LBF) elicited by electrically stimulating the central cut end of the LN to study the effect of sectioning the superior cervical sympathetic nerve (CSN), or the microinjection of nonselective, reversible local anesthesia (lidocaine) into the trigeminal spinal nucleus (Vsp) or the Edinger-Westphal (EW) nucleus on the pupillary dilation. The EW nuclei are known to send the preganglionic parasympathetic axons with the oculomotor nerve to the iris.
The purpose of this study was to examine the central and peripheral efferent pathways of the pupillary dilation and increase in the LBF and to determine whether they are sympathetic or parasympathetic pathways. The cat was selected because its pupil is constricted under -chloralose-urethane anesthesia, which enabled us to obtain measurements of the reflex pupil dilation. The cat was also used because the reflex arcs for somatoparasympathetic reflex vasodilation in the lower lip have been well defined.15 16 17
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Fourteen adult cats approximately 2 to 4 years of age and of both sexes were used. The cats, weighing 2.5 to 4.8 kg, were initially sedated with ketamine hydrochloride (30 mg/kg intramuscularly) and anesthetized with a mixture of -chloralose (50 mg/kg) and urethane (100 mg/kg). A femoral artery was cannulated for the measurement of SABP. The anesthetized animals were intubated and paralyzed by an initial intravenous (IV) injection of 0.4 mg/kg pancuronium bromide (Mioblock; Organon, Teknika, The Netherlands). The paralysis was maintained by supplements of 0.2 mg/kg approximately every hour after testing the level of anesthesia. The animals were artificially ventilated with a mixture of 50% air and 50% O2. The ventilator (model SN-480-6; Shinano, Tokyo, Japan) was set to deliver a tidal volume of 10 to 12 cm3/kg at a rate of 20 breaths/min, and the end-tidal concentration of CO2 was determined by an infrared analyzer (Capnomac Ultima; Datex Co., Helsinki, Finland) and kept at 35 to 40 mm Hg. The rectal temperature was maintained at 37°C to 38°C with a heating pad.
The criterion for an adequate depth of anesthesia was the absence of any significant changes in SABP as reflex responses to a minor noxious stimulus (e.g., a pinch of the upper lip for approximately 2 seconds). If the depth of anesthesia was considered inadequate, additional -chloralose and urethane were administered intravenously in doses of 5 and 10 mg/kg, respectively. Once an adequate depth of anesthesia was attained, supplemental doses of pancuronium were given approximately every 60 minutes to maintain immobilization during periods of stimulation.
Isolation and Electrical Stimulation of the CSN
An operating microscope (OME; Olympus, Tokyo, Japan) was used for the isolation of the CSN from the vagosympathetic nerve bundle, for the insertion of a bipolar electrode into the peripheral cut end of the isolated nerve, and for the insertion of a guide cannula into the Vsp or EW nucleus. The CSN was excited electrically with a stimulator (model SEN-7103; Nihon Kohden, Tokyo, Japan) for 20-second trains (10 V, 0.1–100 Hz, 2-ms pulse duration).
Measurements of Pupil Size
Photographs of pupils were taken with a digital camera (EOS D60; Canon, Tokyo, Japan). A millimeter scale was placed on the forehead of the cat to measure the pupil size. Photographs were taken from 25 to 30 cm, which was the nearest distance at which both pupils could be viewed on one screen. The photographs were taken before and immediately after the stimulation for each stimulus condition.
The photographs were analyzed by the NIH image program (available by ftp at zippy.nimh.nih.gov/ or at http://rsb.info.nih.gov/nih-image; developed by Wayne Rasband, National Institutes of Health, Bethesda, MD). The areas of pupils were outlined manually on a personal computer, and sizes were calculated automatically in pixels. The pupillary areas were then converted into millimeters by using the scale that had been calibrated in pixels. We calculated and plotted the changes in pupillary size as the size after the stimulation minus the size before the stimulation.
Measurements of LBF and SABP
Changes in the blood flow in the lower lip were measured with a laser Doppler flowmeter (model ALF21D; Advance, Tokyo, Japan), as described elsewhere.7 18 19 The probe was placed against the lower lip without exerting any pressure on the tissue. The changes in the blood flow were compared by measuring the height (in millimeters) of the response on the polygraph of the flowmeter. The blood flow levels are expressed in arbitrary units.
The SABP was recorded with the femoral catheter connected to a Statham pressure transducer. A tachograph (model AT-610G; Nihon Kohden) triggered by the arterial pulse was used to monitor heart rate.
Electrical Stimulation of LN and Vsp
The central cut end of the LN was stimulated electrically to simulate somatic stimulation (Fig. 1) . The routine stimulus parameters were a 20-second train of 2-ms rectangular pulses at a frequency of 10 Hz and at supramaximum intensity, usually 30 V, unless otherwise noted, as described elsewhere.7 18 19 A bipolar silver electrode connected to a stimulator (model SEN-7103; Nihon Kohden) was used to stimulate the LN.
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For electrical stimulation of the Vsp, a 20-second train of rectangular pulses generated by a stimulator (model SEN-7103; Nihon Kohden) was delivered through a stimulus-isolation unit (model SS-202J; Nihon Kohden). The usual current was 100 µA, with a pulse duration of 2 ms at a frequency of 10 Hz. The stimulation sites were identified histologically.
Microinjections of Lidocaine
To determine whether the pupillary reflex elicited by LN stimulation was mediated by the Vsp or the EW nucleus, 1.0 µL of lidocaine (2%) was injected into either the Vsp or EW nucleus through an injection cannula (0.50 mm outside diameter) that was inserted through the previously implanted guide cannula. The stimulating electrode was interchangeable with the injection cannula, both were of equal length, and each extended 5.0 mm beyond the tip of the guide cannula. Thus, microinjections and electrical stimulations were performed at the same sites. Saline (1.0 µL) was used for control injections, and the saline injections never induced any significant effect on the LN-evoked pupillary reflex or on the resting cardiovascular parameters. The magnitude of the LN-evoked response obtained after microinjection of a given agent was expressed as a percentage of the control response for LBF or the relative area of the pupillary dilation.
Histologic Examination
At the completion of the experiment, animals were given an overdose of pentobarbital sodium (60 mg/kg) through the ascending aorta followed by 1.0 to 2.0 L of saline (0.9%) and then by 2 L of 10% formaldehyde. The brain stem and upper cervical spinal cord were then removed and stored for 1 to 4 days in buffered 30% sucrose. After storage, 50-µm sections were cut on a freezing microtome and collected in 0.1 M phosphate buffer (pH 7.4). The sections were mounted on gelatin-coated slides and stained with thionin. Photomicrographs of representative coronal sections showing the sites of the electrical stimulation or microinjection of lidocaine into the Vsp or EW nucleus are shown in Figure 2 .
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From these reports and our results, three possible mechanisms for mediating the bilateral pupillary dilation induced by stimulation of the central cut end of the LN in cats should be considered: (1) humoral dilation evoked by the release of adrenaline by activation of the sympathetic nerves, (2) neural dilation induced by inhibition or stimulation of the parasympathetic preganglionic cell body (EW nucleus), or (3) neural dilation induced by excitation of the CSN. We will discuss first the possible involvement of humoral substance(s) and sympathetic and parasympathetic nerves as the mechanisms by which LN stimulation evokes the ipsilateral pupillary dilation, and second, the possible pathways of the contralateral pupillary dilation. The latter mechanism is important because mutual neural networks between the Vsp and EW nucleus are still unclear, and it is not known whether an ipsilateral or contralateral mechanism is involved in the trigeminal–parasympathetic reflex pupillary response.
Mechanism for Ipsilateral Pupillary Dilation
Pharmacologic analysis has been used to determine whether autonomic nerves are involved in the pupillary response after LN stimulation.1 3 5 16 18 19 28 However, pharmacologic agents such as ganglionic blockers (hexamethonium), antimuscarinic agents (atropine), and antiadrenergic agents (phentolamine and propranolol) should not be used in this type of study, because they have direct actions on the iris sphincter muscle and dilator muscles. Furthermore, the blocking of autonomic ganglionic cells by hexamethonium by itself elicits pupil dilation, presumably by inhibiting spontaneous discharge in the ciliary ganglion. For these reasons, we used microinjections into the Vsp and EW nuclei to produce nonselective, reversible local anesthesia (lidocaine). We also examined the effect of sectioning the CSN to examine the contribution made by the superior CSN.15 16 17
The microinjection of lidocaine into the EW nucleus reduced the pupillary dilation induced by stimulation of the LN significantly, but had no effect on the increase in LBF, whereas microinjection of lidocaine into the Vsp abolished both the pupil dilation and the LBF increases. These results suggest that (1) the EW nucleus is a critical bulbar relay site for the LN-induced pupil dilation, but not for the LN-evoked LBF increase; (2) the neural pathways mediating these two responses diverge after nerve fibers derived from nociceptive trigeminal leave the Vsp, and (3) the LN-induced changes in pupil dilation are not due to humoral substance(s) released by activation of the sympathetic nerve after stimulation of presympathetic brain stem–spinal neurons. These findings are supported by reports showing efferent connections to the EW nucleus from the Vsp of the cat.29
Stimulation of the peripheral cut end of the superior CSN elicited a current intensity-dependent pupil dilation only on the ipsilateral side (Fig. 5A) . The pupillary dilation induced by LN stimulation was not affected by sectioning the ipsilateral or both superior CSNs (Fig. 5B) . These results suggest that the pupillary dilation evoked by LN stimulation is not mediated by a somatosympathetic mechanism in the cat. However, the pupil dilation was largely abolished by microinjection of lidocaine into the Vsp or the EW nucleus (Figs. 7 8 9) . These results indicate that the Vsp and EW nuclei participate as bulbar relay stations for the pupillary dilation induced by LN stimulation and suggest that the efferent arc of the response is a parasympathetic pathway. These findings are not in accord with the observation of Yu and Blessing14 who reported that stimulation of the spinal tract of the trigeminal nerve causes a brisk dilation of both pupils that seem to be affected by stimulation of the presympathetic brain stem–spinal neurons in rabbits. These differences may be due to species variation as an earlier study reported a marked species difference in the trigeminal–autonomic reflex responses such as SABP changes and parasympathetic mediated LBF responses.30
Mechanism for Contralateral Pupil Dilation
Unilateral application of 2% lidocaine into the Vsp ipsilateral to the LN-stimulated side produced reversible inhibitory effects on the pupillary dilation and the LBF increases on both sides. These results indicate that the ipsilateral Vsp is an essential bulbar relay for the LN-evoked contralateral pupillary dilation. The data also suggest that pupil dilation evoked by LN stimulation depends on the activation of a pathway originating from the Vsp ipsilateral to the stimulated nerve, which crosses over to the contralateral EW nucleus. In addition, the data indicate that the contralateral Vsp is not involved in the LN-induced contralateral reflex pupillary dilation. The microinjection of lidocaine into the EW nucleus abolished the ipsilateral and contralateral pupil dilation in response to LN stimulation, regardless of the side of LN stimulation in sympathectomized cats (Fig. 9) .
To judge from the results of our and other studies, at least five possible brain-stem pathways may mediate the contralateral pupil dilation induced by LN stimulation (Fig. 10) : (i) the afferent neural input projects directly to the contralateral Vsp, (ii) there are projections from the ipsilateral Vsp to the contralateral Vsp, (iii) efferent fibers project from the EW nucleus contralaterally, (iv) there are projections from the ipsilateral EW nucleus to the contralateral EW nucleus, and (v) fibers project from the Vsp to the contralateral EW nucleus. We will discuss these possibilities in order.
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Second, we examined the possibility that fibers project from the Vsp ipsilateral to the stimulated side across to the contralateral Vsp. Microinjection of lidocaine into the Vsp contralateral to the stimulated side did not affect the LN-induced contralateral pupil dilation (Fig. 8) . This finding suggests that the contralateral Vsp is not involved in the LN-induced contralateral reflex pupillary dilation.
Third, the possibility that parasympathetic pupil-constrictor fibers project from the EW nucleus contralaterally seems to be unlikely. When horseradish peroxidase is injected into the ciliary ganglion, retrogradely labeled cells are found only in the ipsilateral side (1.0 mm laterally from the midplane) of the lateral border zones of the anteromedian, EW nucleus, somatic oculomotor nuclei, and their ventral continuations of the ventral tegmental areas, including the regions among the oculomotor root fibers.34
Fourth, as far as we are aware, no information is available as to whether a connection exists between the parasympathetic preganglionic cells in both hemispheres (e.g., EW nucleus).
Finally, we are left with the fifth possibility: that fibers project from the Vsp to the contralateral EW nucleus. This possibility seems to be the most plausible in view of the present experimental findings: (1) microinjection of lidocaine into the Vsp attenuated the reflex contralateral pupil dilation (Figs. 7 8) , (2) microinjection of lidocaine into the Vsp contralateral to the stimulated side did not affect contralateral pupillary dilation (Fig. 8) , and (3) microinjection of lidocaine into the EW nucleus inhibited contralateral pupillary dilation (Fig. 9) . On the basis of our data, we suggest that contralateral pupillary dilation is mediated (at least in part) by fibers projecting from the Vsp across the midline to the contralateral EW nucleus. These findings are in accord with our previous observations that neural pathways exist between the Vsp and salivatory nucleus in palate blood vessels.17
However, we have not completely ruled out the fourth possibility (that there are projections from the ipsilateral EW nucleus to the contralateral EW nucleus), in view of the following observations. We could not examine the neural pathways between the Vsp and EW nucleus because of technical difficulties. We have reported that the effects produced by a microinjection of lidocaine into the Vsp would be exerted within a 1- to 1.5-mm in radius, judging from our previous histologic examination performed by injecting a histologic marker (horseradish peroxidase) into the brain stem.15 As shown in Figure 10 , the two EW nuclei of cats are not separate, as opposed to those of humans and monkeys, and seemed to be one body, as observed by Loewy et al.23 and Kuchiiwa et al.34 Therefore, it seems it would be difficult to elicit the selective inhibition of one side of the EW nucleus by lidocaine microinjections. Furthermore, the site of origin of the oculomotor parasympathetic preganglionic neurons is still not determined. It has been reported in the cat that the oculomotor parasympathetic preganglionic neurons originate from the area immediately ventral to the oculomotor nucleus in the ventral tegmental area22 34 and that the largest pupilloconstrictor responses elicited by electrical stimulation of the midbrain arise from the area ventral to the EW nucleus, rather than from the nucleus itself.26
In addition, we have not conclusively ruled out the possibility that the dilator muscle can be stimulated by activation of the oculomotor parasympathetic nerve followed by the LN stimulation, given that the dilator muscle receives input from acetylcholinesterase-containing fibers that degenerate after removal of the ciliary ganglion.21 Furthermore, Oono25 suggested that the efferent pathway for somatosensory stimulation in the conscious condition is different from that in the anesthetized condition. Thus, more work is needed to clarify the neural pathway(s) in the brain stem, although the present level of understanding suggests that the Vsp ipsilateral to the stimulated site and the EW nuclei play critical roles for eliciting pupillary dilation in response to LN stimulation. In addition, it should be clarified whether the contralateral pathways for the LN stimulation-induced pupillary dilation is identical with those concerned with the pupillary light reflex, because it is well known that there are the commissural connections between right and left EW nuclei for the pupillary light reflex.
We do not yet know the nature of the adequate stimulus, or indeed the identity of the sensory receptors, of the LN stimulation-induced pupillary dilation, or the characteristics of the afferent fibers that mediate it. However, C-polymodal nociceptors have been put forward as strong candidates for the primary afferents partaking in the parasympathetic-mediated responses such as LBF increase and salivary secretion, because electrical stimulation at higher intensities, topical application of capsaicin, and radiant heat stimulation to the tongue all cause an increase in ipsilateral LBF1 35 and because the LBF increases evoked by all these stimuli are significantly attenuated by pretreatment with an autonomic ganglionic blocker, hexamethonium.1 35 Because these responses can be evoked from all branches of the trigeminal nerve, the afferents are unlikely to arise from a specific cranial organ (e.g., eye, teeth, nasal mucosa, or tongue), and they are not restricted to a specific division of the trigeminal nerve. This suggests that the parasympathetic reflex pupil dilation that occurs in response to trigeminal stimulation does not arise from proprioceptors.
In conclusion, the possibility that fibers project from the Vsp to the contralateral EW nucleus seems to be the most plausible explanation of our results. Further work should clarify the pathways that play roles in eliciting pupillary dilation in response to somatic stimulation.
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Submitted for publication January 25, 2005; revised June 8, 2005; accepted September 9, 2005.
Disclosure: T. Tanaka, None; S. Kuchiiwa, None; H. Izumi, None
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. 
1734 solely to indicate this fact.
Corresponding author: Hiroshi Izumi, Department of Oral Physiology, School of Dentistry, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Hokkaido 061-0293, Japan; izumih{at}hoku-iryo-u.ac.jp.
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; right) elicited by stimulation of the left LN. Electrical stimulations were at (A) various intensities from 1 to 50 V at 10 Hz, 2-ms pulse duration for 20 seconds, and (B) 30 V, at various frequencies from 0.5 to 100 Hz, with a 2-ms pulse duration for 20 seconds. The data are the mean ± SEM (n = 6).
) and right eyes (
) elicited by electrical stimulation of the left LN (30 V, 10 Hz, 2-ms pulse duration for 20 seconds) before and after sectioning the left and/or right CSN. Data are expressed as the mean ± SEM (n = 10).
