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The Production and Cure of Ocular Disturbances in Adult Albino Rats by Adjustment of Vitamin A*
THE PRODUCTION AND CURE OF OCULAR DISTURBANCES IN ADULT ALBINO RATS BY ADJUSTMENT OF VITAMIN A* CLINICAL IMPLICATIONS ARTHUR M. YUDKIN, M.D., ALINE U. ORTEN, M.S., AND ARTHUR H. SMITH, PH.D. New Haven, Connecticut
Much, if not all, of the previous work on vitamin A has been carried out with young animals. This has occurred partly because of the supposed greater susceptibility of younger animals and partly for economic reasons. Since vitamin A, when fed in abundance, is readily stored in the animal body, it has been necessary to conduct experiments on young animals, when storage is at a minimum, in order to bring about the deficiency within a reasonable length of time. Consequently, it has not been determined whether the familiar ocular changes seen in the young rat suffering from acute vitamin-A deficiency, occur in the adult animal under the same circumstances. In the present experimental study, it was observed that normal-appearing adult rats which had been reared to middle age (315 days of age) on an adequate diet supplying, however, only the smallest amount of vitamin A that would permit normal growth and prevent the appearance of any symptoms of vitamin-A deficiency, rapidly developed the deficiency when the vitamin A was entirely removed. The first signs of ocular disturbance in both the young and the adult animal was lacrimation and photophobia; then the normally protruding eyeballs receded, producing an enophthalmos. The lacrimal secretion within a few days changed to a viscid, then to a serosanguinous discharge which adhered to the eyelids and Ire-
* From the Laboratory of Physiological Chemistry and the Department of Surgery, Yale University School of Medicine. Presented before the American Association for Research in Ophthalmology at Atlantic City, June 8, 1937.
quently matted them together. In the meantime the eyeball became slightly congested and the cul-de-sac filled with broken-down cellular material. When this stage was reached the cornea became hazy and lusterless, and often a plaque formed on the exposed cornea (palpebral fissure). In the adult animal deprived of vitamin A an herpetic eruption of the cornea seemed to be the more prevalent lesion. This could not be brushed off the tissue, as was possible with the plaque. This stage was considered to be an advanced condition.and some of the animals were given cod-liver oil for curative purposes; others were killed for pathological study. The ocular lesion produced in the older animal was more destructive than that observed in the younger animal. The type of gross pathological manifestation produced in the adult animal simulated very closely the classical picture repeatedly observed in vitamin-A deficiency in the young animals. Control animals, however, from whose diet the vitamin A was not removed at any time, showed no evidence of ocular changes. A second control group was fed the same diet, which, however, was supplemented by a comparatively large amount of vitamin A until the animals reached middle age (315 days), when they too were deprived of the vitamin. In contrast to those rats which received a small but adequate quantity of vitamin A during the preliminary period, these animals failed to develop any ocular changes or, in fact, any symptoms of the deficiency. From these results it would appear that
1115
ll16
A. M. YUDKIN, A. U. ORTEN, AND A. H. SMITH
when the entire source of vitamin A is removed, an adult animal which has little or no store of vitamin A is fully as susceptible to the deficiency as is a young rat with its low reserve of the vitamin. In other words, a circumstance that brings about an inadequate intake or absorption of the vitamin may cause the development of symptoms of the deficiency in the adult animal provided the former supply, even though adequate at the time, was insufficient to build up a satisfactory reserve. It is fairly well agreed that in vitamin-A deficiency the outstanding change is a substitution of stratified keratinizing epithelium for normal epithelium in vari·ous parts of the respiratory, alimentary, and genito-urinary tracts and in the ocular tissues. In both the young and adult animals there is evidence of poor staining of the superficial layers of the cornea; in the early stages, the basal layer is stilI preserved and well stained. A cellular exudative reaction appears below this layer. Frequently a break in the continuity of the basal layer is followed by reparative proliferation of the cells. The basal cells may respond by active mitotic division. The invaded area below the basal cells show a predominence of polymorphonuclear lymphocytes. There is a polymorphonuclear infiltration of the epithelium above and of the substantia propria below. The substantia propria becomes increasingly thicker because of the cellular reaction and the accumulation of edema. New blood vessels are formed. As the inflammation progresses the epithelial layer of the cornea stains very poorly and finally sloughs off in a limited zone with the production of a superficial ulcer. The spindle-shaped cells of the substantia propria are fewer in number and the interstitial layer has a frayed appearance; it stains more eosinophilic than normal. There is also a tendency for reparative processes to take place. There appear large mononuclear phago-
cytic cells of the endothelial leucocytic variety. The cells that previously formed the inflammatory process now have pyknotic and karyorrhexic nuclei. It appears that the function of the phagocytic cells is to remove the debris. When the eyes of some of the adult experimental animals were in this condition, vitamin A was fed to cure the deficiency disease. A histologic study of the ocular tissue of the cured animals is exceedingly instructive. The healing progresses gradually. The increased vascularity, which first appeared during the acute inflammatory process, begins to decrease. There remain ultimately only a few blood vessels in the substantia propria. The fibroblasts increase in number and lead to a thickened substantia propria. There is an occasional mononuclear cell filled with phagocytosed yellowish pigment. The basal-cell layer of the epithelium in the final stages of repair no longer forms the sharp line of demarcation but is rather piled up in an irregular manner, simulating the "rete pegs" of the skin. There is hyperkeratosis of the corneal epithelium, which again no longer permits a smooth corneal surface. Among the proliferated epithelial cells of the cornea can be found occasional mitotic figures. In some instances the process has subsided with a complete restitution to normal of the corneal structures and it is impossible to distinguish the healed cornea from one that had not previously been involved. Outspoken vitamin-A deficiency in man appears to have been somewhat more common in Europe than in this country. Nevertheless, recent studies in this country would seem to indicate a surprising prevalence of subclinical vitamin-A deficiency, particularly among children. These observations have depended upon instruments supposedly measuring dark adaptation. As yet there has not been established a norm on which variations in
VITAMIN A AND OCULAR DISTURBANCES
dark adaptation can be estimated. Clinical .experience leads us to believe that there are several factors that may influence dark adaptation; for instance, true lightcomplexioned individuals have a different norm from that of the dark-complexioned individuals; prolonged dark adaptation is present in a variety of diseases; for example, retinitis pigmentosa, retinitis albescens, and choroideremia. Under ordinary conditions it appears that the American dietary is reasonably adequate. It should be kept in mind, however, that the absorption of the various dietary constituents is dependent upon satisfactory gastro-intestinal function. Inasmuch as circumstances of modern life may interfere with proper digestion, an individual may conceivably suffer from an incipient lack of anyone of the indispensable food factors, despite the fact that plenty is consumed in the food. The ophthalmologist sees a group of patients with ocular disturbances presenting no obvious other clinical abnormalities, the individuals of which, however, on careful questioning, offer bits of evidence which in the light of the newer knowledge of nutrition point toward a greater or less degree of nutritional deficiency. Although the condition of acute, uncomplicated, vitamin-A deficiency, such as can be produced in young and adult rats, is rarely seen in private or clinical practice, a type of keratitis in adults amenable to vitamin therapy is encountered. Within the last four years 16 cases of keratitis, in which the etiology was not obvious, have been observed. A thorough physical examination revealed no signs nor manifestations of infection. The histories of the patients indicated that the condition was accompanied by loss of appetite, constipation, headache, and general malaise. Invariably these patients had lost their teeth early in adult life. The patients' ages ranged from 45 to 68 years.
1117
The first complaint was that of having something in the eye and photophobia. The cornea appeared normal at first, nevertheless the eye was very painful. The patients noted no visible ocular disturbance. When the eye was examined, there was seen a breaking down of the periphery of the portion of the cornea that was exposed in the palpebral fissure or in the area covered by the lower lid. Very little, if any, congestion was present. The cornea often stained with fluorescein. The lesion did not seem to improve with local treatment and within a few days the ulcer appeared to be more extensive. The invaded cornea presented a shallow, excavated ulceration which frequently spread along the margin of the cornea and extended toward the pupillary area. An examination of this area with the slitlamp revealed a swollen, edematous corneal epithelium and a similar involvement of the substantia propria. The surrounding tissue showed considerable vascularity. The corneal nerve fibers extending into the diseased area were very prominent. In this type of corneal lesion, particularly in the early stages, no definite inflammation of the deeper layers of the cornea and uveal tract could be detected. Often the lesion was described as a catarrhal ulcer, without any definite inflammation of the conjunctiva. In some ,instances the ulceration had the appearance of a lesion of the cornea produced by an injury, but no history of injury could be elicited. Frequently, when first seen, the corneal lesion resembled that of marginal ulcer, indolent ulcer, or rodent ulcer. At this stage the aqueous humor revealed numerous floaters and Descemet's membrane was sometimes even studded with deposits. The uvea often was involved at this stage. The treatment of this type of corneal lesion has been very unsatisfactory. Local medication and heat seemed to aggra-
1118
A. M. YUDKIN, A. U. ORTEN, AND A. H. SMITH
vate and delay the healing process, producing a refractory condition. It was frequently necessary to cauterize the cornea with full-strength tincture of iodine or phenol. In this stage the cornea often became infected and produced a hypopyon ulcer, necessitating more drastic treatment. It was suggested from experience in the laboratory with animals on deficiency diseases that this condition might be a nutritional disturbance; therefore a number of these patients were given cod-liver oil in addition to their regular diet. Some showed improvement in a short period of time, whereas others did not respond. It was evident that some other factor was involved. From the history of the case it appeared that the intestinal tract might be at fault. These patients were given large quantities of vitamin-B complex
before each meal in addition to the codliver oil. In a large proportion of these cases, the systemic and ocular disturbances disappeared. In spite of the fact that certain ocular lesions in animals have been shown to be due to a deficiency of vitamin A, we are not yet warranted in assuming the same course for somewhat similar lesions in man. For example, some ulcers of the cornea may not respond to vitamin A alone, but will respond when vitamin-B complex is administered. Again, undoubtedly many corneal lesions have an etiology entirely apart from nutritional deficiency. The main object of this paper is to emphasize the part which malnutrition may sometimes play in the production of ocular pathology, a fact which perhaps has not been sufficiently stressed.
DISCUSSION DR. CONRAD BERENS: May I ask if you think cod-liver oil is the best preparation to use in those cases? DR. YUDKIN : I prefer a standard brand of cod-liver oil for the treatment of this type of ocular disturbance. Cod-liver oil concentrates and carotene are used by many clinicians. I believe that there is something in cod-liver oil besides vitamin A that aids in repairing the diseased tissue. Frequently cod-liver oil taken internally does not clear up the ocular disturbance. It is my impression that the cod-liver oil is not absorbed properly by the intestinal tract. I therefore prescribe vitarnin-B complex as a supplement, for it seems to help the intestinal tract to function normally. I ordinarily use a standard brand of brewer's yeast for this purpose. From our clinical experience it is evident that vitamins alone will not repair the damaged tissue unless the patient is also given a well-balanced diet, not a fanatic diet, but one which every trained medical practitioner can very easily formulate. DR. VERHOFF : You stated that ulcers definitely belong in this group. You must mean Mooren's ulcer and I would like
to know whether you ever cured Mooren's ulcer by this treatment. DR. YUDKIN: I have had very little personal experience with Mooren's ulcer, but from an interpretation of the literature the rodent lesion may belong to this group that I have described. DR. BERENS : You said you applied the cod-liver oil locally? DR. YUDKIN: No, not locally. DR. BERENS: Would you be willing to express an opinion in regard to that form of therapy? DR. YUDKIN: Cod-liver oil has been used locally in ocular therapy. I have no personal experience with its application. It is my impression that it will irritate the conjunctiva when so used. Cod-liver oil ointment is advocated for ulcers of the skin. DR. BERENS: Is there no experimental work on that? DR. YUDKIN: I have no knowledge of any experimental work. However, some years ago, I received a circular from a pharmacist in Philadelphia who was dispensing cod-liver oil in the form of a salve to several oculists.
Much, if not all, of the previous work on vitamin A has been carried out with young animals. This has occurred partly because of the supposed greater susceptibility of younger animals and partly for economic reasons. Since vitamin A, when fed in abundance, is readily stored in the animal body, it has been necessary to conduct experiments on young animals, when storage is at a minimum, in order to bring about the deficiency within a reasonable length of time. Consequently, it has not been determined whether the familiar ocular changes seen in the young rat suffering from acute vitamin-A deficiency, occur in the adult animal under the same circumstances. In the present experimental study, it was observed that normal-appearing adult rats which had been reared to middle age (315 days of age) on an adequate diet supplying, however, only the smallest amount of vitamin A that would permit normal growth and prevent the appearance of any symptoms of vitamin-A deficiency, rapidly developed the deficiency when the vitamin A was entirely removed. The first signs of ocular disturbance in both the young and the adult animal was lacrimation and photophobia; then the normally protruding eyeballs receded, producing an enophthalmos. The lacrimal secretion within a few days changed to a viscid, then to a serosanguinous discharge which adhered to the eyelids and Ire-
* From the Laboratory of Physiological Chemistry and the Department of Surgery, Yale University School of Medicine. Presented before the American Association for Research in Ophthalmology at Atlantic City, June 8, 1937.
quently matted them together. In the meantime the eyeball became slightly congested and the cul-de-sac filled with broken-down cellular material. When this stage was reached the cornea became hazy and lusterless, and often a plaque formed on the exposed cornea (palpebral fissure). In the adult animal deprived of vitamin A an herpetic eruption of the cornea seemed to be the more prevalent lesion. This could not be brushed off the tissue, as was possible with the plaque. This stage was considered to be an advanced condition.and some of the animals were given cod-liver oil for curative purposes; others were killed for pathological study. The ocular lesion produced in the older animal was more destructive than that observed in the younger animal. The type of gross pathological manifestation produced in the adult animal simulated very closely the classical picture repeatedly observed in vitamin-A deficiency in the young animals. Control animals, however, from whose diet the vitamin A was not removed at any time, showed no evidence of ocular changes. A second control group was fed the same diet, which, however, was supplemented by a comparatively large amount of vitamin A until the animals reached middle age (315 days), when they too were deprived of the vitamin. In contrast to those rats which received a small but adequate quantity of vitamin A during the preliminary period, these animals failed to develop any ocular changes or, in fact, any symptoms of the deficiency. From these results it would appear that
1115
ll16
A. M. YUDKIN, A. U. ORTEN, AND A. H. SMITH
when the entire source of vitamin A is removed, an adult animal which has little or no store of vitamin A is fully as susceptible to the deficiency as is a young rat with its low reserve of the vitamin. In other words, a circumstance that brings about an inadequate intake or absorption of the vitamin may cause the development of symptoms of the deficiency in the adult animal provided the former supply, even though adequate at the time, was insufficient to build up a satisfactory reserve. It is fairly well agreed that in vitamin-A deficiency the outstanding change is a substitution of stratified keratinizing epithelium for normal epithelium in vari·ous parts of the respiratory, alimentary, and genito-urinary tracts and in the ocular tissues. In both the young and adult animals there is evidence of poor staining of the superficial layers of the cornea; in the early stages, the basal layer is stilI preserved and well stained. A cellular exudative reaction appears below this layer. Frequently a break in the continuity of the basal layer is followed by reparative proliferation of the cells. The basal cells may respond by active mitotic division. The invaded area below the basal cells show a predominence of polymorphonuclear lymphocytes. There is a polymorphonuclear infiltration of the epithelium above and of the substantia propria below. The substantia propria becomes increasingly thicker because of the cellular reaction and the accumulation of edema. New blood vessels are formed. As the inflammation progresses the epithelial layer of the cornea stains very poorly and finally sloughs off in a limited zone with the production of a superficial ulcer. The spindle-shaped cells of the substantia propria are fewer in number and the interstitial layer has a frayed appearance; it stains more eosinophilic than normal. There is also a tendency for reparative processes to take place. There appear large mononuclear phago-
cytic cells of the endothelial leucocytic variety. The cells that previously formed the inflammatory process now have pyknotic and karyorrhexic nuclei. It appears that the function of the phagocytic cells is to remove the debris. When the eyes of some of the adult experimental animals were in this condition, vitamin A was fed to cure the deficiency disease. A histologic study of the ocular tissue of the cured animals is exceedingly instructive. The healing progresses gradually. The increased vascularity, which first appeared during the acute inflammatory process, begins to decrease. There remain ultimately only a few blood vessels in the substantia propria. The fibroblasts increase in number and lead to a thickened substantia propria. There is an occasional mononuclear cell filled with phagocytosed yellowish pigment. The basal-cell layer of the epithelium in the final stages of repair no longer forms the sharp line of demarcation but is rather piled up in an irregular manner, simulating the "rete pegs" of the skin. There is hyperkeratosis of the corneal epithelium, which again no longer permits a smooth corneal surface. Among the proliferated epithelial cells of the cornea can be found occasional mitotic figures. In some instances the process has subsided with a complete restitution to normal of the corneal structures and it is impossible to distinguish the healed cornea from one that had not previously been involved. Outspoken vitamin-A deficiency in man appears to have been somewhat more common in Europe than in this country. Nevertheless, recent studies in this country would seem to indicate a surprising prevalence of subclinical vitamin-A deficiency, particularly among children. These observations have depended upon instruments supposedly measuring dark adaptation. As yet there has not been established a norm on which variations in
VITAMIN A AND OCULAR DISTURBANCES
dark adaptation can be estimated. Clinical .experience leads us to believe that there are several factors that may influence dark adaptation; for instance, true lightcomplexioned individuals have a different norm from that of the dark-complexioned individuals; prolonged dark adaptation is present in a variety of diseases; for example, retinitis pigmentosa, retinitis albescens, and choroideremia. Under ordinary conditions it appears that the American dietary is reasonably adequate. It should be kept in mind, however, that the absorption of the various dietary constituents is dependent upon satisfactory gastro-intestinal function. Inasmuch as circumstances of modern life may interfere with proper digestion, an individual may conceivably suffer from an incipient lack of anyone of the indispensable food factors, despite the fact that plenty is consumed in the food. The ophthalmologist sees a group of patients with ocular disturbances presenting no obvious other clinical abnormalities, the individuals of which, however, on careful questioning, offer bits of evidence which in the light of the newer knowledge of nutrition point toward a greater or less degree of nutritional deficiency. Although the condition of acute, uncomplicated, vitamin-A deficiency, such as can be produced in young and adult rats, is rarely seen in private or clinical practice, a type of keratitis in adults amenable to vitamin therapy is encountered. Within the last four years 16 cases of keratitis, in which the etiology was not obvious, have been observed. A thorough physical examination revealed no signs nor manifestations of infection. The histories of the patients indicated that the condition was accompanied by loss of appetite, constipation, headache, and general malaise. Invariably these patients had lost their teeth early in adult life. The patients' ages ranged from 45 to 68 years.
1117
The first complaint was that of having something in the eye and photophobia. The cornea appeared normal at first, nevertheless the eye was very painful. The patients noted no visible ocular disturbance. When the eye was examined, there was seen a breaking down of the periphery of the portion of the cornea that was exposed in the palpebral fissure or in the area covered by the lower lid. Very little, if any, congestion was present. The cornea often stained with fluorescein. The lesion did not seem to improve with local treatment and within a few days the ulcer appeared to be more extensive. The invaded cornea presented a shallow, excavated ulceration which frequently spread along the margin of the cornea and extended toward the pupillary area. An examination of this area with the slitlamp revealed a swollen, edematous corneal epithelium and a similar involvement of the substantia propria. The surrounding tissue showed considerable vascularity. The corneal nerve fibers extending into the diseased area were very prominent. In this type of corneal lesion, particularly in the early stages, no definite inflammation of the deeper layers of the cornea and uveal tract could be detected. Often the lesion was described as a catarrhal ulcer, without any definite inflammation of the conjunctiva. In some ,instances the ulceration had the appearance of a lesion of the cornea produced by an injury, but no history of injury could be elicited. Frequently, when first seen, the corneal lesion resembled that of marginal ulcer, indolent ulcer, or rodent ulcer. At this stage the aqueous humor revealed numerous floaters and Descemet's membrane was sometimes even studded with deposits. The uvea often was involved at this stage. The treatment of this type of corneal lesion has been very unsatisfactory. Local medication and heat seemed to aggra-
1118
A. M. YUDKIN, A. U. ORTEN, AND A. H. SMITH
vate and delay the healing process, producing a refractory condition. It was frequently necessary to cauterize the cornea with full-strength tincture of iodine or phenol. In this stage the cornea often became infected and produced a hypopyon ulcer, necessitating more drastic treatment. It was suggested from experience in the laboratory with animals on deficiency diseases that this condition might be a nutritional disturbance; therefore a number of these patients were given cod-liver oil in addition to their regular diet. Some showed improvement in a short period of time, whereas others did not respond. It was evident that some other factor was involved. From the history of the case it appeared that the intestinal tract might be at fault. These patients were given large quantities of vitamin-B complex
before each meal in addition to the codliver oil. In a large proportion of these cases, the systemic and ocular disturbances disappeared. In spite of the fact that certain ocular lesions in animals have been shown to be due to a deficiency of vitamin A, we are not yet warranted in assuming the same course for somewhat similar lesions in man. For example, some ulcers of the cornea may not respond to vitamin A alone, but will respond when vitamin-B complex is administered. Again, undoubtedly many corneal lesions have an etiology entirely apart from nutritional deficiency. The main object of this paper is to emphasize the part which malnutrition may sometimes play in the production of ocular pathology, a fact which perhaps has not been sufficiently stressed.
DISCUSSION DR. CONRAD BERENS: May I ask if you think cod-liver oil is the best preparation to use in those cases? DR. YUDKIN : I prefer a standard brand of cod-liver oil for the treatment of this type of ocular disturbance. Cod-liver oil concentrates and carotene are used by many clinicians. I believe that there is something in cod-liver oil besides vitamin A that aids in repairing the diseased tissue. Frequently cod-liver oil taken internally does not clear up the ocular disturbance. It is my impression that the cod-liver oil is not absorbed properly by the intestinal tract. I therefore prescribe vitarnin-B complex as a supplement, for it seems to help the intestinal tract to function normally. I ordinarily use a standard brand of brewer's yeast for this purpose. From our clinical experience it is evident that vitamins alone will not repair the damaged tissue unless the patient is also given a well-balanced diet, not a fanatic diet, but one which every trained medical practitioner can very easily formulate. DR. VERHOFF : You stated that ulcers definitely belong in this group. You must mean Mooren's ulcer and I would like
to know whether you ever cured Mooren's ulcer by this treatment. DR. YUDKIN: I have had very little personal experience with Mooren's ulcer, but from an interpretation of the literature the rodent lesion may belong to this group that I have described. DR. BERENS : You said you applied the cod-liver oil locally? DR. YUDKIN: No, not locally. DR. BERENS: Would you be willing to express an opinion in regard to that form of therapy? DR. YUDKIN: Cod-liver oil has been used locally in ocular therapy. I have no personal experience with its application. It is my impression that it will irritate the conjunctiva when so used. Cod-liver oil ointment is advocated for ulcers of the skin. DR. BERENS: Is there no experimental work on that? DR. YUDKIN: I have no knowledge of any experimental work. However, some years ago, I received a circular from a pharmacist in Philadelphia who was dispensing cod-liver oil in the form of a salve to several oculists.