- Email: [email protected]
Regeneration of Nerves in Experimental Corneal Grafts in Rabbits*
R E G E N E R A T I O N O F NERVES IN E X P E R I M E N T A L CORNEAL GRAFTS IN RABBITS* CLINICAL AND HISTOLOGIC STUDY WALTER KORNBLUETH, + M.D.,
AND A. EDWARD MAUMENEE,
M.D.
San Francisco, California AND JANE E. CROWELL
Baltimore, Maryland The various factors which influence the final clarity of a corneal graft cannot be properly evaluated until it has been determined whether the elements of the donor cornea are replaced or survive. In a previous study,1 histologic examinations of clear corneal grafts, stained with hematoxylin and eosin, revealed that the donor's corneal lamellae and Descemet's membrane were not replaced. There was no destruction or replacement of a large number of stromal cells at any one time, but it could not be determined whether there was a gradual replacement of these cells or not. Endothelial cells were always found on the grafts 4 to 5 days after operation, but it was impossible to determine in all cases whether these cells had migrated from the recipient cornea or belonged to the donor cornea. The epithelium of the graft always sloughed and was replaced in 4 to 5 days by cells from the recipient epithelium. The fate of the corneal nerves was not discussed in the previous report because these structures were not visible in routine histologic preparations. The following report is based on a study of the clinical sensitivity of grafts to touch and histologic examinations of silver-impregnated frozen sections of corneal grafts. REVIEW OF LITERATURE
Clinical and histologie reports on corneal grafts both in man and experimental ani* From the Wilmer Ophthalmological Institute of The Johns Hopkins Hospital and University. + Graduate Training Foundation of Hadassah Medical Organization Fellow in Ophthalmology.
mais have been numerous, but regeneration of corneal nerves into the tissue has not been extensively investigated. The sensibility of corneal transplants has been studied by several investigators, but the results of these workers have differed slightly. Ascher 2 found that sensibility returned in two clear grafts but did not return in cloudy grafts. In one patient with a staphyloma of the cornea a whole corneal transplant was done. The graft became completely opaque except for a small translucent area in thecenter. In this case the graft was insensitive except for the small central translucent area. Elschnig 3 stated that complete sensibility of the implant did not develop even in the oldest cases of transparent implants, but a sensibility to heavy touch was observed. Imre 4 noted that sensibility of the graft returned 10 to 12 months after operation. Morone 5 was able to detect the return of sensibility in both clear and cloudy grafts one year after operation. He thought the return of sensibility did not depend on the degree of clarity, course of healing, or vascularization of the graft. He also observed that the host's cornea became hyposensitive following operation. Morone stated that Magitot believed the sensibility of the graft is connected with vascularization. Thomas 6 tested 29 experimental grafts in rabbits and found no return of sensibility in 5 grafts which remained clear, but 21 of the 24 grafts which were cloudy did regain some sensibility. Thomas concluded that the ingrowth of blood vessels was essential for the return of sensibility to the transplant.
651
652
WALTER KORNBLUETH, A. EDWARD MAUMENEE AND JANE E. CROWELL
found a well-developed subepithelial nerve plexus in the graft, but there were no nerves in the deeper structures except in one area where a few blood vessels had entered the margin of the graft. From these studies they concluded that, if a graft does not become vascularized, nerves other than those of the subepithelial plexus do not invade the tissue. They also suggested that the ingrowth of nerves into the subepithelial region of the graft might be essential for the continued clarity of a transplant.
>3 ± > 4
Z
1.
3.
Fig. 1 (Kornblueth, Maumenee,. and Crowell). Test object for determination of corneal sensitivity. (1) Equivalent to pressure of 25 mg. (2) Equivalent to pressure of 100 mg. (3) Equivalent to pressure of 300 mg. (4) Equivalent to pressure of 1,000 mg.
EXPERIMENTAL OBSERVATIONS
All of the grafts used in the present investigation have been partial penetrating, Galante7 observed 8 translucent to opaque full-thickness homografts in rabbits. The heterografts in dogs and rabbits and found grafts were cut with a 4.5-mm. trephine that the transplants became sensitive at the blade attached to a dental drill. The transperiphery 10 days after operation. At the end plants were held in place by continuous crissof one month the sensibility of the grafts cross corneal sutures inserted into the recipient cornea as closely as possible to the was equal to that of the recipient corneas. edge of the graft. The sutures were reHistologie studies for nerves in corneal moved on the 7th postoperative day. grafts have been made less frequently a. Clinical study. The clinical sensibility than clinical observations on the return of of 12 clear and 10 cloudy grafts has been corneal sensibility.* tested by touching the grafts and observing Babel and Campos8 and Franceschetti and the blink reflex. The hairs near the eyes 9 Babel have reported histologie studies of were clipped before examination to avoid a nerves in grafts. In their first publication false blink reflex caused by touching these they examined four opaque human transstructures. The materials used for testing the plants which were removed in order to incorneal reflex were somewhat similar to v. sert second grafts. In one of these grafts, Frey hairs. Pieces of sutures 2 cm. long were removed 36 days after operation, no nerves secured to match sticks and were bent to a were found. The remaining three grafts were right angle at 1 cm. from the tip of the removed from 10 months to 7 years after suture (fig. 1). When the tip of the bent transplantation. In these specimens nerves suture was pressed with maximal force were found entering the grafts adjacent to onto a weighing pan of an analytical scale invading blood vessels deep in the stroma, the first was just strong enough to lift 25 but no nerves were found in the subepithelial mg., the second 100 mg., the third 300 mg., region in any of the grafts. and the fourth, 1 gm. In their second paper they reported a The blink reflex is admittedly not a very study of a clear human transplant obtained accurate test of corneal sensitivity in rab7 years after operation. In this case they bits ; nevertheless, it gave a general idea of * After this report was submitted for publication, the degree of sensibility of the grafts. The 'an excellent study on degeneration and regeneration results obtained were about the same in the of nerves in corneal transplantation by Humberto Escapini was published in the Arch. Ophth., 39 : clear and cloudy grafts. The transplants 135, 1949. were insensitive until about the 4th to 6th
NERVE REGENERATION IN CORNEAL GRAFTS week after operation, then they developed a slight sensibility to pressure (300-mg. test object) in the periphery of the graft. This gradually spread over the graft and by the 11th to 13th week the grafts became sensitive to lighter touch (100-mg. test object). On the whole the grafts did not regain as complete sensibility as the surrounding normal cornea. These clinical observations on corneal transplants correspond well with the recent work of Marcus Jent.10 In 1945 he made a careful study of the regeneration of the corneal nerves in rabbits following an incision through the cornea around the entire periphery down to Descemet's membrane. In these experiments he found that the sensibility of the cornea returned first to the cicatricial ring in about 4 weeks and then gradually progressed over the entire cornea. b. Histologie study. Seventeen clear and 13 cloudy grafts removed from two days to one year after operation were examined. No appreciable difference was found in the regeneration of the nerves in the clear and cloudy grafts. There was a variation of the ingrowth of the nerves from animal to animal but on the whole the invasion of the nerves and further developments coincided within a week to two weeks in all specimens. Perpendicular and horizontal frozen sections were made on equal halves of the grafts and surrounding corneas. The staining method used was essentially the same as Campos's modification11 of the silver impregnation technique of Bielschowsky and Gros. The slight modification of Campos's method devised by one of us (J. E. C.) gave good and uniform staining of the corneal nerves. The details of the technique used follow. DETAILS OF TECHNIQUE
1. Fix the whole eye for 24 hours or longer in neutral formalin, 10 percent (formalin neutralized to pH 7.4 with aqueous calcium oxide not more than 0.1 percent. Filter aqueous calcium oxide before neutralization).
653
2. Remove cornea from globe. 3. Wash cornea in running tap water 1 to 2 hours, then wash in distilled water. Change distilled water every half hour for 6 hours. 4. Cut frozen horizontal sections 50μ and collect in distilled water. Vertical sections should be cut 75 to 80μ. 5. Mordant overnight (about 18 hours) in solution of silver nitrate (10 to 15 percent) in the dark. The following steps are facilitated by placing the sections in 50-cc. beakers. Add and decant the various solutions. 6. Wash in distilled water and decant as quickly as possible (15 to 30 seconds). 7. Wash in 3 changes of neutral formalin (1 percent). Add about 10 cc. of neutral formalin for each washing. Total time, 2 to 3 minutes. Formalin will become brown or turbid occasionally during washing. 8. Wash sections in silver ammonium solution and decant immediately. (This is done to remove any trace of formalin which may have remained in beaker. Formalin in the presence of silver-ammonium solution will cause sections to turn bright yellow.) Add 10 cc. of silver-ammonium solution and allow sections to soak for one hour. Decant. (The silver ammonium solution is prepared as follows: Concentrated ammonium hydroxide is added slowly to 10 to 15-percent silver nitrate until precipitate is completely dissolved. Agitate constantly while adding ammonium hydroxide. After precipitate is completely dissolved, add an excess of one drop of ammonium hydroxide per cc. of solution. Prepare fresh each day and keep in tightly stoppered dark bottle.) 9. Add 10 to 15 cc. of 0.5-percent neutral formalin to sections. Stir or shake until sections become a uniform light yellow-brown color. If color is slow in appearing (longer than one-half minute) decant and add 1-percent neutral formalin. If the proper color still does not appear, decant and add 2-percent neutral formalin. The latter step (2-percent neutral formalin) is seldom necessary. Check sections under microscope. Nerve fibers and nuclei should be dark brown, stroma colorless. 10. When sections have the desired tint, wash them in running water (drop by drop) for a half hour. Cover beaker with layer of gauze to prevent loss of sections. 11. Wash in 20 cc. distilled water for two minutes. 12. Tone with gold chloride (2 drops of a 1percent aqueous solution of gold chloride to 5 cc. of distilled water) until brown nerve fibers and nuclei are colored dark, gray to black. 13. Fix in 5-percent sodium thiosulfate for 1 to 2 minutes. 14. Wash in distilled water. Dehydrate in 80percent, 95-percent, and two changes of absolute alcohol. Clear in thin cedarwood oil and mount in balsam. Histologie
examination
of
the
stained
6S4
WALTER KORNBLUETH, A. EDWARD MAUMENEE AND JANE E. CROWELL
sections revealed the following general pattern of reaction of the corneal nerves after keratoplasty. During the first 3 days after operation the nerves in both the graft and the surrounding cornea began to show signs of degeneration in the form of segmentation, lighter staining and curling of the fibrils, and finally disappearance of the nuclei of Schwann's sheath cells (fig. 2 ) .
penetrating the scar and entering the margin of graft (figs. 4 and 4a). During the following weeks these small fibers became more numerous and penetrated to the center of the graft. The number of fibers in single nerves increased and, about 12 weeks after operation, nerves could be found in the midstroma which contained S to 10 fibers (fig. 5). At about the same time, small fibrils could be seen in the
K
.1 C i* * ' » *' \4* /·■■·»*■ y■,v■·**:.'·· i W * A
>'.
Fig. 2 (Kornblueth, Maumenee, and Crowell). Section of homogenous corneal graft (3 days after operation) showing a degenerating nerve. (Silver impregnation X12S.)
By the end of the second week practically all of the nerves had disappeared from the graft. In the recipient's cornea most of the nerve fibers for a distance of about 2 mm. from the incision disappeared and nerves in the periphery showed signs of ascending degeneration. By 3 weeks newly formed fibers approached the scar on the edge of the graft. These frequently turned back into the recipient cornea or turned and ran parallel to the scar (fig. 3). By 4 to 6 weeks single nerve fibers could be found in the midstroma,
subepithelial and epithelial region in the grafts (figs. 6 and 6a). During the first two months after operation the nerves in the recipient's cornea at the edge of the graft were usually composed of 1 or 2 fibers but, after 3 to 6 months, nerves containing 6 or more fibers reached the scar tissue surrounding the graft (fig. 7). COMMENT
In this study the return of clinical sensibility coincided well with the anatomic findings. One month after operation only the
NERVE REGENERATION IN CORNEAL GRAFTS
S£èsè£! *
0
^ * "
Fig. 3 (Kornblueth, Maumenee, and Crowell). Section of homogenous corneal graft (20 days after operation) showing newly formed nerve fibers in the recipient's cornea approaching the scar on the edge of the graft. (Silver impregnation X125.)
Fig. 4 (Kornblueth, Maumenee, and Crowell). Section of clear homogenous corneal graft (1 month after operation) showing a nerve fiber entering the margin of the graft. Scar at junction of graft and recipient cornea is seen at the lower left. (Silver impregnation χ125.)
655
6S6
WALTER KORNBLUETH, A. EDWARD MAUMENEE AND JANE E. CROWELL
,VT'
Λ' Fig. 4a (Kornblueth, Maumenee, and Crowell). Section of cloudy homogenous corneal graft (6 weeks after operation) showing nerve fibers entering the margin of the graft. Scar at junction of graft and recipient cornea is seen at the lower left. (Silver impregnation χ125.)
Fig. S (Kornblueth, Maumenee, and Crowell). Section of homogenous corneal graft (3 months after operation) showing a thick nerve in the midstroma of the graft. (Silver impregnation X125.)
NERVE REGENERATION IN CORNEAL GRAFTS
Τ&ΜΨί
Fig. 6 (Kornblueth, Maumenee, and Crowell). Section of clear homogenous corneal graft (7 months after operation) showing a small nerve fiber entering the epithelium of the graft. Epithelium is at the upper left. (Silver impregnation X12S.)
Fig. 6a (Kornblueth, Maumenee, Crowell). Section of cloudy homogenous corneal graft (3 months after operation) showing a small nerve fiber entering the epithelium of the graft. Epithelium is at the upper left. (Silver impregnation X400.)
657
658
WALTER KORNBLUETH, A. EDWARD MAUMENEE AND JANE E. CROWELL
Fig. 7 (Kornblueth, Maumenee, and Crowell)^ Section of homogenous corneal graft (7 months after operation) showing a thick nerve in the recipient's cornea entering the scar tissue at the edge of the graft. (Silver impregnation χ125.) margins of the grafts were sensitive to heavy touch (300-mg. test object). During the course of the next few weeks sensitivity to heavy pressure was acquired by the whole graft. This corresponded to the time when only single nerve fibers were found in the midstroma of the transplants. After 3 to 4 months, corneal sensitivity to light touch (100-mg. test object) could be perceived. At this time, on histologie sections, numerous nerves and nerves with multiple fibers were observed in the midstroma of the graft, and small nerve fibers were found in the subepithelial and epithelial regions. The ingrowth of nerves and return of corneal sensibility was approximately the same in clear and cloudy grafts. There was no correlation between the invasion of blood vessels and nerves into the graft as had been suggested by Thomas, Babel and Campos, and Franceschetti and Babel. Blood vessels could be found in cloudy grafts 1 to 2 weeks before the nerves entered the graft, and even then the nerves
did not necessarily take the same course as the invading capillaries. On the other hand, nerves were found in entirely clear grafts which did not contain blood vessels. We were also not able to confirm Franceschetti and Babel's suggestion that the presence of subepithelial nerves in grafts was essential for the final clarity of transplants. In our experiments both clear and cloudy grafts showed thin nerve fibers entering the subepithelial and epithelial region several months after operation (figs. 6 and 6a). SUMMARY
The return of corneal sensitivity was tested in 12 clear and 10 cloudy transplants. The ingrowth of corneal nerves was studied histologically by the use of the silver-impregnation technique in 17 clear and 13 cloudy grafts. The return of deep sensibility in the grafts in 4 to 6 weeks corresponded to the ingrowth of nerves into the midstroma. The acquisition of light sensibility in 3 to 4 months corresponded to the
659
SENSITIVITY TO GOLD-BALL IMPLANT penetration of nerves into the subepithelial and epithelial regions of the grafts. No appreciable difference was found in the regeneration of nerves into clear or cloudy grafts. The ingrowth of nerve fibers was
not dependent on the invasion of the grafts by blood vessels. Stanford University Hospitals (15). The Johns Hopkins Hospital (5).
REFERENCES
1. Maumenee, A. E., and Kornblueth, W. : The physiopathology of corneal grafts. Tr. Am. Acad. Ophth., 1948 (Mar.-Apr.), p. 331 ; Am. J. Ophth., 31:1384, 1948. 2. Ascher, K. W.: The question of keratoplasty II. Arch. f. Ophth., 107:241, 1922; The question of keratoplasty III. Arch. f. Ophth., 107 :439,1922. 3. Elschnig, A. : Keratoplasty. Arch. Ophth., 4:16S, 1930. 4. Imre, J. : Clinical and histological experiences with corneal transplantation. Beiheft, Klin. Monatsbl. f. Augenh., p. 1,1942. 5. Morone, G. : Research on the sensibility of transplants. Ann. d. Ottal. e. Clin. Ocul., 72:279, 1946. 6. Thomas, J. W. T. : On the return of sensitiveness in corneal grafts in rabbits. Proc. Roy. Soc. Med., Series B., 108 :301, 1931. 7. Galante, E. : Experimental research on keratoplasty. Ann. d. Ottal. e. Clin. Ocul., 62:119, 1934. 8. Babel, J., and Campos, R. : On the regeneration of nerves in corneal transplants. Ophthalmologica, 111 :140, 1946. 9. Franceschetti, A., and Babel, J. : Histologie examination of a transparent corneal transplant : the behavior of nerves. Ann. d'Ocul., 180:14S, 1947. 10. Jent, M. : The cornea test for the examination at pursuit of regeneration in the peripheral nerves. Helvet. physiol. acta., 3 :6S, 194S. 11. Campos, R. : Modification of the method of silver impregnation of Bielschowsky-Gros. Acta anat., 2 :75, 1946. A CASE O F SENSITIVITY TO GOLD-BALL ORBITAL IMPLANT* ECZEMATOUS CONTACT-TYPE DERMATITIS DUE TO 14-KARAT GOLD H. WALTER FORSTER, JR., M.D.,
AND ROBERT F. DICKEY,
M.D.
Philadelphia, Pennsylvania The medical literature contains numerous references to dermatitis following parenteral administration of gold compounds in the therapy of arthritis, lupus erythematosus, vitiligo, and so forth. Ophthalmologists and dermatologists frequently see contact dermatitis due to monel metal (an alloy of copper and nickel) and "white gold" (an alloy of gold, copper, and nickel), which are used extensively in spectacle frames1 and jewelry. The offending allergen in these cases is considered to be nickel or its salts. The important synergistic action between pyogen and nickel allergy has been emphasized by Cormia,2 Stokes,3 and others. Gold chloride is said to cause dermatitis among * From the Departments of Ophthalmology and Dermatology, Hospital of the University of Pennsylvania.
photographers, 4 but our review of the literature failed to find any report of contact dermatitis due to gold leaf. Gold, silver, tin, and copper in pure metallic form are not considered primary irritants or sensitizers. Most reports of sensitivity to these metals from their use in industry or in daily life emphasize the fact that usually the offending allergen is a salt of the metal concerned. The subject of this case report showed a marked sensitivity clinically and by patch tests to a 14-karat gold ball which, 5 years previously, had been implanted into Tenon's capsule at the time of enucleation of the left eye. This sensitivity was manifested by a recurrent seropurulent orbital discharge and an eczematous contact-type dermatitis of the lids and adjacent skin.
AND A. EDWARD MAUMENEE,
M.D.
San Francisco, California AND JANE E. CROWELL
Baltimore, Maryland The various factors which influence the final clarity of a corneal graft cannot be properly evaluated until it has been determined whether the elements of the donor cornea are replaced or survive. In a previous study,1 histologic examinations of clear corneal grafts, stained with hematoxylin and eosin, revealed that the donor's corneal lamellae and Descemet's membrane were not replaced. There was no destruction or replacement of a large number of stromal cells at any one time, but it could not be determined whether there was a gradual replacement of these cells or not. Endothelial cells were always found on the grafts 4 to 5 days after operation, but it was impossible to determine in all cases whether these cells had migrated from the recipient cornea or belonged to the donor cornea. The epithelium of the graft always sloughed and was replaced in 4 to 5 days by cells from the recipient epithelium. The fate of the corneal nerves was not discussed in the previous report because these structures were not visible in routine histologic preparations. The following report is based on a study of the clinical sensitivity of grafts to touch and histologic examinations of silver-impregnated frozen sections of corneal grafts. REVIEW OF LITERATURE
Clinical and histologie reports on corneal grafts both in man and experimental ani* From the Wilmer Ophthalmological Institute of The Johns Hopkins Hospital and University. + Graduate Training Foundation of Hadassah Medical Organization Fellow in Ophthalmology.
mais have been numerous, but regeneration of corneal nerves into the tissue has not been extensively investigated. The sensibility of corneal transplants has been studied by several investigators, but the results of these workers have differed slightly. Ascher 2 found that sensibility returned in two clear grafts but did not return in cloudy grafts. In one patient with a staphyloma of the cornea a whole corneal transplant was done. The graft became completely opaque except for a small translucent area in thecenter. In this case the graft was insensitive except for the small central translucent area. Elschnig 3 stated that complete sensibility of the implant did not develop even in the oldest cases of transparent implants, but a sensibility to heavy touch was observed. Imre 4 noted that sensibility of the graft returned 10 to 12 months after operation. Morone 5 was able to detect the return of sensibility in both clear and cloudy grafts one year after operation. He thought the return of sensibility did not depend on the degree of clarity, course of healing, or vascularization of the graft. He also observed that the host's cornea became hyposensitive following operation. Morone stated that Magitot believed the sensibility of the graft is connected with vascularization. Thomas 6 tested 29 experimental grafts in rabbits and found no return of sensibility in 5 grafts which remained clear, but 21 of the 24 grafts which were cloudy did regain some sensibility. Thomas concluded that the ingrowth of blood vessels was essential for the return of sensibility to the transplant.
651
652
WALTER KORNBLUETH, A. EDWARD MAUMENEE AND JANE E. CROWELL
found a well-developed subepithelial nerve plexus in the graft, but there were no nerves in the deeper structures except in one area where a few blood vessels had entered the margin of the graft. From these studies they concluded that, if a graft does not become vascularized, nerves other than those of the subepithelial plexus do not invade the tissue. They also suggested that the ingrowth of nerves into the subepithelial region of the graft might be essential for the continued clarity of a transplant.
>3 ± > 4
Z
1.
3.
Fig. 1 (Kornblueth, Maumenee,. and Crowell). Test object for determination of corneal sensitivity. (1) Equivalent to pressure of 25 mg. (2) Equivalent to pressure of 100 mg. (3) Equivalent to pressure of 300 mg. (4) Equivalent to pressure of 1,000 mg.
EXPERIMENTAL OBSERVATIONS
All of the grafts used in the present investigation have been partial penetrating, Galante7 observed 8 translucent to opaque full-thickness homografts in rabbits. The heterografts in dogs and rabbits and found grafts were cut with a 4.5-mm. trephine that the transplants became sensitive at the blade attached to a dental drill. The transperiphery 10 days after operation. At the end plants were held in place by continuous crissof one month the sensibility of the grafts cross corneal sutures inserted into the recipient cornea as closely as possible to the was equal to that of the recipient corneas. edge of the graft. The sutures were reHistologie studies for nerves in corneal moved on the 7th postoperative day. grafts have been made less frequently a. Clinical study. The clinical sensibility than clinical observations on the return of of 12 clear and 10 cloudy grafts has been corneal sensibility.* tested by touching the grafts and observing Babel and Campos8 and Franceschetti and the blink reflex. The hairs near the eyes 9 Babel have reported histologie studies of were clipped before examination to avoid a nerves in grafts. In their first publication false blink reflex caused by touching these they examined four opaque human transstructures. The materials used for testing the plants which were removed in order to incorneal reflex were somewhat similar to v. sert second grafts. In one of these grafts, Frey hairs. Pieces of sutures 2 cm. long were removed 36 days after operation, no nerves secured to match sticks and were bent to a were found. The remaining three grafts were right angle at 1 cm. from the tip of the removed from 10 months to 7 years after suture (fig. 1). When the tip of the bent transplantation. In these specimens nerves suture was pressed with maximal force were found entering the grafts adjacent to onto a weighing pan of an analytical scale invading blood vessels deep in the stroma, the first was just strong enough to lift 25 but no nerves were found in the subepithelial mg., the second 100 mg., the third 300 mg., region in any of the grafts. and the fourth, 1 gm. In their second paper they reported a The blink reflex is admittedly not a very study of a clear human transplant obtained accurate test of corneal sensitivity in rab7 years after operation. In this case they bits ; nevertheless, it gave a general idea of * After this report was submitted for publication, the degree of sensibility of the grafts. The 'an excellent study on degeneration and regeneration results obtained were about the same in the of nerves in corneal transplantation by Humberto Escapini was published in the Arch. Ophth., 39 : clear and cloudy grafts. The transplants 135, 1949. were insensitive until about the 4th to 6th
NERVE REGENERATION IN CORNEAL GRAFTS week after operation, then they developed a slight sensibility to pressure (300-mg. test object) in the periphery of the graft. This gradually spread over the graft and by the 11th to 13th week the grafts became sensitive to lighter touch (100-mg. test object). On the whole the grafts did not regain as complete sensibility as the surrounding normal cornea. These clinical observations on corneal transplants correspond well with the recent work of Marcus Jent.10 In 1945 he made a careful study of the regeneration of the corneal nerves in rabbits following an incision through the cornea around the entire periphery down to Descemet's membrane. In these experiments he found that the sensibility of the cornea returned first to the cicatricial ring in about 4 weeks and then gradually progressed over the entire cornea. b. Histologie study. Seventeen clear and 13 cloudy grafts removed from two days to one year after operation were examined. No appreciable difference was found in the regeneration of the nerves in the clear and cloudy grafts. There was a variation of the ingrowth of the nerves from animal to animal but on the whole the invasion of the nerves and further developments coincided within a week to two weeks in all specimens. Perpendicular and horizontal frozen sections were made on equal halves of the grafts and surrounding corneas. The staining method used was essentially the same as Campos's modification11 of the silver impregnation technique of Bielschowsky and Gros. The slight modification of Campos's method devised by one of us (J. E. C.) gave good and uniform staining of the corneal nerves. The details of the technique used follow. DETAILS OF TECHNIQUE
1. Fix the whole eye for 24 hours or longer in neutral formalin, 10 percent (formalin neutralized to pH 7.4 with aqueous calcium oxide not more than 0.1 percent. Filter aqueous calcium oxide before neutralization).
653
2. Remove cornea from globe. 3. Wash cornea in running tap water 1 to 2 hours, then wash in distilled water. Change distilled water every half hour for 6 hours. 4. Cut frozen horizontal sections 50μ and collect in distilled water. Vertical sections should be cut 75 to 80μ. 5. Mordant overnight (about 18 hours) in solution of silver nitrate (10 to 15 percent) in the dark. The following steps are facilitated by placing the sections in 50-cc. beakers. Add and decant the various solutions. 6. Wash in distilled water and decant as quickly as possible (15 to 30 seconds). 7. Wash in 3 changes of neutral formalin (1 percent). Add about 10 cc. of neutral formalin for each washing. Total time, 2 to 3 minutes. Formalin will become brown or turbid occasionally during washing. 8. Wash sections in silver ammonium solution and decant immediately. (This is done to remove any trace of formalin which may have remained in beaker. Formalin in the presence of silver-ammonium solution will cause sections to turn bright yellow.) Add 10 cc. of silver-ammonium solution and allow sections to soak for one hour. Decant. (The silver ammonium solution is prepared as follows: Concentrated ammonium hydroxide is added slowly to 10 to 15-percent silver nitrate until precipitate is completely dissolved. Agitate constantly while adding ammonium hydroxide. After precipitate is completely dissolved, add an excess of one drop of ammonium hydroxide per cc. of solution. Prepare fresh each day and keep in tightly stoppered dark bottle.) 9. Add 10 to 15 cc. of 0.5-percent neutral formalin to sections. Stir or shake until sections become a uniform light yellow-brown color. If color is slow in appearing (longer than one-half minute) decant and add 1-percent neutral formalin. If the proper color still does not appear, decant and add 2-percent neutral formalin. The latter step (2-percent neutral formalin) is seldom necessary. Check sections under microscope. Nerve fibers and nuclei should be dark brown, stroma colorless. 10. When sections have the desired tint, wash them in running water (drop by drop) for a half hour. Cover beaker with layer of gauze to prevent loss of sections. 11. Wash in 20 cc. distilled water for two minutes. 12. Tone with gold chloride (2 drops of a 1percent aqueous solution of gold chloride to 5 cc. of distilled water) until brown nerve fibers and nuclei are colored dark, gray to black. 13. Fix in 5-percent sodium thiosulfate for 1 to 2 minutes. 14. Wash in distilled water. Dehydrate in 80percent, 95-percent, and two changes of absolute alcohol. Clear in thin cedarwood oil and mount in balsam. Histologie
examination
of
the
stained
6S4
WALTER KORNBLUETH, A. EDWARD MAUMENEE AND JANE E. CROWELL
sections revealed the following general pattern of reaction of the corneal nerves after keratoplasty. During the first 3 days after operation the nerves in both the graft and the surrounding cornea began to show signs of degeneration in the form of segmentation, lighter staining and curling of the fibrils, and finally disappearance of the nuclei of Schwann's sheath cells (fig. 2 ) .
penetrating the scar and entering the margin of graft (figs. 4 and 4a). During the following weeks these small fibers became more numerous and penetrated to the center of the graft. The number of fibers in single nerves increased and, about 12 weeks after operation, nerves could be found in the midstroma which contained S to 10 fibers (fig. 5). At about the same time, small fibrils could be seen in the
K
.1 C i* * ' » *' \4* /·■■·»*■ y■,v■·**:.'·· i W * A
>'.
Fig. 2 (Kornblueth, Maumenee, and Crowell). Section of homogenous corneal graft (3 days after operation) showing a degenerating nerve. (Silver impregnation X12S.)
By the end of the second week practically all of the nerves had disappeared from the graft. In the recipient's cornea most of the nerve fibers for a distance of about 2 mm. from the incision disappeared and nerves in the periphery showed signs of ascending degeneration. By 3 weeks newly formed fibers approached the scar on the edge of the graft. These frequently turned back into the recipient cornea or turned and ran parallel to the scar (fig. 3). By 4 to 6 weeks single nerve fibers could be found in the midstroma,
subepithelial and epithelial region in the grafts (figs. 6 and 6a). During the first two months after operation the nerves in the recipient's cornea at the edge of the graft were usually composed of 1 or 2 fibers but, after 3 to 6 months, nerves containing 6 or more fibers reached the scar tissue surrounding the graft (fig. 7). COMMENT
In this study the return of clinical sensibility coincided well with the anatomic findings. One month after operation only the
NERVE REGENERATION IN CORNEAL GRAFTS
S£èsè£! *
0
^ * "
Fig. 3 (Kornblueth, Maumenee, and Crowell). Section of homogenous corneal graft (20 days after operation) showing newly formed nerve fibers in the recipient's cornea approaching the scar on the edge of the graft. (Silver impregnation X125.)
Fig. 4 (Kornblueth, Maumenee, and Crowell). Section of clear homogenous corneal graft (1 month after operation) showing a nerve fiber entering the margin of the graft. Scar at junction of graft and recipient cornea is seen at the lower left. (Silver impregnation χ125.)
655
6S6
WALTER KORNBLUETH, A. EDWARD MAUMENEE AND JANE E. CROWELL
,VT'
Λ' Fig. 4a (Kornblueth, Maumenee, and Crowell). Section of cloudy homogenous corneal graft (6 weeks after operation) showing nerve fibers entering the margin of the graft. Scar at junction of graft and recipient cornea is seen at the lower left. (Silver impregnation χ125.)
Fig. S (Kornblueth, Maumenee, and Crowell). Section of homogenous corneal graft (3 months after operation) showing a thick nerve in the midstroma of the graft. (Silver impregnation X125.)
NERVE REGENERATION IN CORNEAL GRAFTS
Τ&ΜΨί
Fig. 6 (Kornblueth, Maumenee, and Crowell). Section of clear homogenous corneal graft (7 months after operation) showing a small nerve fiber entering the epithelium of the graft. Epithelium is at the upper left. (Silver impregnation X12S.)
Fig. 6a (Kornblueth, Maumenee, Crowell). Section of cloudy homogenous corneal graft (3 months after operation) showing a small nerve fiber entering the epithelium of the graft. Epithelium is at the upper left. (Silver impregnation X400.)
657
658
WALTER KORNBLUETH, A. EDWARD MAUMENEE AND JANE E. CROWELL
Fig. 7 (Kornblueth, Maumenee, and Crowell)^ Section of homogenous corneal graft (7 months after operation) showing a thick nerve in the recipient's cornea entering the scar tissue at the edge of the graft. (Silver impregnation χ125.) margins of the grafts were sensitive to heavy touch (300-mg. test object). During the course of the next few weeks sensitivity to heavy pressure was acquired by the whole graft. This corresponded to the time when only single nerve fibers were found in the midstroma of the transplants. After 3 to 4 months, corneal sensitivity to light touch (100-mg. test object) could be perceived. At this time, on histologie sections, numerous nerves and nerves with multiple fibers were observed in the midstroma of the graft, and small nerve fibers were found in the subepithelial and epithelial regions. The ingrowth of nerves and return of corneal sensibility was approximately the same in clear and cloudy grafts. There was no correlation between the invasion of blood vessels and nerves into the graft as had been suggested by Thomas, Babel and Campos, and Franceschetti and Babel. Blood vessels could be found in cloudy grafts 1 to 2 weeks before the nerves entered the graft, and even then the nerves
did not necessarily take the same course as the invading capillaries. On the other hand, nerves were found in entirely clear grafts which did not contain blood vessels. We were also not able to confirm Franceschetti and Babel's suggestion that the presence of subepithelial nerves in grafts was essential for the final clarity of transplants. In our experiments both clear and cloudy grafts showed thin nerve fibers entering the subepithelial and epithelial region several months after operation (figs. 6 and 6a). SUMMARY
The return of corneal sensitivity was tested in 12 clear and 10 cloudy transplants. The ingrowth of corneal nerves was studied histologically by the use of the silver-impregnation technique in 17 clear and 13 cloudy grafts. The return of deep sensibility in the grafts in 4 to 6 weeks corresponded to the ingrowth of nerves into the midstroma. The acquisition of light sensibility in 3 to 4 months corresponded to the
659
SENSITIVITY TO GOLD-BALL IMPLANT penetration of nerves into the subepithelial and epithelial regions of the grafts. No appreciable difference was found in the regeneration of nerves into clear or cloudy grafts. The ingrowth of nerve fibers was
not dependent on the invasion of the grafts by blood vessels. Stanford University Hospitals (15). The Johns Hopkins Hospital (5).
REFERENCES
1. Maumenee, A. E., and Kornblueth, W. : The physiopathology of corneal grafts. Tr. Am. Acad. Ophth., 1948 (Mar.-Apr.), p. 331 ; Am. J. Ophth., 31:1384, 1948. 2. Ascher, K. W.: The question of keratoplasty II. Arch. f. Ophth., 107:241, 1922; The question of keratoplasty III. Arch. f. Ophth., 107 :439,1922. 3. Elschnig, A. : Keratoplasty. Arch. Ophth., 4:16S, 1930. 4. Imre, J. : Clinical and histological experiences with corneal transplantation. Beiheft, Klin. Monatsbl. f. Augenh., p. 1,1942. 5. Morone, G. : Research on the sensibility of transplants. Ann. d. Ottal. e. Clin. Ocul., 72:279, 1946. 6. Thomas, J. W. T. : On the return of sensitiveness in corneal grafts in rabbits. Proc. Roy. Soc. Med., Series B., 108 :301, 1931. 7. Galante, E. : Experimental research on keratoplasty. Ann. d. Ottal. e. Clin. Ocul., 62:119, 1934. 8. Babel, J., and Campos, R. : On the regeneration of nerves in corneal transplants. Ophthalmologica, 111 :140, 1946. 9. Franceschetti, A., and Babel, J. : Histologie examination of a transparent corneal transplant : the behavior of nerves. Ann. d'Ocul., 180:14S, 1947. 10. Jent, M. : The cornea test for the examination at pursuit of regeneration in the peripheral nerves. Helvet. physiol. acta., 3 :6S, 194S. 11. Campos, R. : Modification of the method of silver impregnation of Bielschowsky-Gros. Acta anat., 2 :75, 1946. A CASE O F SENSITIVITY TO GOLD-BALL ORBITAL IMPLANT* ECZEMATOUS CONTACT-TYPE DERMATITIS DUE TO 14-KARAT GOLD H. WALTER FORSTER, JR., M.D.,
AND ROBERT F. DICKEY,
M.D.
Philadelphia, Pennsylvania The medical literature contains numerous references to dermatitis following parenteral administration of gold compounds in the therapy of arthritis, lupus erythematosus, vitiligo, and so forth. Ophthalmologists and dermatologists frequently see contact dermatitis due to monel metal (an alloy of copper and nickel) and "white gold" (an alloy of gold, copper, and nickel), which are used extensively in spectacle frames1 and jewelry. The offending allergen in these cases is considered to be nickel or its salts. The important synergistic action between pyogen and nickel allergy has been emphasized by Cormia,2 Stokes,3 and others. Gold chloride is said to cause dermatitis among * From the Departments of Ophthalmology and Dermatology, Hospital of the University of Pennsylvania.
photographers, 4 but our review of the literature failed to find any report of contact dermatitis due to gold leaf. Gold, silver, tin, and copper in pure metallic form are not considered primary irritants or sensitizers. Most reports of sensitivity to these metals from their use in industry or in daily life emphasize the fact that usually the offending allergen is a salt of the metal concerned. The subject of this case report showed a marked sensitivity clinically and by patch tests to a 14-karat gold ball which, 5 years previously, had been implanted into Tenon's capsule at the time of enucleation of the left eye. This sensitivity was manifested by a recurrent seropurulent orbital discharge and an eczematous contact-type dermatitis of the lids and adjacent skin.