J. COMP.
PATH.
1975.
HISTOPATHOLOGY FATTY LIVER
VOL.
539
85.
AND
OF THE KIDNEY
ADRENAL SYNDROME
GLANDS IN THE OF CHICKENS
BY
P. A. Agricultural
L.
WIGHT
Research Council, Poultry Research Centre, King’s Buildings, Edinburgh EH9 3JS, Scotland
West Mains
Road,
INTRODUCTION
The fatty liver and kidney syndrome (FLKS) is a metabolic disorder of young chickens characterized by the accumulation of abnormally large quantities of lipid in the liver and kidney (Marthedal and Vellinge, 1958 ; Hemsley, 1965). Recent studies (Wight and Siller, 1975) have shown that lipid deposits may involve a wide variety of organs and also that the basophilia of the adrenal medulla is lost. The latter phenomenon did not appear to be directly associated with the presence of lipid accumulations. In the present study the histopathology of the adrenal glands is examined in detail. MATERIAL
AND
METHODS
Groups of sexed, Ross I broiler chicks, housed in deep-litter pens maintained at 2” to 3 “C above the recommended temperature, were fed a wheat-based, commercialtype broiler-grower mash, but the diet designed to induce FLKS had a low protein and low fat content. Details of diets and husbandry have been recorded by Whitehead and Blair (1974) and Whitehead, Bannister, Blair and Evans (1974). Affected birds were killed by decapitation when showing symptoms of the disease, usually between 3 and 5 weeks of age, and their adrenal glands were removed as rapidly as possible. One apparently healthy control bird was killed at the same time as each diseased bird and its adrenals treated similarly. Liver, kidney and heart were taken from both affected and control animals, fixed in formol-saline and frozen sections stained with Sudan IV to confirm histologically the presence or absence of FLKS. One adrenal gland from every bird was fixed in Bouin and stained with HE. In order to distinguish the interrenal (cortical) and chromaffin (medullary) cells more distinctly, the other gland from some birds was fixed in Allen Bouin 15 and stained by a picro-Mallory trichrome method or fixed in Regaud’s dichromate fixative and stained by Schmorl’s ferric-ferricyanide method counterstained with neutral red. The methods used to differentiate noradrenaline and adrenaline-containing cells and also to distinguish medulla from cortex, were the potassium iodate method of Hillarp and H&felt (1955), Giemsa stain following the acetate-buffered, formol-dichromate fixation procedure suggested by Coupland (1961) and the acetate-buffered, eosin yellow-aniline blue stain after buffered-dichromate fixation described by Wood (1963). Catecholamines were specifically identified by a formaldehyde-condensation fluorescent method based on Pearse’s (1972) modification of the technique of Falck and Owman; small pieces of gland about 2 mm3. were rapidly frozen in an ethylene glycol-liquid nitrogen mixture, freeze-dried at -40 “C for 4 to 5 h. and then condensed over preheated paraformaldehyde for 3 h. at 80 “C. It was not necessary to equilibrate the paraformaldehyde in the humidity prevailing in this laboratory.
540
P. A. L. WIGHT
Paraffin sections were mounted in liquid paraffin and examined by WV. blue light (mean excitation 410 nm; K530 barrier filters). Ascorbic acid was detected by a silver nitrate technique based on the histochemical method of Bourne (Pearse, 1968). For electron microscopy animals were anaesthetized with pentobarbitone sodium and the adrenal glands perfused by the aortic arch with an aqueous pre-rinse containing 5 per cent. dextran and 0.9 per cent. sodium chloride, followed by 500 ml of 2.5 per cent. glutaraldehyde in 0.1 M Millonig’s phosphate buffer (pH 7.4 and 360 m-osmol.). The glands were then chopped into 1 mm. cubes and fixation continued by immersion for 1 h. in the same fixative. After washing in 0.2 M Millonig’s buffer and post-fixation in 0.1 M Millonig’s phosphate-buffered 1 per cent. osmium tetroxide, the tissues were dehydrated in graded alcohols and embedded in Araldite. Blocks from a few glands were also fixed in Dalton’s fixative made up with a modified buffer (3 per cent. potassium dichromate, 2.6 per cent. sodium chloride). RESULTS
Light Microscopy Examination of HE. sections of the adrenal glands from 25 birds which had clinical signs of advanced FLKS and characteristic lipid deposits in liver, kidney and heart, revealed loss of basophilia of the chromaffin cells in 18. Adrenals from the 25 control chickens were normal. The picro-Mallory tri-
chrome stained the normal interrenal cells red and the chromaffin cells bluepurple and again the colouration of the latter was lost, all the glandular tissue
Fig.
1. Adrenal gland from normal control cholamines has resulted in distinct Schmorl’s method after dichromate Fig. 2. Adrenal from a chicken affected by Schmorl’s method after dichromate
chicken. Reduction greenish-blue staining fixation. x 33. FLKS shows almost fixation. x 33.
of ferric-ferricyanide by the cateof the columns of chromaffin cells. complete
absence
of reduction
sites.
ADRENALS
IN FATTY
LIVER
AND
KIDNEY
541
SYNDROME
being stained red. Medullary amines have strong reducing properties which, in normal adrenal glands, reduce the ferric-ferricyanide in Schmorl’s method so that the chromaffin cells are coloured dark greenish-blue. In FLKS this reducing capacity appeared to be absent so that very few greenish-blue stained cells were seen (Figs 1 and 2). The methods of Hillarp and Hskfelt (1955)) Coupland (1961) and Wood (1963) confirmed the absence of typical medullary cell staining reactions and they also showed that this abnormality affected both noradrenaline and adrenaline-containing cells (Figs 3 and 4). When the formaldehyde-condensation preparations of control adrenal glands were examined by U.V. light, the interrenal cells appeared a dull green while chromaffin cells fluoresced light or golden-yellow. In FLKS very few yellow fluorescing cells were present
Fig. 3. Noradrenalineand adrenaline-containing Wood’s method. x 324. Fig. 4. Relatively few amine-containing chromaffin Wood’s method. x 324.
chromaffin cells remain
cells in the adrenal in the gland
of a control
of a chicken
with
chicken. FLKS.
(Figs 5 and 6). However, it was usually possible to discern a faintly silveryyellow fluorescent background of cells in similar numbers and occupying a similar area to the chromaffin cells of the normal adrenals. This suggested that the loss of basophilia in HE. stained sections was not due to destruction of the chromaffin cells, but to a depletion of their catecholamine content. Silver granules were present in both cortical and medullary tissue after applying Bourne’s technique. The reactions, supposedly indicative of the presence of ascorbic acid, appeared quantitatively similar in diseased and control birds.
542
P. A. L. WIGHT
of columns of chromaffin Fig. 5. Strong fluorescence condensation. x 90. Fig. 6. Very few chromaffin cells fluoresce strongly escence indicates the position of the columns
Fig. 7. Chromaffin adrenaline
cells in a control adrenal gland granules and strongly electron-dense
cells in a control in FLKS although of cells. Formaldehyde
adrenal
gland.
Formaldehyde
a background of faint fluorcondensation. x 90.
.- .____ - ._.._.-_ -. showing cells with moderately electron-densr noradrenaline granules. x 4480.
ADRENALS
Fig. 8. Chromaffin
Fig. 9. Chromaffin granules.
IN FATTY
cells in FLKS;
cell in a control x 16 800.
adrenal
LIVER
there
AND
KIDNEY
is severe depletion
containing
electron-dense,
SYNDROME
of amine
543
granules.
spherical
x
4480.
or oval noradrenaline
544
P. A.
L.
WIGHT
Fig.
10. Chromaffin cell in an adrenal from a case of FLKS. cytoplasm an empty appearance but mitochondria present. x 8260.
Fig.
11. Chromaffin
cell in ELKS. f xrrL- ---
Amine granules are absent giving are normal. Some lipid droplets
the are
ADRENALS
IN FATTY
LIVER
AND
KIDNEY
SYNDROME
545
Electron Microscopy The normal chromaffin cells of the fowl can be divided into 2 populations on the basis of their specific cytoplasmic granules (Fig. 7). Some contain numerous oval or spherical, membrane-limited granules of moderate electron density and these are considered to be adrenaline-storing granules. In other cells the granules are similar, but are highly electron-dense and these are noradrenalinestoring granules (Fig. 9). Internal sub-unit structure could sometimes be identified in both types of granule. In FLKS the number of granules of both types was much less than in the control birds (Figs 8 and 10). Remaining granules in affected cells were also reduced in size and of irregular, angular shape and the perigranular space beneath the limiting membrane appeared correspondingly wide (Fig. 11). In many cases of the disease, nearly all the chromaffin cells seen were affected and most cells showed an almost complete loss of granules (Figs 8 and 10). Sometimes a few dense-cored granules of very small diameter were present. On the other hand, the nucleus and cytoplasmic organelles were not morphologically abnormal. The cytoplasmic matrix had an empty appearance resulting from the loss of amine-storing granules and large vacuoles containing pale, granularfibrillary material, sometimes referred to as colloid vacuoles in the normal cell, were often unduly numerous (Fig. 11). Mitochondria of normal structure were present (Figs 10 and 11). A few lipid droplets were seen in some medullary cells (Fig. lo), but lipid deposition was not a characteristic feature of chromaffin cell involvement. These changes were present after both glutaraldehyde and Dalton’s fixation. No consistent evidence of morphological change affecting the interrenal cells was found by electron-microscopic examination. Some lipid droplets were of slightly denser electron-translucency in the disease than in the controls, but, by subjective examination, quantitative alterations in the lipid content of the cells was not detected. DISCUSSION
Morphological changes in the adrenal glands in FLKS were reported by Wight and Siller (1975) as a loss of tinctorial distinction between the medulla and cortex. The present study demonstrates both histochemically and ultrastructurally that this phenomenon is due to loss of catecholamines from the chromaffin cells; norepinephrine and epinephrine were both depleted. The ultrastructural study suggests that the changes were an extension of the normal physiological discharge of adrenaline and noradrenaline granules (De Robertis and Vaz Ferreira, 1957). This is indicated by the absence of alteration in any other medullary organelle and in this respect the adrenal lesion is similar to that of other organs in FLKS in which degenerative or inflammatory reactions were generally absent (Siller and Wight, 1975; Wight, 1975). Unlike many organs in the syndrome, lipid was rarely present in the medullary cells and then only in small amounts, so its direct presence does not appear to have caused the loss of amines. Viral and bacterial infections, toxic products of microbes and parasites and
546
P. A.
L. WIGHT
chemical toxins may produce intense general-adaptation-syndrome phenomena in the adrenals of mammals, including the discharge of chromaffin granules (Selye, 1950). Reye’s syndrome is an encephalopathy and visceral fatty degeneration of children with many features in common with FLKS, but the endocrine glands are not abnormal (Reye, Morgan and Baral, 1963). The chromaffinity of the adrenal medulla is considerably decreased in scorbutic guinea-pigs (Selye, 1950). However, the addition of vitamin C to an FLKSinducing diet did not significantly affect the incidence of the disease (Whitehead, Blair, Bannister, Evans and Jones, 1975) nor was a histochemicallydetectable decrease in ascorbic acid found in the glands in the present study. Perhaps the latter finding should be interpreted cautiously because relatively mild stress may cause a fall in biochemically-determined adrenal ascorbic acid levels (Freeman, 1967). On the other hand, the histochemical silver methods are of doubtful specificity (Pearse, 1968) ; in particular, the phosphatide content of mitochondria may reduce acid silver nitrate. Electron microscopy showed that the mitochondria of the adrenal medullary cells were wellpreserved in FLKS. It has been suggested that the signs of FLKS may be due to adrenal malfunction (Payne, Gilchrist, Pearson and Hemsley, 1974), but in the present investigation adrenal involvement was not observed in every case. These apparently-normal adrenals may have recovered their chromaffinity, but it is also possible that they were never morphologically affected and that the adrenal abnormality, even although it may be associated with symptoms in some cases of FLKS, is a secondary response to the original metabolic lesion. Adrenal catecholamines have a role in regulating the metabolism of lipid, carbohydrate and protein in mammals. As regards lipid metabolism, the experimental injection of these amines into fowls induces neither the rise in blood free fatty acid levels nor the lipid accumulation in certain organs which results from a similar procedure in mammals (Wells and Wight, 1971). Lipolysis may be stimulated to a small extent when avian adipose tissue is incubated in vitro with high concentrations of adrenaline and noradrenaline (Goodridge and Ball, 1965; Langslow and Hales, 1969). Although it cannot, therefore, be concluded that these amines have no effect on lipid metabolism in birds (HimmsHagen, 1967), our present knowledge provides no explanation for chromaffin depletion in relation to the tissue triglyceride deposition and elevated plasma free fatty acid concentrations (Evans, Bannister and Whitehead, 1975) which occur in FLKS. However, the known effects of adrenaline and noradrenaline on carbohydrate metabolism in the fowl (Wells and Wight, 1971) do provide a possible explanation for the depletion. These amines accelerate the discharge of liver induce hyperglycaemia and, perhaps acting by the adenosine glycogen, nucleotide cycle, permit accelerated gluconeogenesis. In mammals, adrenaline also acts upon the islets of Langerhans to inhibit the production of insulin and stimulates the secretion of ACTH which in turn stimulates the secretion of adrenal glucocorticosteroids, the latter inducing gluconeogenesis. Low blood sugar, probably acting through receptor centres in the hypothalamus and subsequent neural pathways, stimulates the secretion of these medullary amines.
ADRENALS
IN FATTY
LIVER
AND
KIDNEY
SYNDROME
547
Hypoglycaemia and decreased hepatic gluconeogenesis are important occurrences in FLKS (B annister, Evans and Whitehead, 1975) and it therefore seems possible that attempts to maintain homeostasis by stimulating gluconeogenesis and rectifying this low blood-sugar level may result in excessive secretion and ultimate depletion of adrenal catecholamine stores. This possibility is born out by ultrastructural investigations (Fujita, Kano, Kunishima and Kido, 1959; D’Anzi, 1969) of the fowl and mammalian adrenal in insulin-induced hypoglycaemia in which depletion of amines morphologically similar to that in FLKS was observed.
There
is much
circumstantial
evidence
that stress is involved
in FLKS
(Whitehead and Blair, 1974). Certain experimental stresses have been shown to cause a biochemically-determined decrease in the epinephrine and norepinephrine concentrations of the chicken’s adrenal (Zachariasen and Newcomer, 1974) and it is possible that the morphological depletion demonstrated in the present study may be associated with the adverse effects of environmental stress in FLKS. SUMMARY
Decreased chromaffinity occurred in the adrenal medulla of 60 per cent. of cases of the fatty liver and kidney syndrome which were examined. This abnormality was shown by histochemical and ultrastructural examinations to be due to severe depletion of catecholamines. The finding is discussed in relation to the symptoms, histopathology and biochemical changes which characterize the disease. REFERENCES
Bannister, D. W., Evans, A. J., and Whitehead, C. C. (1975). Fatty liver and kidney syndrome in chicks. Evidence for a lesion in carbohydrate metabolism. Research in Veterinary Science, 18, 149-156. Coupland, R. E. (1961). The distribution of cholinesteraseand other enzymes in the adrenal glands of the ox and man and in a human phaeochromocytoma. Cytolo@ of the .Nervous System, pp. 28-32, Anatomical Society of Great Britain and Ireland Symposium. D’Anzi, F. A. (1969). Morphological and biochemical observations on the catecholamine-storing vesiclesof rat adreno-meduilary cells during insulin-induced hypoglycaemia. American Journal of Anatomy, 125, 381-397. De Robertis, E., and Vaz Ferreira, A. (1957). Electronmicroscopic study of the excretion of catechol-containing droplets in the adrenal medulla. Experimental Cell Research, 12, 568-574. Evans, A. J., Bannister, D. W., and Whitehead, C. C. (1975). Fatty liver and kidney syndrome in chicks. Some aspects of lipid metabolism. Research in Veterinary Science, 18, 26-31. Freeman, B. M. (1967). Effects of stresson the ascorbic acid content of the adrenal gland of Gallus domesticus. Comjarative Biochemistry and Physiology, 23, 303-305. Fujita, H., Kano, M., Kunishima, I., and Kido, T. (1959). Electron microscopic observations on the adrenal medulla of the chick after injection of insulin. Archivum histologicumjaponicum, 18, 411-419. Goodridge, A. G., and Ball, E. G. (1965). Studies on the metabolism of adipose tissue XVIII. In vitro effects of insulin, epinephrine and glucagon on lipolysis and glycolysis in pigeon adipose tissue. Comparative Biochemistry and Physiology, 6, 367-381.
548
P. A. L. WIGHT
Hemsley, L. A. (1965). The fatty liver and kidney syndrome of young chickens. Veterinary Record, 77, 124-126. Hillarp, N. A., and Htikfelt, B. (1955). Histochemical demonstration of noradrenaline and adrenaline in adrenal medulla. Journal of Histochemistry and Cytochemistry, 3, l-5. Himms-Hagen, J. (1967). Sympathetic regulation of metabolism. Pharmacological Reviews, 19, 367-46 1. Langslow, D. R., and Hales, C. N. (1969). Lipolysis in chicken adipose tissue i?z vitro. Journal of Endocrinology, 43, 285-294. hos Kyllinger, Marthedal, H. E., and Vellinge, G. (1958). L ever-og nyrelidelse Nordisk Veterinarmb’tet, Helsinki, 8, 250-255. Payne, C. G., Gilchrist, P., Pearson, J. A., and Hemsley, L. A. (1974). Involvement of biotin in the fatty liver and kidney syndrome of broilers. British Poultry Science,
15, 489-498. Pearse, A. G. E. (1968; 1972). Histochemistry: Theoretical and Applied, 1 and 2, 2nd edit. Churchill-Livingstone, Edinburgh and London. and fatty deReye, R. D. K., Morgan, G., and Baral, J. (1963). Encephalopathy generation of the viscera, a disease entity in childhood. Lance& 2, 749-752. Selye, H. (1950). Stress,pp. 286-349, Acta, Montreal. Siller, W. G., and Wight, P. A. L. (1975). A n ultrastructural study of the fatty liver and kidney syndrome in the fowl. Research in Veterinary Science (in press). Wells, J. W., and Wight, P. A. L. (1971). The adrenal glands. In Physiology and Biochemistry of the Domestic Fowl, 1. D. J. Bell and B. M. Freeman, Eds. pp. 489-520, Academic Press, London. Whitehead, C. C., and Blair, R. (1974). Fatty liver and kidney syndrome in chicks. The involvement of dietary energy-protein ratio and house temperature. Research in Veterinary Science, 17, 86-90. Whitehead, C. C., Bannister, D. W., Blair, R., and Evans, A. J. (1974). Fatty liver and kidney syndrome in chicks. Effects on liver and kidney of diets causing the syndrome. Research in Veterinary Science, 17, 222-225. Whitehead, C. C., Blair, R. B., Bannister, D. W., Evans, A. J., and Jones, R. IM. (1975). The involvement of biotin in preventing the fatty liver and kidney syndrome in broilers. Research in Veterinary Science (in press). Wight, P. A. L. (1975). Th e neuropathology of the fatty liver and kidney syndrome of chicks. Neuropathology and Applied fleurobiolopy, 1 (in press). Wight, P. A. L., and Siller, W. G. (1975). The histopathology of the fatty liver and kidney syndrome in chicks. Research in Veterinary Science (in press). Wood, J. G. (1963). Identification of and observations on epinephrine and norepinephrine containing cells in the adrenal medulla. American Journal of Anatomy,
112, 285-303. Zachariasen, R. D., and Newcomer, W. S. (1974). Phenyl ethanolamine-N-methyltransferase activity in the avian adrenal following immobilization or adrenocorticotropin. General and Comparative Endocrinology, 23, 193-198. [Received for publication,
February 26th, 19751
PATH.
1975.
HISTOPATHOLOGY FATTY LIVER
VOL.
539
85.
AND
OF THE KIDNEY
ADRENAL SYNDROME
GLANDS IN THE OF CHICKENS
BY
P. A. Agricultural
L.
WIGHT
Research Council, Poultry Research Centre, King’s Buildings, Edinburgh EH9 3JS, Scotland
West Mains
Road,
INTRODUCTION
The fatty liver and kidney syndrome (FLKS) is a metabolic disorder of young chickens characterized by the accumulation of abnormally large quantities of lipid in the liver and kidney (Marthedal and Vellinge, 1958 ; Hemsley, 1965). Recent studies (Wight and Siller, 1975) have shown that lipid deposits may involve a wide variety of organs and also that the basophilia of the adrenal medulla is lost. The latter phenomenon did not appear to be directly associated with the presence of lipid accumulations. In the present study the histopathology of the adrenal glands is examined in detail. MATERIAL
AND
METHODS
Groups of sexed, Ross I broiler chicks, housed in deep-litter pens maintained at 2” to 3 “C above the recommended temperature, were fed a wheat-based, commercialtype broiler-grower mash, but the diet designed to induce FLKS had a low protein and low fat content. Details of diets and husbandry have been recorded by Whitehead and Blair (1974) and Whitehead, Bannister, Blair and Evans (1974). Affected birds were killed by decapitation when showing symptoms of the disease, usually between 3 and 5 weeks of age, and their adrenal glands were removed as rapidly as possible. One apparently healthy control bird was killed at the same time as each diseased bird and its adrenals treated similarly. Liver, kidney and heart were taken from both affected and control animals, fixed in formol-saline and frozen sections stained with Sudan IV to confirm histologically the presence or absence of FLKS. One adrenal gland from every bird was fixed in Bouin and stained with HE. In order to distinguish the interrenal (cortical) and chromaffin (medullary) cells more distinctly, the other gland from some birds was fixed in Allen Bouin 15 and stained by a picro-Mallory trichrome method or fixed in Regaud’s dichromate fixative and stained by Schmorl’s ferric-ferricyanide method counterstained with neutral red. The methods used to differentiate noradrenaline and adrenaline-containing cells and also to distinguish medulla from cortex, were the potassium iodate method of Hillarp and H&felt (1955), Giemsa stain following the acetate-buffered, formol-dichromate fixation procedure suggested by Coupland (1961) and the acetate-buffered, eosin yellow-aniline blue stain after buffered-dichromate fixation described by Wood (1963). Catecholamines were specifically identified by a formaldehyde-condensation fluorescent method based on Pearse’s (1972) modification of the technique of Falck and Owman; small pieces of gland about 2 mm3. were rapidly frozen in an ethylene glycol-liquid nitrogen mixture, freeze-dried at -40 “C for 4 to 5 h. and then condensed over preheated paraformaldehyde for 3 h. at 80 “C. It was not necessary to equilibrate the paraformaldehyde in the humidity prevailing in this laboratory.
540
P. A. L. WIGHT
Paraffin sections were mounted in liquid paraffin and examined by WV. blue light (mean excitation 410 nm; K530 barrier filters). Ascorbic acid was detected by a silver nitrate technique based on the histochemical method of Bourne (Pearse, 1968). For electron microscopy animals were anaesthetized with pentobarbitone sodium and the adrenal glands perfused by the aortic arch with an aqueous pre-rinse containing 5 per cent. dextran and 0.9 per cent. sodium chloride, followed by 500 ml of 2.5 per cent. glutaraldehyde in 0.1 M Millonig’s phosphate buffer (pH 7.4 and 360 m-osmol.). The glands were then chopped into 1 mm. cubes and fixation continued by immersion for 1 h. in the same fixative. After washing in 0.2 M Millonig’s buffer and post-fixation in 0.1 M Millonig’s phosphate-buffered 1 per cent. osmium tetroxide, the tissues were dehydrated in graded alcohols and embedded in Araldite. Blocks from a few glands were also fixed in Dalton’s fixative made up with a modified buffer (3 per cent. potassium dichromate, 2.6 per cent. sodium chloride). RESULTS
Light Microscopy Examination of HE. sections of the adrenal glands from 25 birds which had clinical signs of advanced FLKS and characteristic lipid deposits in liver, kidney and heart, revealed loss of basophilia of the chromaffin cells in 18. Adrenals from the 25 control chickens were normal. The picro-Mallory tri-
chrome stained the normal interrenal cells red and the chromaffin cells bluepurple and again the colouration of the latter was lost, all the glandular tissue
Fig.
1. Adrenal gland from normal control cholamines has resulted in distinct Schmorl’s method after dichromate Fig. 2. Adrenal from a chicken affected by Schmorl’s method after dichromate
chicken. Reduction greenish-blue staining fixation. x 33. FLKS shows almost fixation. x 33.
of ferric-ferricyanide by the cateof the columns of chromaffin cells. complete
absence
of reduction
sites.
ADRENALS
IN FATTY
LIVER
AND
KIDNEY
541
SYNDROME
being stained red. Medullary amines have strong reducing properties which, in normal adrenal glands, reduce the ferric-ferricyanide in Schmorl’s method so that the chromaffin cells are coloured dark greenish-blue. In FLKS this reducing capacity appeared to be absent so that very few greenish-blue stained cells were seen (Figs 1 and 2). The methods of Hillarp and Hskfelt (1955)) Coupland (1961) and Wood (1963) confirmed the absence of typical medullary cell staining reactions and they also showed that this abnormality affected both noradrenaline and adrenaline-containing cells (Figs 3 and 4). When the formaldehyde-condensation preparations of control adrenal glands were examined by U.V. light, the interrenal cells appeared a dull green while chromaffin cells fluoresced light or golden-yellow. In FLKS very few yellow fluorescing cells were present
Fig. 3. Noradrenalineand adrenaline-containing Wood’s method. x 324. Fig. 4. Relatively few amine-containing chromaffin Wood’s method. x 324.
chromaffin cells remain
cells in the adrenal in the gland
of a control
of a chicken
with
chicken. FLKS.
(Figs 5 and 6). However, it was usually possible to discern a faintly silveryyellow fluorescent background of cells in similar numbers and occupying a similar area to the chromaffin cells of the normal adrenals. This suggested that the loss of basophilia in HE. stained sections was not due to destruction of the chromaffin cells, but to a depletion of their catecholamine content. Silver granules were present in both cortical and medullary tissue after applying Bourne’s technique. The reactions, supposedly indicative of the presence of ascorbic acid, appeared quantitatively similar in diseased and control birds.
542
P. A. L. WIGHT
of columns of chromaffin Fig. 5. Strong fluorescence condensation. x 90. Fig. 6. Very few chromaffin cells fluoresce strongly escence indicates the position of the columns
Fig. 7. Chromaffin adrenaline
cells in a control adrenal gland granules and strongly electron-dense
cells in a control in FLKS although of cells. Formaldehyde
adrenal
gland.
Formaldehyde
a background of faint fluorcondensation. x 90.
.- .____ - ._.._.-_ -. showing cells with moderately electron-densr noradrenaline granules. x 4480.
ADRENALS
Fig. 8. Chromaffin
Fig. 9. Chromaffin granules.
IN FATTY
cells in FLKS;
cell in a control x 16 800.
adrenal
LIVER
there
AND
KIDNEY
is severe depletion
containing
electron-dense,
SYNDROME
of amine
543
granules.
spherical
x
4480.
or oval noradrenaline
544
P. A.
L.
WIGHT
Fig.
10. Chromaffin cell in an adrenal from a case of FLKS. cytoplasm an empty appearance but mitochondria present. x 8260.
Fig.
11. Chromaffin
cell in ELKS. f xrrL- ---
Amine granules are absent giving are normal. Some lipid droplets
the are
ADRENALS
IN FATTY
LIVER
AND
KIDNEY
SYNDROME
545
Electron Microscopy The normal chromaffin cells of the fowl can be divided into 2 populations on the basis of their specific cytoplasmic granules (Fig. 7). Some contain numerous oval or spherical, membrane-limited granules of moderate electron density and these are considered to be adrenaline-storing granules. In other cells the granules are similar, but are highly electron-dense and these are noradrenalinestoring granules (Fig. 9). Internal sub-unit structure could sometimes be identified in both types of granule. In FLKS the number of granules of both types was much less than in the control birds (Figs 8 and 10). Remaining granules in affected cells were also reduced in size and of irregular, angular shape and the perigranular space beneath the limiting membrane appeared correspondingly wide (Fig. 11). In many cases of the disease, nearly all the chromaffin cells seen were affected and most cells showed an almost complete loss of granules (Figs 8 and 10). Sometimes a few dense-cored granules of very small diameter were present. On the other hand, the nucleus and cytoplasmic organelles were not morphologically abnormal. The cytoplasmic matrix had an empty appearance resulting from the loss of amine-storing granules and large vacuoles containing pale, granularfibrillary material, sometimes referred to as colloid vacuoles in the normal cell, were often unduly numerous (Fig. 11). Mitochondria of normal structure were present (Figs 10 and 11). A few lipid droplets were seen in some medullary cells (Fig. lo), but lipid deposition was not a characteristic feature of chromaffin cell involvement. These changes were present after both glutaraldehyde and Dalton’s fixation. No consistent evidence of morphological change affecting the interrenal cells was found by electron-microscopic examination. Some lipid droplets were of slightly denser electron-translucency in the disease than in the controls, but, by subjective examination, quantitative alterations in the lipid content of the cells was not detected. DISCUSSION
Morphological changes in the adrenal glands in FLKS were reported by Wight and Siller (1975) as a loss of tinctorial distinction between the medulla and cortex. The present study demonstrates both histochemically and ultrastructurally that this phenomenon is due to loss of catecholamines from the chromaffin cells; norepinephrine and epinephrine were both depleted. The ultrastructural study suggests that the changes were an extension of the normal physiological discharge of adrenaline and noradrenaline granules (De Robertis and Vaz Ferreira, 1957). This is indicated by the absence of alteration in any other medullary organelle and in this respect the adrenal lesion is similar to that of other organs in FLKS in which degenerative or inflammatory reactions were generally absent (Siller and Wight, 1975; Wight, 1975). Unlike many organs in the syndrome, lipid was rarely present in the medullary cells and then only in small amounts, so its direct presence does not appear to have caused the loss of amines. Viral and bacterial infections, toxic products of microbes and parasites and
546
P. A.
L. WIGHT
chemical toxins may produce intense general-adaptation-syndrome phenomena in the adrenals of mammals, including the discharge of chromaffin granules (Selye, 1950). Reye’s syndrome is an encephalopathy and visceral fatty degeneration of children with many features in common with FLKS, but the endocrine glands are not abnormal (Reye, Morgan and Baral, 1963). The chromaffinity of the adrenal medulla is considerably decreased in scorbutic guinea-pigs (Selye, 1950). However, the addition of vitamin C to an FLKSinducing diet did not significantly affect the incidence of the disease (Whitehead, Blair, Bannister, Evans and Jones, 1975) nor was a histochemicallydetectable decrease in ascorbic acid found in the glands in the present study. Perhaps the latter finding should be interpreted cautiously because relatively mild stress may cause a fall in biochemically-determined adrenal ascorbic acid levels (Freeman, 1967). On the other hand, the histochemical silver methods are of doubtful specificity (Pearse, 1968) ; in particular, the phosphatide content of mitochondria may reduce acid silver nitrate. Electron microscopy showed that the mitochondria of the adrenal medullary cells were wellpreserved in FLKS. It has been suggested that the signs of FLKS may be due to adrenal malfunction (Payne, Gilchrist, Pearson and Hemsley, 1974), but in the present investigation adrenal involvement was not observed in every case. These apparently-normal adrenals may have recovered their chromaffinity, but it is also possible that they were never morphologically affected and that the adrenal abnormality, even although it may be associated with symptoms in some cases of FLKS, is a secondary response to the original metabolic lesion. Adrenal catecholamines have a role in regulating the metabolism of lipid, carbohydrate and protein in mammals. As regards lipid metabolism, the experimental injection of these amines into fowls induces neither the rise in blood free fatty acid levels nor the lipid accumulation in certain organs which results from a similar procedure in mammals (Wells and Wight, 1971). Lipolysis may be stimulated to a small extent when avian adipose tissue is incubated in vitro with high concentrations of adrenaline and noradrenaline (Goodridge and Ball, 1965; Langslow and Hales, 1969). Although it cannot, therefore, be concluded that these amines have no effect on lipid metabolism in birds (HimmsHagen, 1967), our present knowledge provides no explanation for chromaffin depletion in relation to the tissue triglyceride deposition and elevated plasma free fatty acid concentrations (Evans, Bannister and Whitehead, 1975) which occur in FLKS. However, the known effects of adrenaline and noradrenaline on carbohydrate metabolism in the fowl (Wells and Wight, 1971) do provide a possible explanation for the depletion. These amines accelerate the discharge of liver induce hyperglycaemia and, perhaps acting by the adenosine glycogen, nucleotide cycle, permit accelerated gluconeogenesis. In mammals, adrenaline also acts upon the islets of Langerhans to inhibit the production of insulin and stimulates the secretion of ACTH which in turn stimulates the secretion of adrenal glucocorticosteroids, the latter inducing gluconeogenesis. Low blood sugar, probably acting through receptor centres in the hypothalamus and subsequent neural pathways, stimulates the secretion of these medullary amines.
ADRENALS
IN FATTY
LIVER
AND
KIDNEY
SYNDROME
547
Hypoglycaemia and decreased hepatic gluconeogenesis are important occurrences in FLKS (B annister, Evans and Whitehead, 1975) and it therefore seems possible that attempts to maintain homeostasis by stimulating gluconeogenesis and rectifying this low blood-sugar level may result in excessive secretion and ultimate depletion of adrenal catecholamine stores. This possibility is born out by ultrastructural investigations (Fujita, Kano, Kunishima and Kido, 1959; D’Anzi, 1969) of the fowl and mammalian adrenal in insulin-induced hypoglycaemia in which depletion of amines morphologically similar to that in FLKS was observed.
There
is much
circumstantial
evidence
that stress is involved
in FLKS
(Whitehead and Blair, 1974). Certain experimental stresses have been shown to cause a biochemically-determined decrease in the epinephrine and norepinephrine concentrations of the chicken’s adrenal (Zachariasen and Newcomer, 1974) and it is possible that the morphological depletion demonstrated in the present study may be associated with the adverse effects of environmental stress in FLKS. SUMMARY
Decreased chromaffinity occurred in the adrenal medulla of 60 per cent. of cases of the fatty liver and kidney syndrome which were examined. This abnormality was shown by histochemical and ultrastructural examinations to be due to severe depletion of catecholamines. The finding is discussed in relation to the symptoms, histopathology and biochemical changes which characterize the disease. REFERENCES
Bannister, D. W., Evans, A. J., and Whitehead, C. C. (1975). Fatty liver and kidney syndrome in chicks. Evidence for a lesion in carbohydrate metabolism. Research in Veterinary Science, 18, 149-156. Coupland, R. E. (1961). The distribution of cholinesteraseand other enzymes in the adrenal glands of the ox and man and in a human phaeochromocytoma. Cytolo@ of the .Nervous System, pp. 28-32, Anatomical Society of Great Britain and Ireland Symposium. D’Anzi, F. A. (1969). Morphological and biochemical observations on the catecholamine-storing vesiclesof rat adreno-meduilary cells during insulin-induced hypoglycaemia. American Journal of Anatomy, 125, 381-397. De Robertis, E., and Vaz Ferreira, A. (1957). Electronmicroscopic study of the excretion of catechol-containing droplets in the adrenal medulla. Experimental Cell Research, 12, 568-574. Evans, A. J., Bannister, D. W., and Whitehead, C. C. (1975). Fatty liver and kidney syndrome in chicks. Some aspects of lipid metabolism. Research in Veterinary Science, 18, 26-31. Freeman, B. M. (1967). Effects of stresson the ascorbic acid content of the adrenal gland of Gallus domesticus. Comjarative Biochemistry and Physiology, 23, 303-305. Fujita, H., Kano, M., Kunishima, I., and Kido, T. (1959). Electron microscopic observations on the adrenal medulla of the chick after injection of insulin. Archivum histologicumjaponicum, 18, 411-419. Goodridge, A. G., and Ball, E. G. (1965). Studies on the metabolism of adipose tissue XVIII. In vitro effects of insulin, epinephrine and glucagon on lipolysis and glycolysis in pigeon adipose tissue. Comparative Biochemistry and Physiology, 6, 367-381.
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Hemsley, L. A. (1965). The fatty liver and kidney syndrome of young chickens. Veterinary Record, 77, 124-126. Hillarp, N. A., and Htikfelt, B. (1955). Histochemical demonstration of noradrenaline and adrenaline in adrenal medulla. Journal of Histochemistry and Cytochemistry, 3, l-5. Himms-Hagen, J. (1967). Sympathetic regulation of metabolism. Pharmacological Reviews, 19, 367-46 1. Langslow, D. R., and Hales, C. N. (1969). Lipolysis in chicken adipose tissue i?z vitro. Journal of Endocrinology, 43, 285-294. hos Kyllinger, Marthedal, H. E., and Vellinge, G. (1958). L ever-og nyrelidelse Nordisk Veterinarmb’tet, Helsinki, 8, 250-255. Payne, C. G., Gilchrist, P., Pearson, J. A., and Hemsley, L. A. (1974). Involvement of biotin in the fatty liver and kidney syndrome of broilers. British Poultry Science,
15, 489-498. Pearse, A. G. E. (1968; 1972). Histochemistry: Theoretical and Applied, 1 and 2, 2nd edit. Churchill-Livingstone, Edinburgh and London. and fatty deReye, R. D. K., Morgan, G., and Baral, J. (1963). Encephalopathy generation of the viscera, a disease entity in childhood. Lance& 2, 749-752. Selye, H. (1950). Stress,pp. 286-349, Acta, Montreal. Siller, W. G., and Wight, P. A. L. (1975). A n ultrastructural study of the fatty liver and kidney syndrome in the fowl. Research in Veterinary Science (in press). Wells, J. W., and Wight, P. A. L. (1971). The adrenal glands. In Physiology and Biochemistry of the Domestic Fowl, 1. D. J. Bell and B. M. Freeman, Eds. pp. 489-520, Academic Press, London. Whitehead, C. C., and Blair, R. (1974). Fatty liver and kidney syndrome in chicks. The involvement of dietary energy-protein ratio and house temperature. Research in Veterinary Science, 17, 86-90. Whitehead, C. C., Bannister, D. W., Blair, R., and Evans, A. J. (1974). Fatty liver and kidney syndrome in chicks. Effects on liver and kidney of diets causing the syndrome. Research in Veterinary Science, 17, 222-225. Whitehead, C. C., Blair, R. B., Bannister, D. W., Evans, A. J., and Jones, R. IM. (1975). The involvement of biotin in preventing the fatty liver and kidney syndrome in broilers. Research in Veterinary Science (in press). Wight, P. A. L. (1975). Th e neuropathology of the fatty liver and kidney syndrome of chicks. Neuropathology and Applied fleurobiolopy, 1 (in press). Wight, P. A. L., and Siller, W. G. (1975). The histopathology of the fatty liver and kidney syndrome in chicks. Research in Veterinary Science (in press). Wood, J. G. (1963). Identification of and observations on epinephrine and norepinephrine containing cells in the adrenal medulla. American Journal of Anatomy,
112, 285-303. Zachariasen, R. D., and Newcomer, W. S. (1974). Phenyl ethanolamine-N-methyltransferase activity in the avian adrenal following immobilization or adrenocorticotropin. General and Comparative Endocrinology, 23, 193-198. [Received for publication,
February 26th, 19751
