Exp Brain Res (1996) 109:56-62 �9 Springer-Verlag 1996

S a l v a d o r M a r t i n e z �9 C a r l o s B e l m o n t e

c-Fos expression in trigeminal nucleus neurons after chemical irritation of the cornea: Reduction by selective blockade of nociceptor chemosensitivity

Received: 29 June 1995 / Accepted: 7 November 1995

A b s t r a c t The distribution and number of trigeminal brainstem and higher order sensory neurons expressing the protein product of the proto-oncogene c-fos after noxious stimulation of the cornea was studied in the rat using immunocytochemistry. The possibility that attenu- ation of nociceptive messages from the cornea by diltiaz- em reduced Fos-like immunoreactivity of spinal trigemi- nal neurons was also examined. A group of animals were killed 2-3 h after corneal stimulation. One cornea was stimulated with: a drop of 10 mM acetic acid; with acid plus mechanical scratching of the corneal epithelium; or with a drop of saline at 56 ~ C. Half of the animals treated with acid had been pretreated ipsilaterally with topical diltiazem (10 mM). Control rats received either saline in one eye or no treatment. Another group of animals were killed 7-8 h after stimulation with acetic acid. Fos-like immunoreactive neurons were counted in serial brain- stem sections using an anti-Fos primary antiserum and processed according to the avidin-biotin complex meth- od. In rats killed 2-3 h after corneal stimulation with ac- id, heat, or acid plus mechanical injury, labelled neurons were found in laminae ! and II of the intermediate zone between caudalis and interpolaris subnuclei of the ipsi- lateral spinal trigeminal nucleus and, in a reduced num- ber, in the symmetrical zones of the contralateral side. In animals stimulated with noxious heat or combined me- chanical and chemical injury, a few scattered cells were also labelled in the ipsilateral junction between the cervi- cal spinal cord and the caudalmost part of the trigeminal subnucleus caudalis. In rats killed 7 h after stimulation with acid, stained neurons were observed in the same ar- eas of the trigeminal nucleus as in rats killed at shorter times, but in lower numbers; in these animals, no immu- noreactive cells were found in deeper laminae or in high-

S. Martinez �9 C. Belmonte (~ ) Instituto de Neurociencias, Universidad de Alicante, Apdo. 374, E-03080 Alicante, Spain; Fax: +34-6-5658539

S. Martinez Depto de Ciencias Morfol6gicas, Universidad de Murcia, Murcia, Spain

er sensory relay nuclei. Pretreatment with diltiazem sig- nificantly reduced the number of cells of the spinal tri- geminal nucleus labelled after corneal stimulation with acid. The results indicate that brief noxious stimulation of the cornea evoke expression of c-Fos in neurons of the spinal trigeminal complex. Diminution by diltiazem of the number of immunreactive neurons activated by cor- neal irritation suggests that this drug, by reducing che- mosensitivity of nociceptive terminals, decreases noci- ceptive inflow to central nervous structures involved in ocular pain perception.

Key words Sensory neurons �9 Proto-oncogenes �9 Corneal pain. Calcium channel blockers. Diltiazem �9 Rat

Introduction

The cornea of the eye receives its sensory innervation from type B trigeminal ganglion neurons (Ardvidson 1977; Marfurt 1981; Lehtosalo 1984). Thin myelinated and unmyelinated peripheral prolongations of these neu- rons terminate as free nerve endings in the corneal epi- thelium (Zander and Weddell 1951; Rozsa and Beuer- man 1982). About 20% of A-delta corneal sensory affer- ents of the cat respond only to mechanical stimulation (mechanonociceptors), while the remaining A-delta and most of the C corneal nociceptive fibers are also sensi- tive to noxious heat and chemical irritants; thus, they have been classified as polymodal nociceptors (Belmonte and Giraldez 1981; Belmonte et al. 1991; Gallar etal . 1993). Transduction of the different qualities of stimuli in polymodal nociceptors seems to occur at separate membrane sites (Szolcsanyi 1987; Belmonte et al. 1991; Pozo et al. 1992). In the cornea, chemical excitation of single polymodal nociceptive fibers by acid or by irritant substances was selectively blocked by the Ca 2§ antago- nist diltiazem, without affecting their mechanosensitivity (Pozo et al. 1992; Gallar et al. 1993). This observation implies that nervous inflow evoked by chemical irritation of nociceptive terminals can be reduced by pretreatment with diltiazem.

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Corneal sensory neurons project into the spinal t r igem- inal nucleus (Vsp; Ardv idson 1977; Morgan et al. 1978; Panneton and Burton 1981; Mart in and Dolivo 1983; Mar- furt and del Toro 1987). In the rat, corneal project ions end on a small number of neurons forming two main groups: one p laced at the transit ion zone be tween subnucleus in- terpolaris and subnucleus caudal is at the per iobex level, and the second located caudal ly at the junct ion be tween the subnucleus caudal is and the upper cervical spinal cord (Marfurt and del Toro 1987; Light 1992; Lu et al. 1993; Strassman and Vos 1993). Bra ins tem corneal neurons can be driven by noxious s t imulat ion o f the corneal surface and in some cases also of per iocular skin (Mosso and Kru- ger 1973; Nagano et al. 1975; Pozo and Cervero 1993).

There is ample evidence that, in central sensory neu- rons. express ion of the prote in product of the pro to-onco- gene c-fos (Fos) is associa ted with cell act ivation by phys io log ica l s t imuli (Hunt et al. 1987; Bull i t t 1990; An- ton et al. 1991; Morgan and Curran 1991). Express ion of Fos- l ike immunoreac t iv i ty (FLI) within the spinal t r igem- inal nucleus after noxious thermal and mechanica l st imu- lat ion of the cornea had been repor ted recent ly (Lu et al. 1993; S t rassman and Vos 1993). In the present paper, we invest igated whether different modal i t ies of noxious st im- ulat ion of the cornea (chemical , mechanica l plus chemi- cal, and thermal) lead to c-Fos express ion in second- and higher order sensory neurons. The poss ib i l i ty that Fos im- munoreac t iv i ty induced by corneal irr i tat ion was attenuat- ed by pre t rea tment with d i l t i azem was subsequent ly ex- plored. An abstract of some of these f indings has ap- peared prev ious ly (Mar t inez and Be lmonte 1992).

Materials and methods

Animals and experimental treatments

Experiments were carried out in adult Wistar rats (200-400 g) anesthetized with pentobarbital sodium (Nembutal 5 mg/100 g i.p.). Stimulation was initiated as soon as blinking evoked by light touching of the lid border was abolished. Chemical irritation of the cornea was performed by topical administration of a drop (20 ~1) of 10 mM acetic acid solution. Mechanical injury was produced by scratching the whole cornea with a scalpel blade, removing the epithelium. Noxious thermal stimulation was carried out by ocular application of 2-3 drops of saline at 56 ~ C. The animals were killed between 2 and 3 h after the onset of anesthesia.

Six rats received a drop of isotonic saline in one eye, followed 10 min later by 10 mM acetic acid in the same eye. In one of the six rats, the cornea was scratched mechanically prior to applica- tion of acid. Another six rats received diltiazem (10 raM) instead of saline in the test eye, followed 10 rain later by 10 mM acetic acid. One of the diltiazem-treated animals had been subjected to mechanical injury prior to acidic stimulation of the cornea. Each animal of the diltiazem group was processed in parallel with a rat pretreated only with saline.

Two animals received hot saline in one eye. Rats subjected solely to anesthesia (n=3), treated unilaterally with saline at 33 ~ C (n--l), or treated with 10 mM diltiazem (n=l) without further nox- ious stimulation served as controls.

In a second type of experiment, longer survival times were al- lowed, to make possible the appearance of FLI in neurons distant from areas of primary afferent termination. In seven rats, a drop of 10 mM acetic acid was applied to one eye. These animals were killed 7-8 h later.

Tissue processing

For tissue processing, the protocol described by Anton et al. (1991) was followed. Rats of the first experimental group, pre- treated in one eye with diltiazem followed by noxious stimulation, were paired with animals equally stimulated but treated with sa- line instead of diltiazem and processed in parallel to minimize technical variations. All animals were injected at the end of the ex- periment with a lethal dose of Nembutal and were perfused through the heart with 100 ml of phosphate-buffered saline, con- taining 0.1% sodium nitrite and 1000 units/1 heparin, followed by 250 ml of 4% paraformaldehyde in phosphate-buffered saline. Brains were dissected and post-fixed overnight in the same fixa- tive containing 20% sucrose. They were then stored in a 30% su- crose-phosphate buffer 0.1 M, pH=7.4, at 4 ~ C for 24 h. The side of section was recognized by a hole made with an odontologic needle in the contralateral side of the brainstem, parallel to the midline. Serial coronal brainstem sections (40 pm) were cut in a freezing microtome and processed for immunohistochemistry.

Phosphate-buffered saline with 0.2% Triton X-100 was used as rinsing solution and chilled phosphate-buffered saline with 2% gelatine, as diluent for all immunoreagents. After five rinses in phosphate-buffered saline (10 min each), sections were incubated for 12 h at room temperature in polyclonal sheep antiserum direct- ed against the c-fos protein, diluted 1:1000 (Cambridge Research Biochemicals). Goat antisheep immunoglobulin biotinilated anti- serum diluted 1:200 (Vector) was used as secondary antibody. Thereafter, the tissue was processed using the avidin-biotin com- plex (ABC) method (Hsu et al. 1981). Following a final rinse, the sections were developed in a solution of 0.0l % diaminobenzidine and 0.02% hydrogen peroxide. The staining procedure was stopped by washing the tissue in phosphate-buffered saline. The sections were then mounted on chromoalum-gelatine-coated slides, air dried, and coverslipped. Specificity of the reaction was confirmed because all specific staining was abolished when the primary antibody was omitted. In two rats stimulated with acetic acid (one of each group), a parallel series of hindbrain sections were processed for immunochemistry using polyclonal antiserum directed against calcitonin gene-related peptide (CGRP; Kruger et al. 1988), diluted 1:10000, and the ABC method.

Counting and quantification

Serial transverse sections of the whole brain and cervical spinal cord were examined under a light microscope by an observer that ignored the type of treatment applied. Fos-like immunoreactive cells were mapped with camera lucida drawings and photo- graphed. All immunoreactive cells in alternate sections (every 80 gm) were counted.

Data are expressed as mean_+SEM per 40-gm section. Compar- ison of means was done with Student's t-test.

Solutions

The following solutions were employed: acid solution, 124 mM NaC1, 5 mM KC1, 10 mM acetic acid; control solution, 124 mM NaC1, 5 mM KC1 adjusted at pH 7.5 with 20 mM HEPES; and dilti- azem solution, diltiazem 10 mM (Sigma, St. Louis, USA), dissolved in control solution and adjusted to pH 7.5 with 20 mM HEPES.

Results

FLI cel ls after noxious s t imula t ion of the cornea

FLI neurons could be loca l ized in several b ra ins tem nu- clei o f rats 2 -3 h after app l ica t ion of acet ic ac id to an eye. In the Vsp, Fos -pos i t ive cel ls were ident i f ied in ipsi-

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d

Fig. la-d Expression of c-fos immunoreactive protein in spinal trigeminal nucleus neurons induced by noxious stimulation of the eye surface and its reduction by diltiazem, a Immunoreactive cell nuclei in the superficial laminae of left spinal trigeminal nucleus (Vsp; black arrows) and in the lateral reticular neucleus (LRN; empty arrows) of rats that received, in the left eye, 10 mM acetic acid. b Fos-like immunoreactive (FLI) neurons in the superficial laminae of the caudalmost portion of the subnucleus caudalis of the Vsp (black arrow) after stimulation of the left eye with saline at 56 ~ C. e FLI cells in the superficial laminae of Vsp and LRN when 10 mM diltiazem was applied prior to stimulation with 10 mM acetic acid. d Camera lucida drawings of caudorostral brain- stem sections obtained after noxious thermal stimulation. Dots re- present immunopositive neurons. Box 1 indicates the level of sec- tion b; box 2 marks the level of sections a and e of the stimulated side. Stars mark the trigeminal tract of the stimulated side, as a reference (pt pyramidal tract, IO inferior olivary nucleus)

lateral laminae I and II of the ventrolateral portion of the subnucleus caudalis (Fig. la) and to a lesser degree in the same level of the contralateral side (Fig. ld). They extended rostrally up to the caudal pole of the subnucle- us interpolaris and caudally down to the caudal pole of the inferior olivary nuclear complex (IO), corresponding

to a neural segment extending 1 mm caudal and 1 mm rostral to the obex. Other areas displaying FLI bilaterally corresponded to the nucleus of the solitary tract (NST) and the area postrema (data not shown). Some scattered neurons were observed in the lateral reticular nucleus (LRN) (Fig. 1 a, c). In five rats, the mean number of neu- rons counted in the ipsilateral laminae I and II of the Vsp subnucleus caudalis was 414+48. There were 276_+51 FLI neurons in the same location of the contralateral tri- geminal subnucleus caudalis (n=5, Fig. 2). CGRP-con- taining fibers, stained in alternate sections, terminated in the Vsp in the same areas where FLI cells were found (data not shown).

Two rats, subjected to noxious thermal stimulation with saline at 56 ~ C, showed 564 and 594 FLI neurons in the ipsilateral laminae I and II of the Vsp subnucleus caudalis, and 412 and 248 FLI cells in the symmetrical, contralateral side. Some dispersed FLI neurons were also observed in these animals in the caudal area of the Vsp/cervical spinal cord junction, ipsilateral to the stim- ulus (total number per animal, 42 and 24, respectively; Fig. lb).

ACID

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Fig. 2 Number of FLI neurons counted in the ipsi- and contralat- eral spinal trigeminal nucleus of animals that received 10 mM ace- tic acid (hatched bars), or 10 mM diltiazem followed by acetic ac- id (black bars) in the left eye. Values represent mean+SEM (n=5). * P<0.05, ** P<0.02

Another rat, subjected to scratching of the corneal ep- ithelium followed by topical administration of 10 mM acetic acid, showed a number of FLI neurons in the Vsp that doubled the value found when either acidic or ther- mal stimulation alone was employed (1018 cells in the ipsilateral and 700 cells in the contralateral side to the stimulus). In this experiment, a few scattered FLI cells (total number, 12) were also labeled in the ipsilateral junction between the cervical spinal cord and the caudal- most part of the subnucleus caudalis.

Rats that received pentobarbital anesthesia without further experimental manipulations, showed a sparse number of FLI cells in laminae I and II of the Vsp of both sides (mean 46_+10, n=3). With unilateral instilla- tion of saline at 33 ~ C, performed in one animal, the number of FLI cells was 196 in the ipsilateral to 174 in the contralateral spinal trigeminal nuclei. Application of topical 10 mM diltiazem to one rat showed 173 ipsilater- al and 105 contralateral neurons.

Rats treated with acetic acid and killed 7-8 h later showed a lower number of FLI cells in the Vsp (187_43, n=7), but similar laminar and rostrocaudal distribution as animals killed 2 h after noxious stimula- tion with acetic acid. Specific labeling was not ob- served in sections caudal to the periobex region of Vsp and in the contralateral ventrobasal thalamic complex, where Vsp neurons are known to project (Lund and Webster 1967; Bruce et al. 1987; Kempley and Webster 1989).

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At diencephalic levels, FLI neurons were observed bi- laterally in the intergeniculate leaflet and lateral habenu- lar nuclei (Fig. 3); the numbers were similar in control and experimental animals (data not shown).

Pretreatment with diltiazem

In five rats, topical administration of diltiazem (10 mM) to one eye 10 min before application of acetic acid to the cornea significantly reduced the mean number of FLI neurons in the ipsilateral (230_+49, n=5, P<0.02) and in the contralateral (114_44, n=5, P<0.05) spinal trigeminal nuclei in comparison with rats treated with acid alone and processed in parallel (Figs. 1C, 2). A diltiazem-treated animal, subjected to mechanical injury and acidic stimu- lation, paired off with the corresponding rat, equally stim- ulated but treated with saline, presented a comparatively lower total number of FLI neurons in the stimulated and the contralateral Vsp (800 and 306 cells, respectively).

Discussion

Fos expression after noxious stimulation of the cornea

Fos expression in second-order nociceptive neurons of the spinal cord and brainstem has been evoked by vari- ous types of nociceptive stimuli (Hunt etal . 1987; Men6trey et al. 1989; Williams et al. 1990; Anton et al. 1991; Strassman and Vos 1993; T611e et al. 1994a, b). Our results show that, under our experimental condi- tions, a small number of Vsp cells exhibited FLI without intended manipulation of the cornea and surrounding oc- ular tissues, perhaps due to anesthesia (Takayama et al. 1994). Topical application of isotonic saline or diltiazem, which are known to only briefly excite corneal sensory fibers (Belmonte et al. 1991; Pozo et al. 1992), recruited some FL! neurons in Vsp. This number was increased when an overt noxious stimulation of the cornea was per- formed with acid or noxious heat. Combining mechani- cal damage with chemical stimulation induced a high number of brainstem neurons. This is presumably associ- ated with an intense nociceptive activation when both stimuli were simultaneously applied and perhaps also to the fact that about 20% of corneal nociceptors are purely mechanoreceptive and do not respond to acidic stimuli (Belmonte et al. 1991; Gallar et al. 1993). In contrast, thermal injury and chemical irritation gave comparable quantitative results in laminae I and II of the Vsp subnu- cleus caudalis, thus supporting the interpretation that noxious thermal and chemical stimuli are transduced by the same type of corneal polymodal nociceptors (Bel- monte et al. 1991; Gallar et al. 1993).

Marfurt and del Toro (1987) described numerous pro- jections of primary afferent neurons innervating the cor- nea to the interstitial nucleus of Cajal in the spinal tri- geminal tract, at the level of caudal pars interpolaris and rostral pars caudalis, with no projections in more rostral

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Fig. 3a-f Fos-like immunoreactivity in higher order sensory nu- clei 7-8 h after acidic stimulation of one cornea, a-d Transverse sections of the thalamus (from rostral to caudal) showing the ab- sence of FLI cells in the ventrobasal thalamic complex, contralat- eral to the stimulus. FLI neurons can be seen in the area of the oli- vary pretectal nucleus (OPT), the intergeniculate leaflet (IGL), and lateral habenular nucleus (LH). e, f Expanded photomicrographs of the areas included in the insets in a and d, respectively. Scale bars: a-d 20 ~am; e, f 55 ~m (APT anterior pretectal nucleus, DLG dorsal lateral geniculate nucleus, LD laterodorsal thalamic nuclei, LP lateral posterior nucleus, MG medial geniculate nucleus, MH medial habenular nucleus, ML medial lemniscus, MPT medial pre- tectal nucleus, PC posterior commissure, PO posterior thalamic nuclear group, FR fasciculus retroflexus, PPT posterior pretectal nucleus, PV paraventricular thalamic nucleus, RT reticular thalam- ic nucleus, SC superior colliculus, V ventricle, VLG ventral lateral geniculate nucleus, VL ventrolateral thalamic nucleus, VM ventro- medial thalamic nucleus, VPL ventral posterolateral thalamic nu- cleus, VPM ventral posteromedial thalamic nucleus)

levels of the Vsp and sparse terminal fields in caudal pars caudalis and spinal cord dorsal horn. Presence of FLI cells in this lower level after corneal stimulation has been also reported (Lu et al. 1993). Our results show a concentration of FLI neurons mainly located at the peri- obex region of Vsp and are consistent with the view that they correspond to main monosynaptic projections of corneal and conjunctival afferent neurons to the Vsp, as suggested also by electrophysiological studies in the rat (Pozo and Cervero 1993). This is further supported by the overlapping of CGRP-containing fibers, which are considered trigeminal primary afferents (Tashiro et al. 1991), with FLI neurons in the Vsp.

In our experimental conditions, FL! cells in the most caudal region of the trigeminal subnucleus caudalis ap- peared only when thermal or mechanical plus chemical irritation were combined. This indicates that higher in-

61

tensities of noxious stimulation, leading to longer lasting impulse discharges and perhaps recruitment of mechano- nociceptors (Belmonte and Giraldez 1981; Belmonte et al. 1991), are probably required to activate this subpopu- lation of second-order corneal sensory neurons.

With tracer methods, the existence of corneal primary afferent projections that cross the midline to terminate in the contralateral side has been controversial (Panneton and Burton 1981; Marfurt and del Toro 1987; Jacquin et al. 1990; Marfurt and Rajchert 1991); however, about 17% of corneal-driven neurons recorded in the rat had a contralateral input (Nagano et al. 1975). Our data con- firm the existence of a significant population of second- order trigeminal (and perhaps higher order) neurons, ac- tivated by noxious input from the contralateral anterior segment of the eye. The proportion of FLI contralateral neurons found in this study is higher than when electro- physiological methods were used (Nagano 1975). This is not surprising, considering that in electrophysiological recordings identification of neurons may be limited by their size and location. FLI neurons were also found in the reticular formation and in the nucleus of the solitary tract. This may partly reflect nonspecific effects of anes- thesia and stress (Anton et al. 1991), but also the projec- tion of corneal trigeminal neurons to these structures, as suggested by morphological and electrophysiological studies (Clarke and Bowsher 1962; Mosso and Kruger 1973; Nagano et al. 1975; Marfurt and Rajchert 1991). Moreover, positive cells in the lateral reticular nucleus may correspond to neurons of A5 and A7 catecholamine nuclei in the brainstem, involved in nociceptive modula- tion (Burnett and Gebhart 1991).

It has been reported that noxious stimulation of the skin by heat or subcutaneous injection of formalin in- duced a rapid onset of Fos in neurons of laminae I and II of the spinal cord dorsal horn that reached a maximum in 2 h and decayed gradually in the following 8 h; a "second wave" of c-Fos labeling occurred with cutaneous thermal stimulation in higher order sensory neurons (Williams et al. 1990; Leah et al. 1992). In agreement with these data, the number of corneal brainstem neurons displaying FLI was higher 2 h after application of an acidic stimulus to the eye than when 8 h had elapsed. However, no labelled neurons were observed in deeper laminae of the Vsp or in higher sensory areas of the brain over this longer period of time, in spite of the labeling of other brain nuclei not related to the processing of nociceptive information. It is possible that, in our experimental conditions, injurious stimuli were not strong enough the produce sufficient polysynaptic neuronal activation to evoke this second wave of Fos protein synthesis, which appears to be strongly influenced by the type of noxious stimulus and by anesthesia (Williams et al. 1990; T611e et al. 1994a).

Effects of diltiazem pretreatment

The five animals treated with diltiazem prior to noxious stimulation exhibited a pattern of stained neurons that

was comparable with control animals, except that the number of labelled neurons was significantly reduced. The number of FLI neurons was also lower in rats with combined mechanical and acidic stimulation, after pre- treatment with diltiazem. Differences in number of neu- rons expressing c-Fos have been used to evaluate the ac- tion of drugs on neuronal discharge activity (T611e et al. 1990, 1994a, b; Presley et al. 1990). Topical diltiazem, at the doses employed here, reduced sensory discharges elicited by acetic acid in corneal polymodal nociceptors as well as behavioral signs of discomfort and inflamma- tion induced by chemical irritation of the eye (Pozo et al. 1992; Gonzalez et al. 1993). These effects are presum- ably associated to the specific blockade of ionic channels involved in chemical excitation of nociceptive terminals (Konnerth et al. 1987). It can be assumed that the re- duced number of Fos-labelled neurons lit up by acidic stimulation in the Vsp of eyes pretreated with diltiazem was due to a decrease in peripheral noxious input reach- ing Vsp neurons after topical administration of the drug; this sensory message leads ultimately to pain sensations (Tanelian and Beuerman 1984; Chen et al. 1995). There- fore, selective blockade of chemical activation of noci- ceptors with drugs like diltiazem may be a therapeutic means of reducing peripheral pain.

Acknowledgements We wish to thank Dr. J. Gallar and Dr. G. G. Gonzalez for their collaboration. The technical assistance of Mr. Sim6n Moya is also acknowledged. Supported by the Secretaria General del Plan Nacional de Investigaci6n Cientifica y Desarrollo Tecnol6gico, Comisi6n interministerial de Ciencia y Tecnologla (SAF93-0267).

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