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Ocular Lesion Following Fifth-Nerve Injury in Rats
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AMERICAN JOURNAL OF OPHTHALMOLOGY
ergic mechanisms and the outflow of aqueous humor from the rabbit eye. J. Pharmacol. Exp. Ther. 144:60, 1964. 14. Sears, M., Mizuno, K., Cintron, G, Alter, A. and Sherk, T. : Changes in outflow facility and con tent of norepinephrine in iris and ciliary processes of albino rabbits after cervical ganglionectomy. In vest. Ophth. 5:312, 1966. 15. Sears, M. and Gillis, C. N. : Mydriasis and the increase in outflow of aqueous humor from the rabbit eye after cervical ganglionectomy in relation to the release of norepinephrine from the iris. Biochem. Pharmacol. 16 -.777, 1967. 16. Langham, M. E. and Taylor, C. B. : The in fluence of pre- and post-ganglionic section of the cervical sympathetic on the intraocular pressure of rabbits and cats. J. Physiol. 152:437, 1960. 17. · : The influence of superior cervical ganglionectomy on intraocular dynamics. J. Physiol. 152:447, 1960. 18. Langer, S. Z. and Trendelenburg, U. : The
DECEMBER, 1969
onset of denervation supersensitivity. J. Pharma col. Exp. Ther. 151:73, 1966. 19. Trendelenburg, U. : Supersensitivity to nor epinephrine induced by continuous nerve stimula tion. J. Pharmacol. Exp. Ther. 151:95, 1966. 20. Langer, S. Z., Draskóczy, P. R., and Tren delenburg, U. : Time course of the development of supersensitivity to various amines in the nictitat ing membrane of the pithed cat after denervation or decentralization. J. Pharmacol. Exp. Ther. 157 : 255, 1967 21. Jonnesco, T. : Die Resection des Halssympathicus in der Behandlung des Glaukoms. Wien. Klin. Wschr. 12 :483, 1899. 22. Linksz, A. : Der Einflass Der Sympathicusausschaltung Auf Die Blut-Kammerwasser schranke. Klin. Wschr. 10:839, 1931. 23. Langham, M. E. and Carmel, D. D. : The ac tion of protriptyline on adrenergic mechanisms in rabbit, primate, and human eyes. J. Pharmacol. Exp. Ther. 163 :368, 1968.
OCULAR LESION FOLLOWING F I F T H - N E R V E INJURY IN RATS ROBERT A.
MOSES, M.D.,
AND MARVIN F.
FELDMAN
St. Louis, Missouri
Magendie 1 ' 2 noted that a few days after neurotomy of the fifth cranial nerve in the rabbit, the ipsilateral eye became purulent. In man, interruption of the ophthalmic divi sion of the fifth nerve may cause the disease called neuroparalytic keratitis. A large body of literature on the experimental and clinical aspects of this condition has appeared. In the human disease the central corneal epithelium desquamates, the cornea becomes cloudy and may perforate. If the eye lids are closed in the early stage of the disease, the cornea re-epithelializes but may or may not ulcerate again when the eye is opened. Com plete eye lid closure is often unnecessary to maintain corneal integrity, and the patient may be allowed a slit between tarsorraphy adhesions for viewing. From the Department of Ophthalmology, Wash ington University School of Medicine. This study was supported in part by USPHS Grants NB04774-06 and NB-03639-07 from the National Insti tutes of Health. Reprint requests to Robert A. Moses, M.D., De partment of Ophthalmology, Washington Univer sity School of Medicine, 660 South Euclid Avenue, St. Louis, Missouri 63110.
Sigelman and Friedenwald3 devised a procedure for interruption of the fifth nerve in rats in which a diathermy electrode pierced the roof of the rat's mouth and base of the skull to burn the ophthalmic division of the trigeminal nerve. The emphasis of Si gelman and Friedenwald's work was on the decrease in mitosis rate of the corneal epi thelium found after operation but, in pass ing, they mentioned that polymorphonuclear leukocytes were found in the cornea and that the cornea became necrotic two or three days after surgery. We have employed the surgical method of Sigelman and Friedenwald in an attempt to explore further the ocular disease following nerve injury. It must be emphasized that, in our hands, it has not been possible to limit the injury to the first division of the fifth nerve. Invariably, the pupil on the operated side was dilated at the end of surgery, indi cating third nerve damage, and it is likely that other structures were injured as well. nevertheless, an ocular disease developed which we have studied in some detail. Three sets of experiments are reported
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In all cases, the second eye was the con trol. Rats were killed at various times fol lowing operation. The eyes were enucleated and the brain was removed for inspection of the diathermy puncture. The experiments reported here are of MATERIALS AND METHODS three types : experiment 1 is concerned with Locally obtained white rats, of both sexes the cohesion of the corneal epithelium and its adhesion to the stroma ; experiment 2 is a weighing 250-325 grams were used. Basal anesthesia was induced with intra- study of the histologie nature of the ocular peritoneal injection of 6.0 mg of pentobarbi- disease ; and experiment 3 is an attempt to tal (0.1 ml 60 mg/ml solution). The fur modify the course of the disease. In experiment 1, epithelial adhesion was over the back was wet with soap solution and the rat was restrained supine on a brass tested by a method modified from Buschke.4 plate which served as the diathermy ground The two enucleated eyes of one rat were electrode. The jaws were held widely apart placed in small cups in a plastic block, cor by rubberband traction behind the incisor nea upward. Suction at the bottom of the teeth. The cheeks were spread apart by a re cups kept the eyes in place and, by distorting tractor of thin plastic bent in a U-shape. the eyes, kept the eyes firm. A moistened Open-drop ether was used to supplement the glass microscope slide was placed in contact basal anesthesia. A diathermy electrode fash with the corneas and slid back and forth at a ioned from a screwdriver and insulated to rate of 40-60 cycles/min. The machine was the tip, according to the directions of Sigel- stopped every 30 seconds so that the corneas man and Friedenwald, was plunged through could be observed with a low-power micro the roof of the mouth to its full depth of 4.0 scope. Fluorescein and rose-bengal solutions mm, 3.0 mm from the midline and 3.0 mm were used as surface stains. Eyes of 40 rats behind the most posterior deep furrow of the were examined this way four to 48 hours hard palate while coagulating current was after surgery. The eye lids had been sewn applied. On withdrawal of the electrode closed on the operated side in all cases. there was some bleeding from the rat's In experiments 2 and 3, the eyes were mouth and nose, but about 60% of the rats fixed in formalin, embedded in paraffin (Tissurvived with acceptable lesions of the fifth suemat) and sectioned. The sections were nerve, as demonstrated at autopsy. It was stained with hematoxylin-eosin. The 10 eyes difficult to keep rats alive more than two of five rats were fixed in osmic acid or gludays after surgery as infection ascended to teraldehyde and osmic acid and epoxy-resin the brain from the nasopharynx. embedded. Sections were stained with toluiOn recovery from anesthesia, the blink re dine blue for light microscopy.* flex was tested by touching the cornea with a RESULTS wisp of cotton. If the reflex remained, it was Experiment 1. The eye on the side of the assumed that the fifth nerve had not been af fected and the animal was "discarded. On the neurotomy was sewn closed in all cases. In other hand, several animals with absent blink those eyes which had been closed a day or reflex and definite histologie ocular changes more, a ridge could be seen across the cornea were found at autopsy to have little or no parallel to the palpebrai fissure when the eye gross interruption of the nerve. Apparently, was opened. The ridge took a light supravithe nerve function may be altered without tal stain with rose bengal. The epithelium of gross evidence of a burn. In some experi ments, the eye lids on the operated side were * The epon embedding and sectioning were done in the laboratory of Dr. Jack Kayes. closed with two 6-0 silk mattress sutures.
here : ( 1 ) test of the effect of denervation of adhesion of the epithelium to the stroma of the cornea; (2) histologie studies of the course of the ocular lesion; (3) attempts to suppress the ocular lesion.
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both eyes was resistant to shear. Although the cornea was seen to wrinkle with each stroke of the glass slide, epithelium pulled loose only in small tags after several minutes of shearing. Even in rats operated upon more than two days before testing, no difference was noted in the effect of shear on the intact and denervated corneas. Flat prep arations of these corneas5 were made, but no difference between control and denervated eyes was detected. Mitosis counts were not made. Experiment 2. In this group, used to study the time course of histologie ocular change, the eyelids of the operated side were sutured in all cases. Six to eight hours after diathermy, proteinaceous material was found in the ante rior chamber and the superficial limbal re gion was infiltrated with polymorphonuclear leukocytes. There were often a few polymor phonuclear leukocytes in the anterior cham ber, a few in the iris and a clump in the cil iary body adjacent to the sclera. A striking effect was invasion of the cornea by the leu kocytes, which occurred as early as any of the changes noted and appeared to be a mi gration from the superficial limbal tissues, often predominantly into the anterior onethird of the corneal stroma. The corneal epi thelium was not invaded (fig. 1). In some cases the leukocytic invasion of the cornea was massive with the histologie appearance of ring abscess and corneal necrosis. The posterior portions of the eye were not involved. No change was found in the sclera, choroid or retina. Occasionally, a few poly morphonuclear leukocytes and some acidophilic material were found in the posterior chamber, but little evidence of vitreous change could be adduced. The changes, graded from 0 to -4—I—!—l·, are summarized in Table 1. The histologie effect of eyelid closure alone was studied in epon-embedded eyes and in paraffin sections of eyes of animals in which the diathermy operation on the fifth nerve had failed. Little change in the eye was noted in paraffin sections. In the epon-
DECEMBER, 1969
embedded material (48 or 72 hours, one eye each), only epithelial edema was noted (fig. 2)· Experiment 3. In part of this group, atro-
Fig. 1 (Moses and Feldman). Anterior segment of rat eye six hours after diathermy to the oph thalmic division of the trigeminal nerve. The eye lids were sewn closed immediately following sur gery. (A) There is marked infiltration of the subconjunctival tissue and anterior corneal lamellas by polymorphonuclear leukocytes. Proteinaceous ma terial fills the anterior chamber. (B) Opposite side of the same section shows much less leukocytic in filtration. (Paraffin, hematoxylin-eosin, χ150.)
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TABLE 1 TIME COURSE OF HISTOLOGIC OCULAR CHANGES (OPERATED EYE*) Hours After Operation
Cornea
Anterior Chamber
Iris
Ciliary Body
1
0
0
0
0
2
0 0
0 0
0 0
0 0
3
0
0
0
0
0 0
0 0
0 0
0 0
4 6
8
10 14 27
0
+++ ++++ +++ + ++ ++ ++ + +++
0
+++ ++++ +++
0
0
++ +++ +
++ +++ +
+ +++ +++ +++ +++
0
0
++ + + +++
++ ± ± +++
0
0
0
Severity of B u m
+++ + ++ + + ++ ++++ + + ++ + ++++ ++
* In all cases the control eye was negative. Changes are graded from 0 to + + + + (most severe).
Fig. 2 (Moses and Feldman). Rat cornea after eyelids have been sewn closed for 48 hours; no other surgery was performed. There is edema of the basal layers of the corneal epithelium, but no cellular infiltration of the stroma. (Epon, toluidine blue, X350.)
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TABLE 2 TIME COURSE OF HISTOLOGIC OCULAR CHANGES (ATROPINE-TREATED EYES AND SEWED EYE LIDS)
Hours After Surgery
Cornea
Anterior Chamber
Iris
Ciliary Body
12
16 19
0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
0
0 0
0 0
0 0
++ 20
0 0
0 0
0 0
0 0
24
++ ++
0 0
0 0
0 0
28
0 0
0 0
0 0
0 0
32
++
0
0 0 0
+
0
0
++
36
0 0 0 0 0
0 0 0 0
0 0 0 0 0
40
0 0 0
0 0 0
0 0 0
0 0 0
44
0 0
0 0 0
0 0 0
0 0 0
0 0
0 0 0
0 0 0
0 0 0
0
0
0 0
0 0
48 SO
+ ++ +++
++
pine sulfate ointment ( 1 % ) , or drops ( 3 % ) , was applied to each eye once daily for three days before surgery. The rest of the group was originally to be part of experiment 2, but atropine sulfate 0.006 mg (0.024 mg/k) was given intraperitoneally as preoperative medication in these rats. Both methods of atropine administration resulted in marked decrease of ocular reaction if the eye lids were sewn closed (table 2). However, if the eyelids were left open, most eyes showed in flammatory signs similar to those following operation in the closed, unmedicated eye
0 0 0 0 0
Severity of Burn
++ ++++ ++ +++ ++ +++ ++ ++ +++ ++++ ++++ ++ ++ +++ ++++ ++++ ++ +++ ++ +++ ++++ ++++ +++ ++++ ++++ +++ +++ ++ ++ ++++ ++++ ++++
(table 3). A few open eyes of atropinetreated animals developed little or no reac tion. DISCUSSION
The appearance of protein in the anterior chamber and the migration of polymorphonuclear leukocytes into the limbal region and cornea following diathermy to the fifth nerve and adjacent structures in rats suggest a mechanism involving vasodilatation in the an terior segment of the eye. However, gross vasodilatation of the limbal and iris vessels
SEQUELA OF FIFTH-NERVE INJURY
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TABLE 3 T I M E COURSE OF HISTOLOGIC OCULAR CHANGES (ATROPINE-TREATED EYES AND OPEN EYE LIDS)
6
++ ++
8
0 0
9
++
Ciliary Body
Severity of Burn
+ ++
0 0
+ ±
0 0
0 0
+++
+
+++
++++ ++++ ++++ ++++ ++++ +++ +++ ++++ ++ +++ + ++++ +
Anterior Chamber
+++ +++
Iris
10
0
0
0
0
11
0
0
0
0
12
++ ++ 0
0
+++
16
+
0 0
20
0
0
28
0 31 32 36 40 44 48
+++ ++++ ++++ +++ +++ ++++ 0
++++
++
25
+++ +++ +++ +++ +++ ++++
++ +++ 0
+++ ++ ++++ ++++ +++ ++++ 0
++++
is not prominent as a slit lamp finding in op erated animals. Invasion of the cornea by leukocytes and increased permeability of an terior uveal vessels were also observed by Zaiko6 after fifth-nerve section in the rabbit, but these features are mild or absent in the cat (Rodger 7 and personal unpublished ob servations). The human shows evidence of increased vascular permeability after fifthnerve section.8 There are several agents stored in the an terior segment of the eye that may act on blood vessels. Histamine and serotonin are
0
++
0
0 0
0
0
++
24
0
+ 0
++ ++ 0
++ + +++ ++ ++ ++++ 0
++++
+++ +++ +++ ++++ +++ +++ 0
++ ++ +++ ++ ++ +++ 0
+++
++ +++ ++
Cornea
1 +++
Hours After Operation
++++ ++ +++ + ++++ ++++ ++++ + + ++++ ++++ ++++
found in the mast cells of the rat limbus. Acetylcholine has been demonstrated in the rabbit corneal epithelium,9 and may very well occur in the rat epithelium. Prostaglandins have been found in the iris of sheep10 and rabbits.11 At the present time, it is not possible to say if any of these vasoactive substances is implicated in the anterior-segment reaction we have described. The reaction is blocked by atropine if the eyelids are closed. Atro pine is a powerful competitive blocker of acetylcholine, but it is nonspecific and also
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inhibits responses to histamine and serotonin at somewhat higher dosage levels.12 The ocu lar reaction to nerve section is not regularly impeded by atropine if the eye lids are left open, suggesting that oxygen may play some part in development of the reaction. An attempt was made to eliminate seroto nin as a possible mediator of the ocular reac tion by pretreatment of rats with reserpine, but all of these rats died following operation, probably because of depletion of norepinephrine. Attempts to modify the leukocytic ele ment of the response by pretreatment with busulfan were also unsuccessful ; the treated rats bled persistently from the small wounds inflicted for leukocyte counts and were therefore not subjected to major surgery. Cervical sympathectomy was performed on a number of rats in order to test the findings of Baker and Gottlieb,13 but these rats also received prediathermy atropine injections and had the eyelids sewn. The lack of ocular lesions in this group cannot therefore, be ex plained. The reaction we have observed is limited to the anterior segment of the eye and does not involve the retina, choroid or sclera. The choroid is well innervated but pain sensation is absent. The cornea and iris, on the other hand, have pain fibers derived from the fifth nerve. Dilatation of the iris and limbal ves sels secondary to irritation of the cornea is well known, and is attributed to antidromic conduction in branches of pain fibers.14 Diathermy burn of the ophthalmic division of the fifth nerve may well initiate similar antidromic impulses. SUMMARY
The corneal epithelium remains normally adherent in eyes protected by eyelid suture. Histologically, the ocular lesion is charac terized by the appearance of protein in the aqueous and infiltration of the cornea, iris
DECEMBER. 1969
and ciliary body by polymorphonuclear cells six to eight hours following surgery. Development of the reaction is blocked by the combination of atropine premedication and eye lid suturing, but not by either alone. REFERENCES
1. Magendie, F.: De l'influence de la cinquième paire de nerfs sur la nutrition et les functions de l'oeil. J. Physiol. (Paris) 4:176, 1824. 2. ■ : Suite des expériences sur les func tions de la cinquième paire de nerfs. J. Physiol. (Paris) 4:302, 1824. 3. Sigelman, S. and Friedenwald, J. S. : Mitotic and wound-healing activities of the corneal epithe lium. Arch. Ophth. 52:46, 1954. 4. Buschke, W. : Studies on intercellular cohesion in corneal epithelium. J. Cell. Comp. Physiol. 33:145, 1949. 5. Buschke, W., Friedenwald, J. S. and Fleisch mann, VV. : Studies on the mitotic activity of the corneal epithelium. Bull. Johns Hopkins Hosp. 73:143, 1943. 6. Zaiko, N. N. : Morphologic observations in ex perimental neurodystrophic (neuroparalytic) keratitis. Izv. Akad. Nauk SSSR (Mosk.) 2:99, 1954. Abstracted number 1874 in Excerpta Med. Ophth. 8:445, 1954. 7. Rodger, F. C. : Source and nature of nerve fibers in cat cornea. Arch. Neurol. Psychiat. 70:206, 1953. 8. Duke-Elder, S. and Leigh, A. G. : In System of Ophthalmology. (S. Duke-Elder, ed.) St. Louis, Mosby, 1965, v. 3, part 2, pp. 803-810. 9. Brücke, H. V., Hellauer, H. F. and Umrath, K. : Azetylcholin—und Aneuringehalt der Hornhaut und sein Beziehungen zur Nervenversorgung. Ophthalmologica 117:19, 1949. 10. Änggard, E. and Samuelsson, B. : Smooth muscle stimulating lipids in sheep iris. The identi fication of prostaglandin F20. Biochem. Pharmacol. 13:281, 1964. 11. Waitzman, M. B., Bailey, W. R., Jr., and Kirby, C. G. : Chromatographie analysis of biologi cally active lipids from rabbit irides. Exp. Eye Res. 6:130, 1967. 12. Innes, I. R. and Nickerson, M. : In The Phar macologie Basis of Therapeutics. (L. S. Goodman and A. Gilman, eds.) New York, Macmillan, 1965, ed. 3, p. 524. 13. Baker, G. S. and Gottlieb, C. M. : The pre vention of corneal ulcération in the denervated eye by cervical sympathectomy : An experimental study in cats. Proc Mayo Clinic 34:474, 1959. 14. Adler, F. H.: Physiology of the Eye. St. Louis, Mosby, 1965, ed. 4, p. 213.
AMERICAN JOURNAL OF OPHTHALMOLOGY
ergic mechanisms and the outflow of aqueous humor from the rabbit eye. J. Pharmacol. Exp. Ther. 144:60, 1964. 14. Sears, M., Mizuno, K., Cintron, G, Alter, A. and Sherk, T. : Changes in outflow facility and con tent of norepinephrine in iris and ciliary processes of albino rabbits after cervical ganglionectomy. In vest. Ophth. 5:312, 1966. 15. Sears, M. and Gillis, C. N. : Mydriasis and the increase in outflow of aqueous humor from the rabbit eye after cervical ganglionectomy in relation to the release of norepinephrine from the iris. Biochem. Pharmacol. 16 -.777, 1967. 16. Langham, M. E. and Taylor, C. B. : The in fluence of pre- and post-ganglionic section of the cervical sympathetic on the intraocular pressure of rabbits and cats. J. Physiol. 152:437, 1960. 17. · : The influence of superior cervical ganglionectomy on intraocular dynamics. J. Physiol. 152:447, 1960. 18. Langer, S. Z. and Trendelenburg, U. : The
DECEMBER, 1969
onset of denervation supersensitivity. J. Pharma col. Exp. Ther. 151:73, 1966. 19. Trendelenburg, U. : Supersensitivity to nor epinephrine induced by continuous nerve stimula tion. J. Pharmacol. Exp. Ther. 151:95, 1966. 20. Langer, S. Z., Draskóczy, P. R., and Tren delenburg, U. : Time course of the development of supersensitivity to various amines in the nictitat ing membrane of the pithed cat after denervation or decentralization. J. Pharmacol. Exp. Ther. 157 : 255, 1967 21. Jonnesco, T. : Die Resection des Halssympathicus in der Behandlung des Glaukoms. Wien. Klin. Wschr. 12 :483, 1899. 22. Linksz, A. : Der Einflass Der Sympathicusausschaltung Auf Die Blut-Kammerwasser schranke. Klin. Wschr. 10:839, 1931. 23. Langham, M. E. and Carmel, D. D. : The ac tion of protriptyline on adrenergic mechanisms in rabbit, primate, and human eyes. J. Pharmacol. Exp. Ther. 163 :368, 1968.
OCULAR LESION FOLLOWING F I F T H - N E R V E INJURY IN RATS ROBERT A.
MOSES, M.D.,
AND MARVIN F.
FELDMAN
St. Louis, Missouri
Magendie 1 ' 2 noted that a few days after neurotomy of the fifth cranial nerve in the rabbit, the ipsilateral eye became purulent. In man, interruption of the ophthalmic divi sion of the fifth nerve may cause the disease called neuroparalytic keratitis. A large body of literature on the experimental and clinical aspects of this condition has appeared. In the human disease the central corneal epithelium desquamates, the cornea becomes cloudy and may perforate. If the eye lids are closed in the early stage of the disease, the cornea re-epithelializes but may or may not ulcerate again when the eye is opened. Com plete eye lid closure is often unnecessary to maintain corneal integrity, and the patient may be allowed a slit between tarsorraphy adhesions for viewing. From the Department of Ophthalmology, Wash ington University School of Medicine. This study was supported in part by USPHS Grants NB04774-06 and NB-03639-07 from the National Insti tutes of Health. Reprint requests to Robert A. Moses, M.D., De partment of Ophthalmology, Washington Univer sity School of Medicine, 660 South Euclid Avenue, St. Louis, Missouri 63110.
Sigelman and Friedenwald3 devised a procedure for interruption of the fifth nerve in rats in which a diathermy electrode pierced the roof of the rat's mouth and base of the skull to burn the ophthalmic division of the trigeminal nerve. The emphasis of Si gelman and Friedenwald's work was on the decrease in mitosis rate of the corneal epi thelium found after operation but, in pass ing, they mentioned that polymorphonuclear leukocytes were found in the cornea and that the cornea became necrotic two or three days after surgery. We have employed the surgical method of Sigelman and Friedenwald in an attempt to explore further the ocular disease following nerve injury. It must be emphasized that, in our hands, it has not been possible to limit the injury to the first division of the fifth nerve. Invariably, the pupil on the operated side was dilated at the end of surgery, indi cating third nerve damage, and it is likely that other structures were injured as well. nevertheless, an ocular disease developed which we have studied in some detail. Three sets of experiments are reported
VOL. 68, NO. 6
SEQUELA OF FIFTH-NERVE INJURY
1083
In all cases, the second eye was the con trol. Rats were killed at various times fol lowing operation. The eyes were enucleated and the brain was removed for inspection of the diathermy puncture. The experiments reported here are of MATERIALS AND METHODS three types : experiment 1 is concerned with Locally obtained white rats, of both sexes the cohesion of the corneal epithelium and its adhesion to the stroma ; experiment 2 is a weighing 250-325 grams were used. Basal anesthesia was induced with intra- study of the histologie nature of the ocular peritoneal injection of 6.0 mg of pentobarbi- disease ; and experiment 3 is an attempt to tal (0.1 ml 60 mg/ml solution). The fur modify the course of the disease. In experiment 1, epithelial adhesion was over the back was wet with soap solution and the rat was restrained supine on a brass tested by a method modified from Buschke.4 plate which served as the diathermy ground The two enucleated eyes of one rat were electrode. The jaws were held widely apart placed in small cups in a plastic block, cor by rubberband traction behind the incisor nea upward. Suction at the bottom of the teeth. The cheeks were spread apart by a re cups kept the eyes in place and, by distorting tractor of thin plastic bent in a U-shape. the eyes, kept the eyes firm. A moistened Open-drop ether was used to supplement the glass microscope slide was placed in contact basal anesthesia. A diathermy electrode fash with the corneas and slid back and forth at a ioned from a screwdriver and insulated to rate of 40-60 cycles/min. The machine was the tip, according to the directions of Sigel- stopped every 30 seconds so that the corneas man and Friedenwald, was plunged through could be observed with a low-power micro the roof of the mouth to its full depth of 4.0 scope. Fluorescein and rose-bengal solutions mm, 3.0 mm from the midline and 3.0 mm were used as surface stains. Eyes of 40 rats behind the most posterior deep furrow of the were examined this way four to 48 hours hard palate while coagulating current was after surgery. The eye lids had been sewn applied. On withdrawal of the electrode closed on the operated side in all cases. there was some bleeding from the rat's In experiments 2 and 3, the eyes were mouth and nose, but about 60% of the rats fixed in formalin, embedded in paraffin (Tissurvived with acceptable lesions of the fifth suemat) and sectioned. The sections were nerve, as demonstrated at autopsy. It was stained with hematoxylin-eosin. The 10 eyes difficult to keep rats alive more than two of five rats were fixed in osmic acid or gludays after surgery as infection ascended to teraldehyde and osmic acid and epoxy-resin the brain from the nasopharynx. embedded. Sections were stained with toluiOn recovery from anesthesia, the blink re dine blue for light microscopy.* flex was tested by touching the cornea with a RESULTS wisp of cotton. If the reflex remained, it was Experiment 1. The eye on the side of the assumed that the fifth nerve had not been af fected and the animal was "discarded. On the neurotomy was sewn closed in all cases. In other hand, several animals with absent blink those eyes which had been closed a day or reflex and definite histologie ocular changes more, a ridge could be seen across the cornea were found at autopsy to have little or no parallel to the palpebrai fissure when the eye gross interruption of the nerve. Apparently, was opened. The ridge took a light supravithe nerve function may be altered without tal stain with rose bengal. The epithelium of gross evidence of a burn. In some experi ments, the eye lids on the operated side were * The epon embedding and sectioning were done in the laboratory of Dr. Jack Kayes. closed with two 6-0 silk mattress sutures.
here : ( 1 ) test of the effect of denervation of adhesion of the epithelium to the stroma of the cornea; (2) histologie studies of the course of the ocular lesion; (3) attempts to suppress the ocular lesion.
1084
AMERICAN JOURNAL OF OPHTHALMOLOGY
both eyes was resistant to shear. Although the cornea was seen to wrinkle with each stroke of the glass slide, epithelium pulled loose only in small tags after several minutes of shearing. Even in rats operated upon more than two days before testing, no difference was noted in the effect of shear on the intact and denervated corneas. Flat prep arations of these corneas5 were made, but no difference between control and denervated eyes was detected. Mitosis counts were not made. Experiment 2. In this group, used to study the time course of histologie ocular change, the eyelids of the operated side were sutured in all cases. Six to eight hours after diathermy, proteinaceous material was found in the ante rior chamber and the superficial limbal re gion was infiltrated with polymorphonuclear leukocytes. There were often a few polymor phonuclear leukocytes in the anterior cham ber, a few in the iris and a clump in the cil iary body adjacent to the sclera. A striking effect was invasion of the cornea by the leu kocytes, which occurred as early as any of the changes noted and appeared to be a mi gration from the superficial limbal tissues, often predominantly into the anterior onethird of the corneal stroma. The corneal epi thelium was not invaded (fig. 1). In some cases the leukocytic invasion of the cornea was massive with the histologie appearance of ring abscess and corneal necrosis. The posterior portions of the eye were not involved. No change was found in the sclera, choroid or retina. Occasionally, a few poly morphonuclear leukocytes and some acidophilic material were found in the posterior chamber, but little evidence of vitreous change could be adduced. The changes, graded from 0 to -4—I—!—l·, are summarized in Table 1. The histologie effect of eyelid closure alone was studied in epon-embedded eyes and in paraffin sections of eyes of animals in which the diathermy operation on the fifth nerve had failed. Little change in the eye was noted in paraffin sections. In the epon-
DECEMBER, 1969
embedded material (48 or 72 hours, one eye each), only epithelial edema was noted (fig. 2)· Experiment 3. In part of this group, atro-
Fig. 1 (Moses and Feldman). Anterior segment of rat eye six hours after diathermy to the oph thalmic division of the trigeminal nerve. The eye lids were sewn closed immediately following sur gery. (A) There is marked infiltration of the subconjunctival tissue and anterior corneal lamellas by polymorphonuclear leukocytes. Proteinaceous ma terial fills the anterior chamber. (B) Opposite side of the same section shows much less leukocytic in filtration. (Paraffin, hematoxylin-eosin, χ150.)
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TABLE 1 TIME COURSE OF HISTOLOGIC OCULAR CHANGES (OPERATED EYE*) Hours After Operation
Cornea
Anterior Chamber
Iris
Ciliary Body
1
0
0
0
0
2
0 0
0 0
0 0
0 0
3
0
0
0
0
0 0
0 0
0 0
0 0
4 6
8
10 14 27
0
+++ ++++ +++ + ++ ++ ++ + +++
0
+++ ++++ +++
0
0
++ +++ +
++ +++ +
+ +++ +++ +++ +++
0
0
++ + + +++
++ ± ± +++
0
0
0
Severity of B u m
+++ + ++ + + ++ ++++ + + ++ + ++++ ++
* In all cases the control eye was negative. Changes are graded from 0 to + + + + (most severe).
Fig. 2 (Moses and Feldman). Rat cornea after eyelids have been sewn closed for 48 hours; no other surgery was performed. There is edema of the basal layers of the corneal epithelium, but no cellular infiltration of the stroma. (Epon, toluidine blue, X350.)
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TABLE 2 TIME COURSE OF HISTOLOGIC OCULAR CHANGES (ATROPINE-TREATED EYES AND SEWED EYE LIDS)
Hours After Surgery
Cornea
Anterior Chamber
Iris
Ciliary Body
12
16 19
0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
0
0 0
0 0
0 0
++ 20
0 0
0 0
0 0
0 0
24
++ ++
0 0
0 0
0 0
28
0 0
0 0
0 0
0 0
32
++
0
0 0 0
+
0
0
++
36
0 0 0 0 0
0 0 0 0
0 0 0 0 0
40
0 0 0
0 0 0
0 0 0
0 0 0
44
0 0
0 0 0
0 0 0
0 0 0
0 0
0 0 0
0 0 0
0 0 0
0
0
0 0
0 0
48 SO
+ ++ +++
++
pine sulfate ointment ( 1 % ) , or drops ( 3 % ) , was applied to each eye once daily for three days before surgery. The rest of the group was originally to be part of experiment 2, but atropine sulfate 0.006 mg (0.024 mg/k) was given intraperitoneally as preoperative medication in these rats. Both methods of atropine administration resulted in marked decrease of ocular reaction if the eye lids were sewn closed (table 2). However, if the eyelids were left open, most eyes showed in flammatory signs similar to those following operation in the closed, unmedicated eye
0 0 0 0 0
Severity of Burn
++ ++++ ++ +++ ++ +++ ++ ++ +++ ++++ ++++ ++ ++ +++ ++++ ++++ ++ +++ ++ +++ ++++ ++++ +++ ++++ ++++ +++ +++ ++ ++ ++++ ++++ ++++
(table 3). A few open eyes of atropinetreated animals developed little or no reac tion. DISCUSSION
The appearance of protein in the anterior chamber and the migration of polymorphonuclear leukocytes into the limbal region and cornea following diathermy to the fifth nerve and adjacent structures in rats suggest a mechanism involving vasodilatation in the an terior segment of the eye. However, gross vasodilatation of the limbal and iris vessels
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TABLE 3 T I M E COURSE OF HISTOLOGIC OCULAR CHANGES (ATROPINE-TREATED EYES AND OPEN EYE LIDS)
6
++ ++
8
0 0
9
++
Ciliary Body
Severity of Burn
+ ++
0 0
+ ±
0 0
0 0
+++
+
+++
++++ ++++ ++++ ++++ ++++ +++ +++ ++++ ++ +++ + ++++ +
Anterior Chamber
+++ +++
Iris
10
0
0
0
0
11
0
0
0
0
12
++ ++ 0
0
+++
16
+
0 0
20
0
0
28
0 31 32 36 40 44 48
+++ ++++ ++++ +++ +++ ++++ 0
++++
++
25
+++ +++ +++ +++ +++ ++++
++ +++ 0
+++ ++ ++++ ++++ +++ ++++ 0
++++
is not prominent as a slit lamp finding in op erated animals. Invasion of the cornea by leukocytes and increased permeability of an terior uveal vessels were also observed by Zaiko6 after fifth-nerve section in the rabbit, but these features are mild or absent in the cat (Rodger 7 and personal unpublished ob servations). The human shows evidence of increased vascular permeability after fifthnerve section.8 There are several agents stored in the an terior segment of the eye that may act on blood vessels. Histamine and serotonin are
0
++
0
0 0
0
0
++
24
0
+ 0
++ ++ 0
++ + +++ ++ ++ ++++ 0
++++
+++ +++ +++ ++++ +++ +++ 0
++ ++ +++ ++ ++ +++ 0
+++
++ +++ ++
Cornea
1 +++
Hours After Operation
++++ ++ +++ + ++++ ++++ ++++ + + ++++ ++++ ++++
found in the mast cells of the rat limbus. Acetylcholine has been demonstrated in the rabbit corneal epithelium,9 and may very well occur in the rat epithelium. Prostaglandins have been found in the iris of sheep10 and rabbits.11 At the present time, it is not possible to say if any of these vasoactive substances is implicated in the anterior-segment reaction we have described. The reaction is blocked by atropine if the eyelids are closed. Atro pine is a powerful competitive blocker of acetylcholine, but it is nonspecific and also
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inhibits responses to histamine and serotonin at somewhat higher dosage levels.12 The ocu lar reaction to nerve section is not regularly impeded by atropine if the eye lids are left open, suggesting that oxygen may play some part in development of the reaction. An attempt was made to eliminate seroto nin as a possible mediator of the ocular reac tion by pretreatment of rats with reserpine, but all of these rats died following operation, probably because of depletion of norepinephrine. Attempts to modify the leukocytic ele ment of the response by pretreatment with busulfan were also unsuccessful ; the treated rats bled persistently from the small wounds inflicted for leukocyte counts and were therefore not subjected to major surgery. Cervical sympathectomy was performed on a number of rats in order to test the findings of Baker and Gottlieb,13 but these rats also received prediathermy atropine injections and had the eyelids sewn. The lack of ocular lesions in this group cannot therefore, be ex plained. The reaction we have observed is limited to the anterior segment of the eye and does not involve the retina, choroid or sclera. The choroid is well innervated but pain sensation is absent. The cornea and iris, on the other hand, have pain fibers derived from the fifth nerve. Dilatation of the iris and limbal ves sels secondary to irritation of the cornea is well known, and is attributed to antidromic conduction in branches of pain fibers.14 Diathermy burn of the ophthalmic division of the fifth nerve may well initiate similar antidromic impulses. SUMMARY
The corneal epithelium remains normally adherent in eyes protected by eyelid suture. Histologically, the ocular lesion is charac terized by the appearance of protein in the aqueous and infiltration of the cornea, iris
DECEMBER. 1969
and ciliary body by polymorphonuclear cells six to eight hours following surgery. Development of the reaction is blocked by the combination of atropine premedication and eye lid suturing, but not by either alone. REFERENCES
1. Magendie, F.: De l'influence de la cinquième paire de nerfs sur la nutrition et les functions de l'oeil. J. Physiol. (Paris) 4:176, 1824. 2. ■ : Suite des expériences sur les func tions de la cinquième paire de nerfs. J. Physiol. (Paris) 4:302, 1824. 3. Sigelman, S. and Friedenwald, J. S. : Mitotic and wound-healing activities of the corneal epithe lium. Arch. Ophth. 52:46, 1954. 4. Buschke, W. : Studies on intercellular cohesion in corneal epithelium. J. Cell. Comp. Physiol. 33:145, 1949. 5. Buschke, W., Friedenwald, J. S. and Fleisch mann, VV. : Studies on the mitotic activity of the corneal epithelium. Bull. Johns Hopkins Hosp. 73:143, 1943. 6. Zaiko, N. N. : Morphologic observations in ex perimental neurodystrophic (neuroparalytic) keratitis. Izv. Akad. Nauk SSSR (Mosk.) 2:99, 1954. Abstracted number 1874 in Excerpta Med. Ophth. 8:445, 1954. 7. Rodger, F. C. : Source and nature of nerve fibers in cat cornea. Arch. Neurol. Psychiat. 70:206, 1953. 8. Duke-Elder, S. and Leigh, A. G. : In System of Ophthalmology. (S. Duke-Elder, ed.) St. Louis, Mosby, 1965, v. 3, part 2, pp. 803-810. 9. Brücke, H. V., Hellauer, H. F. and Umrath, K. : Azetylcholin—und Aneuringehalt der Hornhaut und sein Beziehungen zur Nervenversorgung. Ophthalmologica 117:19, 1949. 10. Änggard, E. and Samuelsson, B. : Smooth muscle stimulating lipids in sheep iris. The identi fication of prostaglandin F20. Biochem. Pharmacol. 13:281, 1964. 11. Waitzman, M. B., Bailey, W. R., Jr., and Kirby, C. G. : Chromatographie analysis of biologi cally active lipids from rabbit irides. Exp. Eye Res. 6:130, 1967. 12. Innes, I. R. and Nickerson, M. : In The Phar macologie Basis of Therapeutics. (L. S. Goodman and A. Gilman, eds.) New York, Macmillan, 1965, ed. 3, p. 524. 13. Baker, G. S. and Gottlieb, C. M. : The pre vention of corneal ulcération in the denervated eye by cervical sympathectomy : An experimental study in cats. Proc Mayo Clinic 34:474, 1959. 14. Adler, F. H.: Physiology of the Eye. St. Louis, Mosby, 1965, ed. 4, p. 213.