Sensory mediation of the ocular response to neutral formaldehyde

Exp. Eye Res. (19i9)28,577-589

Sensory Mediation of the Ocular Responseto Neutral Formaldehyde JOHN M. BUTLER,~ILLIAM Im+itute

G.UNGERANDBRIAX

R.HAMMOND

of Oph~thnlwwlogy, Judd Street, London WClH

(Received 4 October 1978 and ,iu revised form 3 Jnnunry

9&S, U.K. 1979, London)

Topical application of 100 or 25Opg neutral formaldehyde to the rabbit eye elicits an acute inflammatory response consisting of a raised intraocular pressure (IOP), anterior uveal vasodilation. miosis and an increase in protein in the aqueous humour. Prior treatment with 0.4”, benoxinate (12 drops over 30 min) inhibited these ocular changes whereas svstemic indomethacin or atropine pretreatment had no notable effect. No raised levels of prostaglandin-like activity were observed in any samples of aqueous humour withdrawn 15 min after stimulation with formaldehvde. The maior site of breakdown of the bloodaqueous barrier was shown by fluoresckin angiographv and colloidal carbon vascular - - _ . “labelling” to occur in the ciliary processes. The IOP resnonse to 6-50 IugY formaldehvdeI administered intracamerallv Y durinn 0 closed I circuit perfusion was greatly reduced by benoxinate or intracamerally infused tetrodotoxin. Furthermore the response was essentially abolished in eyes in which the ipsilateral sensory nerve supply had been destroyed by diathermic coagulation of the ophthalmic branch of the trigeminal nerve. The response to submaximal doses of formaldehyde was greater in the unilaterally sympathectomized eye than in the fellow control eye. These observations indicate that the ocular response to this form of chemical irritation is dependent largely, if not entirely, upon a non-prostaglandinergic and non-cholinergic excitatory pathway most likely occurring in sensory nervous elements. Key words: sensory denervation; sympathectomy; tetrodotoxin; benoxinate; indomethacin ; rabbit eye; blood-aqueous barrier; intraocular pressure: ciliary processes; prostaglandin; pupil constriction.

1. Introduction The acute response of the rabbit eye to various noxious stimuli seems generally to be by prostaglandins (PGs) and by an atropine-resistant, neurogenic pathway, the relative contribution of the two depending upon the type of stimulus applied (Duke-Elder and Duke-Elder, 1931; Neufeld, Jampol and Sears, 1972 ; Cole and Unger. 1973; Unge r, Perkins and Bass, 1974; Unger, Cole and Hammond, 1975; Eakins. 197T; Unger and Bass, 19’77; Unger, Cole and Bass, 1977). The response to mechanical irritation (e.g. scratching the iris) is associated primarily with PGs (Amhache. Kavanagh and Whiting, 1965) and is greatly reduced by indomethacin (Cole and Unger, 1973) an inhibitor of PG synthesis (Vane, 1971). Chemical irritants such as neutral formaldehyde appear to act through the nervous pathway, believed to mediated

involve

antidrornic

stimulation

or axonal

reflexes in sensory elements

(Bruce,

1913;

Davson and Huber, 1950; Perkins, 195’7; Sears, 1960; Cole and Unger, 1973). The response to ruby or argon laser irradiation of the pigmented iris seems to include both mechanisms. being partly inhibited by indomethacin or by local anaesthetics and Ijeing abolished by the combination of both drugs (Unger et al., 1974, 1977a, b). Intracranial mechanical stimulation of the Vth nerve elicits a strong ipsilateral inflammatory-type

response (Perkins,

1957; Cole and Unger, 1973). Moreover,

Perkins

(1957) also observed that following Vth nerve section (ostensibly distal to the Gasserian ganglion) no notable change in intraocular pressure (IOP) is obtained on mechanical stimulation of the nerve trunk proximal to the point of severance, thus OOL-4835/79/050577+13 $Ol.OO/(~

0 1979 Academic Press Inc. (London) Limited 517

573

J. hf. BUTLER,

IV. G. CNGER

AND

B. R. HAMMOND

suggesting that the excitatory pathway depends upon antidromic impulses and excluding the involvement of central reflex pathways. Butler and Hammond (1977) suggested that functional sensory ~~crve endings were necessary for the manifestation of the response to both chemical and mechanical irritation and it is likely that sensor,~ denervation would render the eye insensit,ive to chemical irritation with formaldehyde. Although prohahly not directly involved with mediation. adrenergic nervous acti\-it,! is likely to exert an inhibitory influence during an ocular reaction to injury so t,hat sympathetic denervation would result in a greater responsiveness (Unger. 1977). In the present paper the effect of a unilateral spmpathectotny or of sensory denervat,ion on the acute response of the rabbit eye t*o small doses of neutral formaldehyde was investigated. The effects of indomethacin and nerve-blocking agents were also studied. 2. Materials and Methods Adult New Zealand rabbits (2-35 kg) of either sex were annesthetizetl hy :LII initial intravenous injection of 30 mg/kg sodium pentobnrbitone, supplemented during the course of the experiment to maintain a respiration rate of 25-30 per min.

Neutral formaldehyde in Krebs physiological solution (10 or 2.5~1 of l”/b or 50 ,uI of 5”,,) MYMapplied topically to the upper quadrant of the eye and allowed to run over the corue;l into the lower conjunctival sac. The eyelids were held closed for about a minute to enable adequate contact and absorption of the irritant. In eight acute experiments, formaldehyde was administered simultaneously to both eves. one of which had been treated for 30 inin previously with 0.4 O’ , 0 benoxinate (Xovesine; 2 drops/5 min). The intraocular pressure wits measured at intervals of 2-3 min by applanution tonometry (Perkins, 1968) and the pupil size determined using fine dividers. At 15 nlin aqueous humour samples were withdra\vn for analysis. In other acut,e experiments the response to forma.ldehyde of one eye \v:ts determined and aqueous humour withdrawn as above: 20 mg of indomethacin (it Wsynthetase inhibitor, Vane, 1971) was t,hen injected intravenously as a divided dose and the contralateral eye was then similarly stimulated with formaldehyde 30 min later. 111 four experiments 5 mg atropine was injected intravenously 5 min autl again 1 miu before application of 250 pg formaldehyde. Inhxumeral

administration

of ,formcrlde?~~p!e

Neutral formaldehyde was instilled into the mterior chamber of a rabbit eye during closed circuit perfusion as described by Hammond (1977). The head was supported ill a restraining cradle and the cornea transfixed by a special needle which enabled the anterior chamber to be conne&ed into the perfusion circuit. Drugs for infusion were placed in parallel 25 ~1 loops of tubing on the afferent side of the eye unt’il connected into the perfusion circuit,. A pressure transducer was joiued to the circuit near the eye to enable 1977, modification of NagLcontinuous measurement of the IOP ( see Hammond, subramauinu, 1974). The femoral vein and artery were cammlated for systemic inject.iull of drugs and the measurement of blood pressure respectively. Both eyes were simultaneously perfused using independent circuits so that the IOP response of a control eye to formaldehyde could be compared directly with that in a sympathectomized or it sensorily denervated eye (see below). Similarly the effect of indomethacin, henoxinate or tetrodoxin on the response was studied using a test and a contralateral control eye. During perfusion experiments, the pupil size was measured periodically.

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t,Tnilateral sympathetic denervation of the right eye was effected by surgical excision of the right, superior cervical ganglion along with an ascending segment of the cervical nerve (about 1 cm) and the descending post-ganglionic branches as far as possible (up to 0.5 cm). At least 8 days were allowed for nerve degeneration to occur and the respective eyes were tested for supersensitivity to a topical drop of 0.1% noradrenalin 48 hr before an experilllellt. .41~inmlsnot showing supersensitivity were rejected.

ITnilat,eral sellsorv denervation of the left eye was achieved by stereotaxic location and cliathermic destruction of an intracranial segment of one Vth cranial (trigeminal) nerve. The head was positioned in a stereotaxic clamp in a laterally horizontal plane with lamhda 1.5 mu below bregma. A 3 mm hole was trephined through the skull 2.5 mm lateral and 1.5 mm posterior to bregma and a nylon-sheathed needle electrode introduced vertically through the cortex to co-ordinates obtained from a previously constructed data-chart I~,sed on the animal’s body weight. With an anodal plate on a shaved patch of skin on the ahdomc~n, electrical current was passed through the animal at various positions in the estimated region of the Vth nerve (5-7 applications, 10 set each: 33 000 V, 0.1 mA, 50 Hz). .1nt,ibiotic ointment was afterwards applied to the wound and the skin sutured. In a successful operation the lids and cornea were completely insensitive to touch mtl the hlinking reflex lost. although the light reflex remained. The eyelids were sewn together to avoid cornea1 damage and the development of a neuroparalytic keratitis. The alli n& \verc used sis to nine days later.

In order to demonstrate the site of disruption of the blood-aqueous barrier, a flnorescein angiographic procedure (Unger et al., 1974, 1975; Edwards, 1975) was carried out 10 min after adntinistration of 250 pg formaldehyde topically (three experiments) or 25 pg infused intracatnerally (two experiments). Sodium fluorescein (2O%, ; 0.1 ml/kg) was injedted into the marginal ear vein and photographs of the dye fluorescence in the anterior segment of the eye were taken at 1 min intervals for the succeeding 15 min. In four other experiments 5 ml of Uno india ink (colloidal carbon) diluted with an equal \-olume of physiological saline was injected intravenously 10 min and the same volume 2() min after topical or intracnmeral administration of formaldehyde. The animal was killed 30 min later and the iris with ciliary processes attached was excised and placed posterior surface upwards on a microscope slide. Photomierographs of the blood vessels in representative areas of the iris and ciliary processes were taken at x 10 and x 40 magnification (on the film) on Tri-X (Kodak, London) 35 mm film using a Leit,z microscope with an “Ort’hoplan” at,tachment.

111experiments involving topical application of formaldehyde PG-like material in acidlipid extracts (Unger, Stamford and Bennett, 1971) of aqueous humour were bioassayetl ~)IIa11isolated rat stomach fundus strip using authentic PGE, as a reference standard, The rotlcentration of aqueous protein was determined in 10 or 25 ~1 aliquots by a nlo&ficatioll of the method by Lowry, Rosebrough, Fnrr and Randall (1951) against bovine serllnl albumin standards. 3. Results Administration of 100 pg neutral formaldehyde topically to an untreated rabbit eye caused a pupil constriction, a transient increase in IOP and a raised protein concentration in the aqueous humour, all of which were significantly inhibited by prior treatment,

560


W. G. USGEK

AND

B. R. HAMMOND

with benoxinate (five experiments; Table I). After administration of 250 pg formaldehyde similar ocular changes were all significantly reduced by prior treatment with benoxinate (eight experiments) but not by indomethacin (five experiments) or atropine (four experiments) (see Table I). B enoxinate had only a slight effect on the expected IOP rise following 2.5 mg formaldehycle. No abnormally hi& levels of PGactivity (2.5 ks.E.O.9 rig/ml as PGE,; 10 experiments) were found in any aqueous samples from untreated eyes exposed to formaldehyde. TAKLE 1

Ocdar

DOSO*

100 tLg

350 pg

7.5 mg

changes i~nduced by topically

(So.)

DlWg

JIOI’ (mm Hg)

” (0)

23 ‘) JR 0

(1) (0) (4) (0)

20 (2)

ss None Ben

rteutral formaldehyde

3l’upil (--mm)

1.0 o.:! I’ < 0.9 0.3 0.8 0.2

“IJ (1)

(5) (a) (5) (h) (a & h) (11) (cl c+) Cd) (7) (e) (4 (4 (4) (g) (c & d) (C & c)

(C & f) (C $6) (4) (h) C-i) (i) (h & i)

rtpplied

xs “X (2) I9 (2) 1’ < 0.05

(0.3) (0.1) om (02) (O-l) (0.1) (0.1)

0.7 (0.2) I’ < 0~005 R’s I’ < 0.005 KS 4.3 (025) 4.1 (0-t) KS

Protein (mp/ml)

6.1 (1.2) 1.i (0.2) P < 041 7.2 (04) 143 (0.1) 5.8 1.3 6.9 1’ <

(0%) (0.2) (0.7) 0.0035 NS I’ < 040”5 NS 12.9 (1.3) 9.7 (1.“) X-S

* Duse = neutral formaldehyde; (No.) = number of espariment~s; Drugs = O-40, benoxinate (13en), JO mg/kg indomethacin (IM), and 10 mg at,ropine (Atr) as given in Methods; Protein = soluble aqueous protein. The letters are given to facilitate the statistical comparisons. NS = not significant. Values are given as mean (&S.E.).

Intmcameral

formaldehyde

in the sympathectomized

eye

The introduction of small amounts (3-25 pg) of neutral formaldehyde into the rabbit anterior chamber during a closed-circuit perfusion caused a transient ipsilateral elevation of the IOP which was dose dependent both in the normal (left) and the sympathectomized (right) eye. The rise was generally more intense in the latter (see Fig. 1 and Table II). The IOP response usually recovered by about 20 min but in some cases the IOP tended to assume a higher baseline than initially, particularl! with higher doses of formaldehyde. Because the experiments were acute and terminal it was considered acceptable nevertheless to instill two or three doses of formaldehyde during a given experiment in order to assess more readily the responsiveness of the eye and to evaluate the effectiveness of certain drugs. For purposes of analysis the IOP responses were therefore measured in the following two ways: (1) as the rise in IOP from the initial baseline to the peak (baseline to peak) which accordingly incorporated any gain in baseline and would most likely represent an overestimate in the TOP response for the second and third application; (2) as the rise in IOP from the level immediately prior to administration of formaldehyde to the peak of the response

I)C’GLAR

RESPONSE

TO FORMALDEHYDE

581

(l-1 45

PIG. I. ‘rhr rrsporw of intraocular pressure (1OP) induced by intracameral infusion of 1’3pg neutral f~wnaltlehytlt~ was slightly grester in the right unilaterally sympathectomized eye thau in the conttalatSeral rontrol epe.

(peak rise) which by comparison probably represented an underestimate. The greater responsiveness of the sympathectomized eyes over their fellow control eyes increased with increasing amounts of formaldehyde, between 3 and 6 ,ug of formaldehyde. but, l)ecamr less striking with higher doses (12.~50pg) (see Table Ii). Formaldehyde usually caused the pupil to constrict intensely in both normal anti ~vrnpat,lrectoluizerl eyes in a way which was not dose dependent’ and rarelg recovered

Pd intraocular pressure changes following intracanaeral administration of rtcuh-al forwddeh~yde to sympthectomized right eyes and normal left eyes

Uaseline to peak LJow

(SO.)

Left. (mmHg)

(3) (9) (12) (8) (4)

3 11 IX 29 4%

(W!)

3 3 6 I:! “5

(1) (3) (3) (6) (11)

Right (mm%)

7 20 33 36 48

(1) (2) (4) (6) (9)

Peak rise (R-L) (mm Hg)

4 9 13 7 6

(1) (:3) (4) (2) (6)

P

om WOO5 0.005 .1’s ,

Left (mmHg)

3 11 18 24 94

(1) (3) (3) (5) (6)

Right (mmHg)

1 1x 28 28 33

(1) (3) (4) (5) (9)

(R-L)

4 I 11 4 -2

(0)

(3) (4) (1) (4)

1)

.-

0.05 0.05 04l5 Sk3

Rascline to peak is the difference between the 1OP at the peak of a response and the initial baseline whereas “peak rise” is the difference between the peak IOP and that just prior to stimulation (see Results section). Xo. =: number of observations; R--L = difference in IOP: p = the significance calculated by a pairrd I-test: NH : not significant. Values are given as mean ( *S.E.).

585

.J. M. BUTLER,

I\:. G. L’NGER

ASD

U. X. HAMMOND

to the initial size. The miosis was generally greater in the svillpathectorllized eye. IJut this was not consistent nor statistically significant. The results have not therefore Iwen presented here. Z,,hihitio~l

by benoxinnte

In two experiments topical pretreatment, with 04’);, henoxinat’e retluceci the 101 respoIlse of 17 and 38 mmHg to an intracameral close of 12 and 35 pg formalcleh~tl~~ t)y 100 and SW!{, (iSo,‘, measured from inihial t)aseline) respectively. Intracamcrxl infusion of 2.5 pg benoxinate per min fur 60 min in addition to 100 pg. 10 min l)ricu to st,imulation, prevented an IOY response to 25 or 50 tug fomddehytlc (five eqwimerits; see Table 111) in all hut one experiment (91:/l, reduction; see Fig. 2).

-125 F E E -75 2I E (RI 5 - (L) ;- SO-

;

--

40 -3o-20

FIG. 3. The normal intraocular pressure (IUP) response to neutral formaldehyde by treatment with benoxinate. (F, formaldehyde: 04q, Ben. 04’,,, benoxinstc; eye; B.P., blood pressures.

was grostly inhibited (R, L). right and left

OCULAR

I&ibition

RESPOSSE

683

TO FORMALDEHYDE

by tdrodotoxin

Introduction of 2-5 g tetrodotoxin into the anterior chamber of the right eye after a first stimulation and 20 min before a second reduced the response (to 6; 12 and 25 pg formaldehyde; one experiment each) by 75%lOOq/, (measured as peak rise) or 55 to lOO”;, (measured from initial baseline). By comparison t.he response to a second dose of formaldehyde was reduced in only one experiment’ (by 200{,) in the cont,raIaters I wntrcd cycs (we Table IV and Fig. 3).

- (RI I” E 2 IO ;

30

IO

F CR.L)

TTX CL)

F (R,L)

, F (R,L)

of the normal intraocular response (IOY) t,n Ij pg intrscamornlly infused formaldeh~tln I%:. 3. Inhibition by trtrorlotoxin. F, formaldehyde: TTS, tetrodotosin: (R, L). right and left eyes.

In five experiments the eye subjected to sensory denervation failed to respond t.o 6 pg (two experiments) or to 12 pg formaldehyde (three of five experiments) and produced only a small, delayed rise in two experiments after 12 pg or 25 ~*,g(one experiments) as compared to a normal level of response in the contralateral control

584

J. 31. l3I!TLER,

W. G. UKGIG,K

.5X1,

H. K. HAXMOKU

eyes (P = --< 0.005 to 0.01; see Trtl~le V ant1 Fig. 1). Only a small pupil response occurred in four out of eight formaltlehytle infusiow in the denervated eyes ( -0*3&0*1 mm) whereas that in the norn~al fellow eyes WLS more intecse ( -3.8,:: O-3 mm ; P -:I 0.001). In one additional animal tested 1S hr after the operation. liefore t,he nerve degenerat’ion was complete. the IOP response to 25 pg forn~alrlehydr wm only 35”; less than in the contralateral control eye. Site of lmddom

of tlw blood-rrqwous

h~irrirr

Pluorescein injected intravenously aft,er t,he topical application of furrualtlehyde was observed to enter the anterior chanber rnost~l~ through the pupil from t.hr

* I’ = paired l-teat whew I’ z value given ; t IOP peak response in sensory denervatrvl slowly; $ experiment carried out 18 hr after intracranial diathermy. vahes are given as mean (&SE.). Baseline to peak and Peak rise, see Kesults section or legend for Table II.

rye tlevclopcll

50

Normal CR)

10 tted

,v-

, 6r3 F (RI

u 619 F CL1

2mg Pbf

l&g F IRI

I&, F (Li

25’L F (RI

25:g F IL1

FIG. 4. The intraocular pressure (IOP) response of the normal eye fails to occur in the sensory ,denervated eye following infusion of 6 or 12 pg of formaldehyde. A slow resporlsc to 25 pg formaldehyde ,did occur in the denervated eye. F, formaldehyde: (K, L), right normal and left denervated eyes; Pbt. sod&m pentobarbitone onaesthetic injected intravenously; B.P., blood pressure.

OCULAR

RESPONSE

TO FORMALDEHYDE

Pro. 5. Fluorescein was injected intravenously 10 min after topical application of 250 pg (colmnn A) or intracamwal infusion of 25pp (column 13). The dye associated with dense, apparently plasmoid, aciueous entered the anterior chamber mostly through the pupil. Photographed at (column 9) 4.5 min, IO min and 15 min and (column H) I.5 min, 4 min and 8 min (from top to hottom) after fluoreswin Injection.

posterior chamber, although some diffuse fluorescent material did appear to arrive from the anterior surface of the iris (see Fig. 5). Marked vasodilation in the ciliary processes. particularly the long, anterior (iridial) processes is indicated by the “cartwheel” pattern of fluorescence visible through the non-pigmented iris. In the rabbits receiving india ink intravenously, colloidal carbon particles were found marking the

586

J. &I. BUTLER,

W. G. UNGER

AND

B. R. HAMMOND

capillary beds of some ciliary processes (see Fig. 6) but not in any vessels of the iris indicating that increased permeability occurred in the ciliary capillaries but little if any in the iris. The appearance of some diffuse fluorescein from the surface of the iris might represent a diffusion of free fluorescein (not associated with plasma protein) from the dilated iris vessels or possibly dye associated with protein which might have entered t’he stroma of the iris directly from the anterior (iridial) ciliary processes by diffusion across the iris-ciliary interstromal connections, an anatomical feature peculiar to the rabbit (see Prince, 1964).

FIG. 6. (.‘olloidal carbon injected intravenously after topics1 spplication formaldehyde lnbelled the capillary vessels in many of the iridial processes.

of %opg

of neutral

4. Discussion Bethel and Eakins (1972) reported that the ocular response to formaldehyde is sensitive to treatment with indomethacin, thus implicating PGs in its mediation. Cole and Unger (1973), h owever, observed no involvement of $Gs and postulated that the response to chemical irritation is almost, entirely nerve-mediated. The present experiments showing that benoxinate drops, unlike indomethacin, inhibit the formaldehyde-induced ocular response substantiate the latter hypothesis and suggest

OCULAR

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that the mediation has little or no dependence upon PG synthesis or release. Further weight to this theory is provided by the considerable reduction in the response after infusion of novesine or tetrodotoxin and in sensorily denervated eyes. In assessing the role of the autonomic nervous tracts in the response to formaldehyde two main possibilities exist. Firstly formaldehyde may directly stimulate or inhibit peripheral autonomic elements or their receptors. Secondly central reflex excitation or suppression of autonomic fibres may be produced by orthodromic transmission in sensory nerves. Considering the latter, the observations of Perkins (1957) and Butler and Hammond (1977) would tend to discount the involvement of central reflex pathways. Parasympathetic activity alone could not account for the severity of the response, but it was considered that a subsidiary cholinergic component contributing to aspects of the response is partly or wholly masked by cwcurrcnt sympathetic activity. However our findings that syst)emic mtroduction or topical application of atropine. in concentrations which blocked the light reflex and produced extreme mydriasis. (lid not reduce the response to formaldehyde woultl t,end to discount the inprtame of. or at least any dependence upon parasympathetic activit,y. Any I)arasynlpathetic contribution to the response must be subsidiary to the sewor! nclmponent as its total abolition after Vth nerve section shows. This subst,antiatts t,he olwrvations of Cole and Unger (1973) and a similar conclusion has been drawn 1)~ ringer et al. (1974. 1977a. b) for the laser-induced response. The role of the sympathetic nervous system is more complex. Sympathetic activit,v. 1)~ v&u.e of its rnpdriatic and vasoconstrictor effects. could be expected to exert, d,rl inhibitory influence during an injury response. Although Perkins (1957) reported t,hat swlpat~hcctomy did not affect the response to Vth nerve stimulation, IJnger (1977) lr:w shown that the IOP and pupil response to ruby laser irradiation of the iris are greatly augmentetl in the sympathectomized rabbit e?e. anti a similar effect following chemical irritation is suggested ly the present studies. Just MC:t,hev operate in the Irornlal eye, so c~i autonomic elements undoubtedly exert, to a greater or lesser (sstent, some influence on the vascular and pupillary mechanisms of the inflamed eve. Such activitv however appears to be of secondary importance thwgh sympatlletic, tone piwl)ably moderates by its vasuconstrictor actions the pot~rtnt~ialseverity of t,lle ~ensoril,v mediatetl injury response. The responw to intracameral infusion of formaldehyde was greatly decrease(l 1,~ \wloxinate and t’ctrodotoxin and essentially abolished in the eyes which unclcrwtllt Vth nerve degeneration, confirming the findings of Bruce (1913). Perkins (1957) atIt ISutlw and Hanmlond (1977) that’ a nerve pathway is essential for the mediation of tllr respo~~scand is probably identical to t’llat wit’11sensor\’ function. BlockatIe of the &ots of’ topically applied formaldehyde 1)~ novesine intllcat,etl t,tkat,the response to t,his mode of irrit,ation most likely acts through st~imulation of superficial nerv(,llh c~lwlents. The failure to block the effects of 2.5 mg formaldehyde suggest an extrenlr (tegrec of irritation wherein non-specific perhaps direct toxic actions on various cellular rlenwnt,s in the anterior uvea may predominate. Similarly a variable but incomplete inhibition following intracamerally infused formaldehyde may also reflect some direct t&c effects of the irritant. Tetrodotoxin, a more specific nerve blocking agent (Kao. 196i) &an local anaesthetics, abolished the response to 6 and 1%yg for-naldehyde but (lit1 not completely prevent that to 35 pg. The formaldehyde-induced response was essentially abolished in eyes which suffered Vth nerve coagulation, but even in some animals, which on necroscopy were found to he completely denervated, a delayed residual TOP (and slight rniotic) response occurred. In experiments with normal rabbit

588

J. BI. HVTLER,

N. C;. I’SGEH.

AND

13. 11.. HAMJlOST)

eyes (unpublished observation of Butler) the formaldehyde-induced miosis does not recover as rapidly or as fully as that induced by capsaicin (a pepper extract which appears to act specifically on sensory nerve endings; Jansco. 1960; Arvier, C’hahl and Ladd, 1977) or by antidrornic stimulation of the Vth nerve. Diathermic coagulation of the Vt,h nerve usually produced a milt1 inflammatory reactiou consisting of a moderate weal vasodilation and an EbqlleCJuS flare which persisted for about 12 hr. l’hc possibility that tachyphplaxis was tlue to residual inflammatory changes might account for the insensitivity to snhsequent formaldehyde stimulation. However this m-adiscounted since. at S-6 days after the operation. the eyes, apart from some CcJrned changes, appeareti nornml on slit-lamp examination. and on Jnicrosx~pic exanJinat.iorl of the excised tissue:: the iridial and ciliar\- vascular beds were neither dilated IICJI congested. Vascular congestion is unlikely to 1Jrevent the pupil front constrict#ing. Moreover, the resl)onse to formalcl~hytle ~a’: much less reduced in a tlenervatctl cyc tested 18 hr after coagulation when nerve degeneration was probably incomplete ant1 t,hc vascular congestion more likely to be prrscnt. Prom the foregCJil>gcomment:: it wodd appew t#hat the response to fonnaldehyc 1~ irritat,ion depentls primarily upon intact sensory uerves and that mediation is presunlablv effected through such neural elements. However. owing to a probal)le now specific, toxic action on the tissue, formnltlehvtle would appear to bean inferior nlethclel of sti tnrdetkn for the pwpose of elucidating thr pht;xiokJgical ~~lechnnisni of the’ ocular response to injury. Nevertheless. the clbservatiorls made in the present, st~ucI\ are consistent with previous findings. Tetrotloboxin alone complet~ely abolishetl. Lvltilht ~)enoxinate only partly blocked t,he I(>P and ])uJd res[mJlsc to rubYlaSer irradiatic~ll of the iris (Ung-er et, al., 1977). Butler and Hammond (1977) have shown that, tlrc, sewory denervated eye loses the abilit!. to IW~JOI~ tu E-type PG. or to In-ad!-kinill. On W11S(JI’\’ It thns seems pwsilJle that such chcnlical nrwliators III~! act ~Jrirllaril\nerve endings either to release a lJutat,ive infhwlmat~orv tra~~smit~ter substa,nce or to (i.e. i)? initiaOe nnot,her excitatory pathwn> which is lwopagat~ecl antirlromicall~ asonal reflex) to ncighlwuring tissue element,s (as ill t)hc triple response defined II!. Ferris, 1927) thus augmenting the response, 1ulii)~itir~ii cjf this nerve propagation I J! local anaesthet,ics would only prevent the ‘.sprewl” of the inflammatory stiniulw. \vhilst 1uorc effective nerve blocka~lc or complete tlestruction of the sensory ner\-cs and terminals would abolish the reaction. In cor:clnsion. local aclminifit,rat,ion of nwderate amounts of neutral formaldehytlr elicits z;cutr ocular inflammatory changes \rhich appear to be mediated through sensory nervous elements and att,enuated by sympathetic nervous activity. PG seems unlikely t)o be involved in the mediation of the response.

The authors wish t,o thank Professor D. F. Cole for valued discussions and Q. Oliver for her assistance in the preparation of the nlunuscript.. One of us (J. M. But,ier) also wishrs to t,hank the Medical Research Council for financial support.

Ambache, K., Kavanagh. L. and Whit,ing. *J. (1965). Effert of mechanical stimulation on rabbits’ eyes; release of active subst,ances in anterior chambw perfusstes. J. Physiol. (Land.) 176, 37%4(18. Arvier, P. T.. C’hahl, L. A. and Ladd. 14. J. (1977). Modification of capsaicin and compound 48/X0 of dye leakage induced by irritants in the rat. Hr. J. Phowmcol. 59, 61-8.

OC’CLAIZ

RESPOPU’SE

TO FORMALDEHYDE

5x9

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,soc. H. 109, 19-2x. I~MGns. I<. E. (1977). Prostaglandin and rlorr-prost,aglantlin mediated brc&down of’ the blood :L~IINNIS harrier. /C.z:p.Eye Res. 25, 48R-9% 15tl~~:trtls. .I. ( 1975). A new apparatus for fluorescein angiography of thr anterior scgrrle ;t of the c*,v,~..J. f’h!/.siol. ( Jmd.) 246, 39-401’. H;~n~mo~~tl, I<. I’,. (I 977). Perfusion of the rabbit anttrior chamber. E.yv. I’;~P Hes. 24, 5X-4. .I:~IIcw. N. ( IMO). Role of the nerve termina!s in the mechanism of inflammatory reactio::s. Hull. .I/ illrcrd L’i/~t~Kwc flosp. 7, 53-77. I\;:Io. (I. Y. (1967). Tctrodoxin, saxitoxin and their significance in the study of excitation pheno-

rbnc~tut~. /‘/~rcr~~mo/.Ret*. 18, 997-104ti. Lewis. ‘I’. ( lW7). ‘/‘h/z IZloorl l’~.weE.~@ the //w~r~a Skin rrnrl 7’heir It’espo~w~. I,ondon : Shah and SOIW. Lovry. 0. I+.. liosebrough. N. J.. Farr, A. J. and Randall, K. *J. (1951 ). Protein measurenlent \vit Ir Folin phenol reagent. J. Riol. Chem. 193, X5-75. S;trr;lslll)l.;lr,l~Ini~~l~, S. (1974). The eflect of vasoprcssin on the facilitv of aqueo,w hnmour outtlo\\ iI1 t IIC wbbit. Ophihd. Iloa. 6, ?d-7. Nvufvltl. .I. II.. .Jnmpol. L. RI. and Sears. 11. L. (1972). Aspirin prevents the disruption of’ the blootl :I(~IWOIISbarrier in the rabbit, eye. Xrrture (Lo&on) 238. 158-O. I+rl;ilrs. 11:.S. (1957). Influence of the fifth nerve on the intra-ocular pressure of the rabbit C~,VC. /jr. .J. Ophthnlrttol. 41, 257-801). I’wkills. 15. S. (l!NiX). In Ernluntion qf Drug /~~&ts oz the Eye. (Ed. Pigott, I’. V.). Assoriation of t~~ctliwl adI-isws in the pharmaceutical industry, London. l’I~iI,Cf~ , .I. If. (I 964). 111The Rabbit ilz /Cye Kesertrch. Charles (:. Thomas, Springfield, Illinois. SCWX. 81. I,. ( 19tiO). bliosis and intraornlar prwsure rhartges during manomet.ry. d ~ch. ~ph~?l(/Z~l,/~l. ((‘hicccgo) 63, 707-14. 1.ngc.r. I!‘. (:. (1977). The effect of unilateral sympathertomy on the ocular response of the rabbit c:!~c to lawr irralliat,ion of the iris. Tmns. Ophthrrl. Sot. L’.K. 97, 674-S. 1-ngrr. i\‘. (:. and Unss, M. S. (1977a). Prostaglandin and nerve-mecliated response of the rahl,it v,ve to argon laser irradiat’ion of the iris. O~hthnlnzoloyicrc (Bnsel) 175, 153-8. I’lLper, \\‘. (i., (‘ale, D. F. and Bass, M. S. (19771)). Prostaglandin and ncurogenically mediate,1 ocldar rwponse to laser irradiation of the rabbit iris. I’:xp. E:ye lies. 25, 209-20. I~trgvr. \V. C:.. Colt, D. F. and Hammond, B. K. (1975). Disruption of the blood-aqueous barrier following pnrawntesis in the rabbit. Err/. fSyc IZes. 20, %5-70. I.npcr, \V. (i.. Perkins, E. S. and Bass, Jl. S. (1974). The response of the rabbit eye to lawr irratliatiolt ot the iris. L;Xp. E’yr KPR. 19, 367-77. 1’1,s~. \V. (1.. Stamford, 1. F. and Bennett., A. (1971). Extraction of prostagfandin from human Irlootl. :\‘rrtzrre(Loi,don) 233, X%-i. \.a~. .l. I-C,.(1971). Inhibition of prostaglar,din synthetasn as a mwhanism of action for aspirinlike drus~. Suture (3-e~ Rio!.) 231, 232.-5.