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Clinical signs and morphologic abnormalities in Gunn rats treated with sulfadimethoxine
1070
December 1969 T h e J o u r n a l o[ P E D I A T R I C S
Clinical signs and morphologic abnormalities in Gunn rats treated Mth sulfadimethoxine Gunn rats treated with sul[adimethoxine between 18 hours alter birth and 33 days o[ age developed acute neurologic signs and increased yellow staining o[ their brains. In animals treated between the ages o[ 4 and 22 days, the neurologic abnormalities were proportional to the serum bilirubin levels. Animals treated from 18 to 48 hours alter birth develop more severe signs than older animals with similar bilirubin levels, and those older than 22 days develop mild but definite neurologic signs. This study confirms the importance o[ the albumin binding o[ biIirubin as a protective [actor in hyperbilirubinemia. We conclude that it is superfluous to postulate a blood-brain barrier to bilirubin to explain kernicterus, and that anoxia is not essential [or its development. BiIirubin has access to the central nervous system beyond the newborn period. The reasons for the extreme toxicity of sul[onamides to Gunn rats in the first 2 days o[ life and the relatively mild effects in animals treated at 28 and 33 days are discussed. It is suggested that the characteristic pattern of yellow discoloration o[ the brain in kernicterus could be largely accounted [or by differences in regional cerebral blood flow.
Henry S. Schutta, M.D., and Lois Johnson, M.D. PHILADELPttIA~
PA.
I W I S now accepted that bilirubin is toxic to the central nervous system, ~-a but conclusions derived from various clinical observations and some experimental models regarding the mechanism by which bilirubin encephalopathy (kernicterus) is produced are not uniform. G u n n rats, which are a strain of Wistar rats, 4 have nonhemolytic jaundice as a result of fi-glucuronyl transFrom the Department o[ Neurology and Pediatrics, University o[ Pennsylvania Medical School and the Pennsylvania Hospital. Supported by National Institutes o[ Health Grant No. NB-06899-03 and the Pennsylvania Hospital General Research Support Grant No. 5S01-FR05590-02.
Vol. 75, No. 6, part 1, pp. 1070-1079
ferase deficiency, s a condition which corresponds to the Crigler-Najjar 6 syndrome in humans. F r o m 3 days of life Gunn rats begin to show abnormalities of the central nervous system, which consist of neuronal intraeytoplasmic membranous inclusions and mitochondrial changes. T h e Purkinje cells of the cerebellum are particularly affected and some become necrotic. T h e membranous inclusions can be recognized in the phase contrast microscope as osmiophilic intracytoplasmic granules. 7 Sulfadimethoxine, a long-acting sulfonamide, can be conveniently used to displace bilirubin from its albumin binding sites in
Volume 75 Number 6, part 1
SuI[adimethoxine
Gunn rats. Young Gunn rats injected with sulfadimethoxine develop acute neurologic signs; the brains of such animals have a distinct yellow discoloration, s We have studied the symptomatology and morphologic changes in Gunn rats following administration of sulfadimethoxine in relation to age and serum bilirubin levels. The data are discussed with reference to differing opinions regarding the mode of entry of bilirubin into the central nervous system, the pathogenesis of bilirubin encephalopathy, and the reasons for the preferential accumulation of bilirubin in certain areas of the brain. MATERIALS
AND
METHODS
Sixty-eight homozygous Gunn rats aged from 8 hours to 33 days were studied. Thirty-five animals were given 100 to 150 rag. per kilogram of body weight of sulfadimethoxine subcutaneously and were killed 18 to 24 hours after treatment. A jaundiced littermate which had received a dummy injection and a heterozygous littermate which had been given the same dose of sulfadimethoxine were killed at the same time. The serum bilirubin was estimated by tile diazo method ~ at the time of death in both tile treated rat and the untreated jaundiced littermate. Since the variation in serum bilirubin levels among jaundiced littermates is usually insignificant, providing the colony is healthy, the animals, which are very small and delicate, were bled only once (at the time of death). Tile serum bilirubin concentration measured in the littermate which had received a dummy injection was taken as the approximate pretreatment serum bilirubin of the animal given sulfadimethoxine. The symptomato!ogy before and after the injection of sulfadimethoxine was recorded as follows : 1+, minimal ataxia or minimal decrease in tone; 2 ~-, definite but mild ataxia or mild hypotonia and failure to gain weight; 3+, moderate ataxia or moderate hypotonia with lethargy and failure to gain weight;
1071
4+, severe ataxia or lethargy,, severe hypotonia and weight loss; 5+, severe ataxia and lethargy with alternating erratic hyperactivity; 6+, opisthotonus, convulsions, and death. Thirty-five animals were anesthetized with ether or chloral hydrate and killed by perfusion with 10 per cent formaldehyde in a 0.44M phosphate buffer. The brains and spinal cords were removed. The degree of yellow discoloration of the central nervous system was graded as mild (1+), moderate (2+), or severe (3+). Thirty-three animals from 15 experiments were perfused either with 4 per cent glutaraldehyde in 0.44M phosphate buffer or with Karnovsky's fixative ~~ diluted 1:4 with 0.2M sodium cacodylate buffer. In contrast to glutaraldehyde, this fixative does not obscure the yellow staining of the brain and preserves tissue better for morphologic studies than does formaldehyde. Small blocks of tissue from the vermis of tile cerebellum, the cerebral cortex, and hippocampus, and also from the colliculi, spinal cord gray matter, and the brainstem, were washed in buffer, placed in buffered 1 per cent osmium for one hour, and embedded in araldite. 11 Sections 1 /, thick were cut on a Porter-Blum microtome with glass knives and examined unstained with a phase contrast microscope, or with a conventional microscope after staining with 1 per cent crystal violet in 1 per cent borax. 1~ To estimate the proportion of damaged cells, 400 to 600 Purkinje cells were counted from 12 treated animals and 10 untreated littermates. In addition we have counted the Purkinje cells in the vermis of 34 untreated jaundiced animals between the ages of 11 and 33 days from previous studies. OBSERVATIONS
Serum bilirubin levels. The jaundice in the Gunn rat is lifelong, but in terms of molar equivalents it approaches that of the serum albumin concentration only during tile first 2 ~ weeks of life (Figs. 1 and 2). With rare exceptions damage to the brain probably occurs only during this period, r, s The serum bilirubin level rises after birth
10 7 2
Schutta and Johnson
The Journal of Pediatrics December 1969
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Volume 75 Number 6, part 1
to r e a c h a p e a k of 10 to 13 rag. p e r cent at 14 to 16 days, after which it declines, reaching the m e a n a d u l t level of 4 to 6 rag. per cent by 30 days. G u n n rats born of h o m o zygous m o t h e r s ( j j / j j matings) h a v e higher serum bilirubin levels at birth (1 to 2 rag. per cent) than those born of non j a u n d i c e d (Jj) m o t h e r s (0.5 rag. p e r cent). F r e q u e n t l y such animals die 4 to 5 days after birth (Fig.
1). T h e animals treated with sulfadimethoxine, regardless of age or initial serum bilirubin level, h a d serum bilirubin concentrations 18 hours after injection of 1.5 mg. p e r cent or less, except in 2 instances. These exceptions were in the 2 oldest animals, treated at age 33 days, which h a d bilirubin levels of 2.5 a n d 2.0 mg., respectively, 18 hours after the administration of sulfadimethoxine (Table I). At autopsy the fat and small intestines of the s u l f o n a m i d e - t r e a t e d rats were b r i g h t yellow, strikingly different from those of their j a u n d i c e d littermate controls, whose bilirubin h a d r e m a i n e d p r i m a r i l y in the bloodstream. S y m p t o m a t o l o g y . I n healthy litters, 70 to 75 p e r cent of u n t r e a t e d G u n n rats develop signs of central nervous system disease during the first 18 days of life. A b n o r m a l neurologic signs usually a p p e a r between 8 and 12 days after birth, when m o t o r function is sufficiently developed to allow recognition of ataxia, generally the first sign of central nervous system dysfunction. I n animals younger t h a n 8 days, hypotonia is the first indication of bilirubin e n c e p h a l o p a t h y , followed by lethargy, feeding difficulty, a n d weight loss. I n severely affected o l d e r animals a t a x i a is followed by hypotonia, after which l e t h a r g y a n d feeding difficulties occur. Finally, the animals display erratic hyperactivity, increasing lethargy, opisthotonus, convulsions, a n d death. I n an optimally h e a l t h y colony none of the homozygous animals die, b u t 40 to 50 per cent have various degrees of a t a x i a in a d u l t life. Thus, in 20 to 30 p e r cent the ataxia is transient a n d confined to the first 3 or 4 weeks of life (see also J o h n s o n a n d associatesS).
Sulfadimethoxine
10 73
T a b l e I. A g e of animals at the time of injection, a n d severity of brain d a m a g e (0 to 6+) before a n d after injection. All animals were killed 18 to 24 hours after t r e a t m e n t (Rx) with 150 rag. p e r k i l o g r a m of s u l f a d i m e t h o x i n e by subcutaneous injection. L i t t e r m a t e s given d u m m y injections served as controls. A n i m a l s m a r k e d G A were perfused with g l u t a r a l d e h y d e , discoloration of the b r a i n due to bilirubin could not be assessed in them. N o t e that severity of sulfonamideinduced d a m a g e was directly r e l a t e d to preinjection serum bilirubin level Abnormal CNS Age in signs days at _ _ ___ in)ec- Be[ore Alter lion Rx I Rx 18 hr. 0 4+ 1 1 1 1
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~1.0 ~1.0 ~1.0 ~1.0
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1 0 74
Schutta and Johnson
The Journal o[ Pediatrics December 1969
Fig. 3. Cerebellar cortex from a jaundiced rat, aged 16 days. The Purkinje cells marked P~ and P, are normal. Those marked P,, P,, and P~ show osmiophilic inclusions. There are no abnormalities in the molecular or granule cell layers (Original magnification •
In this experiment, a striking increase in brain damage followed treatment with sulfadimethoxine. Animals treated at age 18 to 48 hours, when serum bilirubin levels of littermates ranged between 5.0 and 6.5 rag. per cent at death 18 hours later, developed more severe signs of damage (3+ to 5+) than older treated animals with serum bilirubin levels in the same range (2+ to 3+). Otherwise the severity of damage related closely to the pretreatment serum bilirubin concentration (Table I ) . Histologic observations. Until 10 days after birth the Purkinje cells in the rat are immature and at times difficult to identify, but thereafter they can be recognized with ease, and accurate counts of normal and abnormal Purkinje cells can be made (Fig. 3). In untreated animals, between days 11 and 13 the number of abnormal Purkinje cells varied from 54 to 71 per cent (10 animals) ; at 14 to 16 days the proportion of abnormal Purkinje cells varied from 52 to 82 per cent (19 animals); thereafter the counts varied between 60 and 80 per cent up to day 22 (11 animals). From day 22 to 33 th~ counts varied considerably, from between 27 and
75 per cent (10 animals). The explanation for the variability is the resorption of many necrotic cells, which decreases the number of abnormal cells. T h a t there is loss of Purkinje cells and other neurons in the cerebellar folia is evident from the considerable atrophy which is present in many animals, particularly those which have ataxia. Asymptomatic animals examined between 11 and 21 days tended to have less than 70 per cent abnormal Purkinje cells, whereas those with ataxia had 70 to 82 per cent. There was thus a considerable variation in the abnormal cell counts from different litters, but the counts among littermates varied not more than 5 per cent, provided that the symptomatology prior to death was similar. Typical abnormalities were found in all animals, including those which had no clinical signs of central nervous system damage at any time. In all instances animals treated with sulfadimethoxine had more abnormal Purkinje cells in the vermis than untreated littermates with identical clinical status before treatment. One animal treated at 21 days had 1+ ataxia before and 3+ ataxia 24 hours after treatment. When the cell count of that ani-
Volume 75 Number 6, part 1
SuI[adimethoxine
%
of
abnormal
Purkinje
1075
cells
I
Jaund i c e d "
+
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,o % 80
70
60
50
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11
II
12 age
13 -
-
16
21
22
28
33
33
days
Fig. 4. Histograms showing the proportions of abnormal cells in treated and untreated animals. The animal treated at day 21 was compared with an untreated littermate which had 2 to 3+ ataxia; this explains why the counts are similar.
m a l was c o m p a r e d with that of an uninjected l i t t e r m a t e with 2-3+ ataxia, the ceil counts were almost identical. H e r e the discrepancy in the symptomatology before t r e a t m e n t explains the similar p r o p o r t i o n of a b n o r m a l cells in both animals (Fig. 4 ) . I n rats which were treated at an age when Purkinje cell counts were impossible to accomplish accurately, the degree of d a m a g e was j u d g e d by the presence or absence of abn o r m a l P u r k i n j e cells in the plastic-embedd e d sections. I n the past a n d in this study we were u n a b l e to see any abnormalities in animals y o u n g e r t h a n 64 hours. T r e a t e d rats which were killed before t h a t age h a d m a n y a b n o r m a l Purkinje cells. I n the animals given sulfadimethoxine 18 to 24 hours after birth, necrotic Purkinje cells were also present. Necrotic cells do not occur in u n t r e a t e d rats until 10 to 11 days. I n treated animals which d i d not convulse, the n a t u r e of the morphologic changes as observed in the light microscope were identical to those f o u n d in u n t r e a t e d animals; however, the a b n o r m a l i ties were m o r e p r o n o u n c e d a n d necrosis of
Purkinje cells was more common. I n the cerebral cortex of u n t r e a t e d rats affected neurons occur very rarely, whereas in sulfad i m e t h o x i n e - t r e a t e d G u n n rats cortical neurons with intracytoplasmic inclusions were relatively common. I n the colliculi a n d tile cerebellar nuclei, neurons with osmiophilic cytoplasmic inclusions were also more comm o n in the treated animals. Evidence of acute cell necrosis was not present in these areas unless the animals were opisthotonic or h a d convulsed, when petechial h e m o r rhages were also present throughout the brains and m a n y areas showed swelling of gIial cells a n d neuronal shrinkage or swelling. W e believe that these changes are more likely the expression of terminal respiratory failure than acute bilirubin intoxication. Brain discoloration. I n u n t r e a t e d j a u n diced animals m i l d diffuse staining of the brain was obvious 4 to 8 hours after b i r t h ; staining r e m a i n e d diffuse u p to age 36 hours. Thereafter, the basal ganglia, the cerebellar cortex, the cerebellar nuclei, the colliculi, the eighth nerve nuclei, a n d also the gray
10 7 6
Schutta and Johnson
matter of the spinal cord were more deeply stained than the rest of the brain. In the cerebellum the vermis was distinctly more stained than the hemispheres. In cerebellar slices the yellow discoloration was visible only in the Purkinje cell and granule cell layers, the molecular and external granular layers appearing unstained. From 16 days on the intensity of staining progressively diminished; by 21 days no staining was apparent in untreated animals. It must be stressed that mild degrees of staining are very difficult to appreciate in fresh brains or those preserved by immersion fixation and may be overlooked unless the remaining blood, which obscures the yellow color of bilirubin, is washed out. In the animals treated with sulfadimethoxine from 18 to 24 hours after birth, diffuse yellow staining of the brains was more apparent than in untreated rats. Beyond that age all animals which were given sulfadimethoxine showed distinctly more marked localized yellow staining of the brain as compared with the untreated jaundiced rats. The increase in yellow staining was most marked in animals which were treated 12 to 16 days after birth. In animals treated as late as 21 to 33 days after birth, moderate staining of the vermis, the colliculi, and the cerebellar nuclei was noted (Table I ) . DISCUSSION
The deleterious effects of sulfonamides in jaundiced babies have been well documented? 3 This syndrome has been reproduced in Gunn rats, which upon administration of certain sulfonamides become sick and have increased bilirubin staining of their brains.14, 15 In the present study all animals treated with sulfadimethoxine developed acute signs referable to the central nervous system and had more yellow staining of the brains than untreated littermates. Sulfadimethoxine efficiently displaced bilirubin from its albumin binding sites, thereby allowing the unbound bilirubin to diffuse freely into the tissues, including the brain. T h e less marked reduction of serum bilirubin by sulfadimethoxine in the 33-day-old"animals is probably the result of the higher serum
The ]ournal o[ Pediatrics December 1969
albumin concentration of older animals and the greater capacity of older rats to excrete sulfonamides. The capacity of albumin to bind bilirubin has been known for some time, 16 but its importance in protecting the central nervous system from bilirubin toxicity was not suggested until 1954 by Lathe 17' is and subsequently by Brown and associates? 9 It was experimentally demonstrated in 1959 by Odell,20, ~1 whose observations have been repeatedly confirmed by others? 4' is, 22-24 The natural course of the encephalopathy in Gunn rats provides further confirmation of the importance of bilirubin binding to albumin. Central nervous system damage occurs during the first 2 to 2 ~ weeks of life when the bilirubin-albumin molar ratio is close to unity. T h e percentage of abnormal Purkinje cells in untreated animals was found to be at a maximum at day 16 (52 to 82 per cent), after which time there was a gradual decline. We believe that this decline is due to the death of severely injured cells and absence of further injury beyond that age in healthy animals. Additional neurologic damage can be induced by displacement of bilirubin from its binding sites. In Gunn rats which were treated with sulfadimethoxine between 4 and 22 days after birth, the severity of acute bilirubin encephalopathy was related to the serum bilirubin concentration present before treatment, suggesting a relationship between the amount of available bilirubin and central nervous system signs. Clinical signs of acute central nervous system intoxication occurred in animals treated up to 33 days after birth, at which time the animals are quite mature (21 days is the usual weaning time for laboratory rats). This suggests that unbound bilirubin has access to the central nervous system beyond the newborn period, which is in accord with previous observations: Johnson and associates s have shown that adult Gunn rats injected with bilirubin and sulfonamides develop ataxia; Diamond and Schmid 2~ demonstrated that 14C-labeled bilirubin enters the central nervous system in adult Gunn rats treated with sulfisoxazole. The extreme toxicity of sulfonamides to
Volume 75 Number 6, part 1
the jaundiced rat in the first two days of life and the relatively mild symptomatology in animals treated at 28 and 33 days deserve further comment. The total serum protein concentration of the rat on day 1 is approximately one quarter that at age 4 weeks. Furthermore, the excretory capacity of the newborn animal for drugs is low, so the levels of drugs in the body after a standard close are almost certainly higher in younger animals. These factors would bring about a more complete displacement of bilirubin from ali extracellular binding sites in the very young rats. Also, the volume of tissue other than brain capable of taking up bilirubin must be very much smaller in the immature rats than in older animals, which means that relatively more bilirubin is available to enter the brain in the young animals. p H homeostasis is also more precarious in very immature rats, probably resulting in an earlier onset of acidosis during the course of bilirubin intoxication. Acidosis is profound in kernicteric animals as they approach death (central nervous system gradings of 4+ to 6+). We believe that these factors account for the differences in the severity of symptomatotogy between the very young and somewhat older Gunn rats treated with sulfadimethoxine. It has been observed that serum bilirubin levels in premature babies may remain relatively low and yet severe bilirubin encephalopathy may develop. This is especially true in babies with respiratory distress. ~s, 26 The adult human brain does not stain with bilirubin even in the presence of severe jaundice, except in areas of necrosis. In some experimental models it is impossible to produce yellow staining of the brain by administering bilirubin intravenously in the absence of anoxia. Therefore, the occurrence of kernicterus has been explained by postulating a blood-brain barrier to bilirubin, which was thought to be either imperfectly developed in the young or damaged by anoxia. 27"32 However, recent studies of the blood-brain barrier to protein have failed to demonstrate differences between mature and immature brains, and the notion that the blood-brain barrier systems are more
Sut/adimethoxine
10 77
permeable in young brains is probably not correct. 3a A blood-brain barrier to free bilirubin (i.e., unbound to protein) has never been demonstrated. Our results suggest that bilirubin enters the central nervous system of quite mature animals after it has been displaced from its albumin binding sites. The low serum bilirubin levels in association with severe central nervous system disturbance can be explained by low serum albumin levels or the dissociation of bilirubin from available albumin binding sites by acidosis a n d / o r competing anions.S, 2o, a7 Even in the presence of relatively high serum bilirubin concentrations, unbound bilirubin can exist in appreciable quantities only in the presence of acidosis or competing anions such as free fatty acids, sulfonamides, or salicylates when there as an abundance of serum protein, as is tile case in the older healthy human baby and in tile mature Gunn rat. The fact that bilirubin discoloration in adult brains occurs only in the presence of pre-existing damage has little relevance to the problems of bilirubin encephalopathy. In necrotic areas the bloodbrain barrier to protein is destroyed and the bilirubin albumin complex has no more difficulty in entering the brain than the Evans blue-albumin complex or other markers used to demonstrate the state of the bloodbrain barrier to protein. '~~ These considerations and the results of the present study suggest that it is superfluous to postulate a blood-braln barrier for bilirubin to explain the incidence of kernicterus, and that the brain is protected from the entry of bilirubin by binding of bilirubin to protein. Petechial hemorrhages, neuronal swelling or shrinkage, and swelling of glial cells occurred only in animals which had convulsed or which were opisthotonic. Such animals were anoxic and acidotic, as judged by the strikingly dark appearance of their blood at the time of death. (In animals whose symptomatology did not exceed 3+, the blood always appeared bright red.) There can be no doubt that the cytotoxicity of bilirubin can be enhanced by anoxia, through its direct action on the neurons and also by
10 78
Schutta and Johnson
virtue of the c o n c o m i t a n t acidosis, since the binding of bilirubin to protein is less efficient at a lower p H ? s I t seems reasonable to assume that a neuron d a m a g e d by a n o x i a will be m o r e severely i n j u r e d a n d have less chance of recovering after exposure to bilirubin than an otherwise healthy cell. F u r thermore, acidosis in itself a p p e a r s to enhance the toxicity of bilirubin, as j u d g e d by its effects on cell cultures of mouse fibroblasts ~6 a n d organ cultures of cerebellumW T h e m a r k e d yellow discoloration of basal ganglia associated with h y p e r b i l i r u b i n e m i a gave rise to the term kernicterus? s I n our animals the cerebral cortex was v e r y definitely stained, p a r t i c u l a r l y a f t e r t r e a t m e n t with sulfadimethoxine, although the intensity of the discoloration was less t h a n in the basal ganglia, the cerebellar cortex, the cerebellar nuclei, the eighth nerve complex, o r the colliculi. I t has been suggested t h a t the differential staining occurs because of preexisting bilirubin d a m a g e s9-41 in n e u r o n a l groups which are exceedingly vulnerable to bilirubin. 22 However, we know of no convincing evidence that a d a m a g e d n e u r o n will take u p m o r e bilirubin t h a n u n d a m a g e d cells. T h e r e is no d o u b t that the Purkinje cells of the young G u n n rat are more easily d a m a g e d by bilirubin t h a n most o t h e r n e u r o n s / T h e colliculi a n d cerebellar nuclei, which show relatively little evidence of d a m age in the G u n n rat, are the most intensely discolored areas. Yellow areas in kernicterus are those which have high regional blood flow; a u t o r a d i o g r a p h s p r e p a r e d for the est i m a t i o n of regional blood flow 42, 43 show a striking resemblance to the p a t t e r n of discoloration f o u n d in kernicterus. I t is therefore possible that the intensity of bilirubin staining in the central nervous system is principally related to regional differences in cerebral blood flow, which m u s t p l a y an i m p o r t a n t role in d e t e r m i n i n g the q u a n t i t y of bilirubin deposited in various areas. T h e m e c h a n i s m of the high vulnerability of some neurons to bilirubin remains to be d e t e r m i n e d . However, since it is suspected t h a t regional blood flow parallels regional m e t a b o l i c rates, 42 it m a y well be tha~ vuln e r a b i l i t y to bilirubin d e p e n d s on the m e t -
The lournal o[ Pediatrics December 1969
abolic rate. T h i s also seems to be the case in e x p e r i m e n t a l n e o n a t a l asphyxia, in which the distribution of d a m a g e 44 closely resembles t h a t of bilirubin e n c e p h a l o p a t h y . This suggestion is f u r t h e r s u p p o r t e d by in vitro studies which show that bilirubin affects respiratory enzyme systems 2' as, 42, 4s and uncouples oxidative phosphorylation, s' 46 H o w ever, the possibility of inhibition by bilirubin of a specific enzyme system in vivo, u p o n which certain neurons m a y d e p e n d m o r e t h a n other cells, is w o r t h further study, because some deeply stained areas do not app e a r to be severely d a m a g e d . We wish to thank Marlene Catalde and Dennis Martell for help in preparing tissues for morphological study, Sandra Valenta, Anthony Mignano, and Edward Smith for help with bilirubin and serum protein estimations, and Muriel Schwartz and Sharon Oaks for secretarial assistance.
REFERENCES 1. Claireaux, A. E , Cole, P. G., and Lathe, G. H.: Icterus of the brain in the newborn, Lancet 2: 1226, 1953. 2. Day, R. L.: Inhibition of brain respiration in vitro by bilirubin reversal of inhibition by various means, Proc. Soc. Exper. Biol. & Med. 85: 261, 1954. 3. Kortsak, A. S., editor: Kernicterus, Toronto, 1961, University of Toronto Press. 4. Gunn, C. K.: Hereditary acholuric jaundice in a new mutant strain of rats, J. Hered. 29: 137, 1938. 5. Sehmid, R., Axelrod, J., Hammaker, L., and Swarm, R. L.: Congenital jaundice in rats due to a defect in bilirubln glucuronide formation, J. Clin. Invest. 37: 1123, 1958. 6. Crigler, J. F., and Najjar, V. A.: Congenital familial nonhemolytic jaundice with kernicterus, Pediatrics 10: 169, 1952. 7. Schutta, H. S., and Johnson, L.: Bilirubin eneephalopathy in the Gunn rat, J. Neuropath. & Exper. Neurol. 26: 377, 1967. 8. Johnson, L., Garcia, M. L., Figueroa, E., and Sarmiento, F.: Kernicterus in rats lacking glucuronyI transferase. II. Factors which alter bilirubin concentration and frequency of kernicterus, Am. J. Dis. Child. 101: 322, 1961. 9. Malloy, H. T., and Evelyn, K. A.: The determination of bilirubin with the photoelectric colorimeter, J. Biol. Chem. 119: 481, 1937. 10. Karnovsky, M. J.: A formaldehyde-glutaraldehyde fixative of high osmolarity for use in electron microscopy, J. Cell. Biol, 27: 270, 1965.
Volume 75 Number 6, part 1
11. Luft, J. H.: Improvement in epoxy resin embedding methods, J. Biochem. & Biophys. Cytol. 9: 409, 1961. 12. Guyer, S.: Personal communication, 1966. 13. Silverman, W. A.: The effects in premature infants of sulfisoxazole in the neonatal period, in Kortsak, A. S., editor: Kernicterus, Toronto, 1964, University of Toronto Press, p. 196. 14. Johnson, L., Sarmento, F., Blanc, W. A., and Day, R.: Kernicterus in rats with an inherited deficiency of glucuronyl transferase, Am. J. Dis. Child. 97: 591, 1959. 15. Blanc, W. A., and Johnson, L.: Studies in kernicterus. Relationship with sulfonamide intoxication. Report on kernicterus in rats with glucuronyl transferase deficiency and review of pathogenesis, J. Neuropath. & Exper. Neurol. 18" 165, 1959. 16. Martin, N. H.: Preparation and properties of serum and plasma proteins. XXI. Interactions with bilirubin, J. Am. Chem. Soc. 71: 1230, 1949. 17. Lathe, G. H.: The chemical pathology of bile pigments, Biochem. Soc. Symp. 12: 34, 1954. 18. Lathe, G. H.: Exchange transfusion as a means of removing bilirubin in haemolytic disease of the newborn, Brit. M. J. 1: 192, 1955. 19. Brown, A. K., Zuelzer, W. W., and Robinson, M. S.: Studies in hyperbilirubinemia, Am. J. Dis. Child. 93: 274, 1957. 20. Odell, G. B.: Studies in kernieterus. I. The protein binding of bilirubin, J. Clin. Invest. 38: 823, 1959. 21. Odell, G. B.: The dissociation of bilirubin from albumin and its clinical implications, J. PEDIAT. 55: 268, 1959. 22. Diamond, I., and Schmid, R.: Experimental bilirubin encephalopathy. The mode of entry of bilirubin 14C into the nervous system, J. Clin. Invest. 45: 678, 1966. 23. Menken, M., Barrett, P. V. D., Swarm, R. L., and Berlin, N. I.: Kernicterus development of an experimental model using bilirubin 14C, Arch. Neurol. 15: 68, 1966. 24. Ostrow, J. D., and Schmid, R.: The protein binding of 14C bilirubin in human and murine serum, J. Clin. Invest. 42: 1286, 1963. 25. Harris, R. C., Lucey, J. F., and MacLean, J. R.: Kernicterus in premature infants associated with low concentration of bilirubin in plasma, Pediatrics 21: 875, 1958. 26. Stern, L., and Denton, R. L.: Kernicterus in small premature infants, Pediatrics 35: 483, 1965. 27. Rozdilsky, B., and Olszewski, J.: Experimental study of the toxicity of bilirubin in newborn animals, J. Neuropath. & Exper. Neurol. 20: 193, 1957. 28. Chen, H., Lien, I. N., and Lu, T. C.: Kernicterus in newborn rabbits, Am. J. Path. 46: 331, 1965. 29. Swinyard, C. A., editor: Kernicterus and its importance in cerebral palsy, Springfield, Ill., 1961, Charles C Thomas, Publisher.
Sulfadimethoxine
10 79
30. Waters, W. J., and Bowen, W. R.: Bilirubin encephalopathy. Preliminary studies related to production, Pediatrics 15: 45, 1955. 31. Zuelzer, W. W.: Bilirubin and mental retardation, A. Res. Nerv. & M e n t . Dis. 3 9 : 1 8 3 1962. 32. Ernster, L., Herlin, L., and Zetterstrom, R.: Experimental studies on the pathogenesis of kernicterus, Pediatrics 20: 647, 1957. 33. Dobbing, J.: The development of the blood brain barrier, in Lajtha, A., editor: Brain barrier systems, Amsterdam, 1968, Elsevier Publishing Company, p. 417. 34. Bakay, L.. In Lajtha, A., editor: The blood brain barrier, Springfield, Ill., 1968, Charles C Thomas, Publisher, p. 78. 35. Odell, G. B., and Cohen, S.: The effect of pH on the protein binding of bilirubin, Am. J. Dis. Child. 100: 525, 1960. 36. Cowger, M. L., Igo, R. P., and Labbe, R. F.: The mechanism of bilirubin toxicity studied with purified respiratory enzyme and tissue culture systems, Biochemistry 4: 2763, 1965. 37. Silberberg, D., and Johnson, L.: Personal communication. 38. Schmorl, G.: Zur Kenntniss Des Ikterus Neonatorum Insbesondere Derbei Autretender Gehirn Veranderungen, Verhandl. deutsch. path. Gesellsch. 6: 109, 1903. 39. Lucey, J. F., Hibbard, E., Behrman, R. E., de Gallardo, E., and Windle, W. F.: Kernicterus in asphyxiated newborn rhesus monkeys, Exper. Neurol. 9: 43, 1964. 40. Day, R. L.: Kernicterus: Further observations on toxicity of bile pigments, Pediatrics 17: 925, 1956. 41. Haymaker, W., Margoles, C., Pentschew, A., Jacob, H., Lindenberg, R., Arroyo, L. S., Stochdorph, O., and Stowens, D.: Pathology of kernicterus and posticteric encephalopathy, in Swinyard, C. A., editor: Kernicterus and its importance in cerebral palsy, Springfield, Ill., 1961, Charles C Thomas, Publisher, p. 21. 42. Sokoloff, L.: Local cerebral circulation at rest and during altered cerebral activity induced by anaesthesia and visual stimulation, in Ketty, S. S., and Elkes, J., eds.: The regional chemistry, physiology and pharmacology of the nervous system, Oxford, 1961, Pergamon Press, p. 107. 43. Reivich, M., Isaachks, G., Evarets, E., and Ketty, S. S.: The effect of slow wave sleep and R E M sleep on regional cerebral blood flow in cats, .J Neurochem. 15: 301, 1968. 44. Rank, J. B., and Windle, W. F.: Brain damage in the monkey Macaca Mulatta by asphyxia neonatorum, Exper. Neurol. 1: 130, 1959. 45. Waters, W. J., and Bowen, W. R.: Bilirubin encephalopathy: Studies related to cellular respiration, Am. J. Dis. Child. 90: 603, 1955. 46. Zetterstrom, R., and Ernster, L.: Bilirubin, an uncoupler of oxidative phosphorylation in isolated mitochondria, Nature 178: 1335. 1956.
December 1969 T h e J o u r n a l o[ P E D I A T R I C S
Clinical signs and morphologic abnormalities in Gunn rats treated Mth sulfadimethoxine Gunn rats treated with sul[adimethoxine between 18 hours alter birth and 33 days o[ age developed acute neurologic signs and increased yellow staining o[ their brains. In animals treated between the ages o[ 4 and 22 days, the neurologic abnormalities were proportional to the serum bilirubin levels. Animals treated from 18 to 48 hours alter birth develop more severe signs than older animals with similar bilirubin levels, and those older than 22 days develop mild but definite neurologic signs. This study confirms the importance o[ the albumin binding o[ biIirubin as a protective [actor in hyperbilirubinemia. We conclude that it is superfluous to postulate a blood-brain barrier to bilirubin to explain kernicterus, and that anoxia is not essential [or its development. BiIirubin has access to the central nervous system beyond the newborn period. The reasons for the extreme toxicity of sul[onamides to Gunn rats in the first 2 days o[ life and the relatively mild effects in animals treated at 28 and 33 days are discussed. It is suggested that the characteristic pattern of yellow discoloration o[ the brain in kernicterus could be largely accounted [or by differences in regional cerebral blood flow.
Henry S. Schutta, M.D., and Lois Johnson, M.D. PHILADELPttIA~
PA.
I W I S now accepted that bilirubin is toxic to the central nervous system, ~-a but conclusions derived from various clinical observations and some experimental models regarding the mechanism by which bilirubin encephalopathy (kernicterus) is produced are not uniform. G u n n rats, which are a strain of Wistar rats, 4 have nonhemolytic jaundice as a result of fi-glucuronyl transFrom the Department o[ Neurology and Pediatrics, University o[ Pennsylvania Medical School and the Pennsylvania Hospital. Supported by National Institutes o[ Health Grant No. NB-06899-03 and the Pennsylvania Hospital General Research Support Grant No. 5S01-FR05590-02.
Vol. 75, No. 6, part 1, pp. 1070-1079
ferase deficiency, s a condition which corresponds to the Crigler-Najjar 6 syndrome in humans. F r o m 3 days of life Gunn rats begin to show abnormalities of the central nervous system, which consist of neuronal intraeytoplasmic membranous inclusions and mitochondrial changes. T h e Purkinje cells of the cerebellum are particularly affected and some become necrotic. T h e membranous inclusions can be recognized in the phase contrast microscope as osmiophilic intracytoplasmic granules. 7 Sulfadimethoxine, a long-acting sulfonamide, can be conveniently used to displace bilirubin from its albumin binding sites in
Volume 75 Number 6, part 1
SuI[adimethoxine
Gunn rats. Young Gunn rats injected with sulfadimethoxine develop acute neurologic signs; the brains of such animals have a distinct yellow discoloration, s We have studied the symptomatology and morphologic changes in Gunn rats following administration of sulfadimethoxine in relation to age and serum bilirubin levels. The data are discussed with reference to differing opinions regarding the mode of entry of bilirubin into the central nervous system, the pathogenesis of bilirubin encephalopathy, and the reasons for the preferential accumulation of bilirubin in certain areas of the brain. MATERIALS
AND
METHODS
Sixty-eight homozygous Gunn rats aged from 8 hours to 33 days were studied. Thirty-five animals were given 100 to 150 rag. per kilogram of body weight of sulfadimethoxine subcutaneously and were killed 18 to 24 hours after treatment. A jaundiced littermate which had received a dummy injection and a heterozygous littermate which had been given the same dose of sulfadimethoxine were killed at the same time. The serum bilirubin was estimated by tile diazo method ~ at the time of death in both tile treated rat and the untreated jaundiced littermate. Since the variation in serum bilirubin levels among jaundiced littermates is usually insignificant, providing the colony is healthy, the animals, which are very small and delicate, were bled only once (at the time of death). Tile serum bilirubin concentration measured in the littermate which had received a dummy injection was taken as the approximate pretreatment serum bilirubin of the animal given sulfadimethoxine. The symptomato!ogy before and after the injection of sulfadimethoxine was recorded as follows : 1+, minimal ataxia or minimal decrease in tone; 2 ~-, definite but mild ataxia or mild hypotonia and failure to gain weight; 3+, moderate ataxia or moderate hypotonia with lethargy and failure to gain weight;
1071
4+, severe ataxia or lethargy,, severe hypotonia and weight loss; 5+, severe ataxia and lethargy with alternating erratic hyperactivity; 6+, opisthotonus, convulsions, and death. Thirty-five animals were anesthetized with ether or chloral hydrate and killed by perfusion with 10 per cent formaldehyde in a 0.44M phosphate buffer. The brains and spinal cords were removed. The degree of yellow discoloration of the central nervous system was graded as mild (1+), moderate (2+), or severe (3+). Thirty-three animals from 15 experiments were perfused either with 4 per cent glutaraldehyde in 0.44M phosphate buffer or with Karnovsky's fixative ~~ diluted 1:4 with 0.2M sodium cacodylate buffer. In contrast to glutaraldehyde, this fixative does not obscure the yellow staining of the brain and preserves tissue better for morphologic studies than does formaldehyde. Small blocks of tissue from the vermis of tile cerebellum, the cerebral cortex, and hippocampus, and also from the colliculi, spinal cord gray matter, and the brainstem, were washed in buffer, placed in buffered 1 per cent osmium for one hour, and embedded in araldite. 11 Sections 1 /, thick were cut on a Porter-Blum microtome with glass knives and examined unstained with a phase contrast microscope, or with a conventional microscope after staining with 1 per cent crystal violet in 1 per cent borax. 1~ To estimate the proportion of damaged cells, 400 to 600 Purkinje cells were counted from 12 treated animals and 10 untreated littermates. In addition we have counted the Purkinje cells in the vermis of 34 untreated jaundiced animals between the ages of 11 and 33 days from previous studies. OBSERVATIONS
Serum bilirubin levels. The jaundice in the Gunn rat is lifelong, but in terms of molar equivalents it approaches that of the serum albumin concentration only during tile first 2 ~ weeks of life (Figs. 1 and 2). With rare exceptions damage to the brain probably occurs only during this period, r, s The serum bilirubin level rises after birth
10 7 2
Schutta and Johnson
The Journal of Pediatrics December 1969
9 .....
rag% serum bilirubin jj rots born of jj mother
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Fig. 1. S e r u m bilirubln levels in j a u n d i c e d (jj) a n i m a l s obtained by cesarean section a n d from b i r t h to 72 h o u r s f r o m j j / j j m a t i n g s a n d f r o m J j / j j m a t i n g s a n d f r o m h e t e r o z y g o u s (Jj) G u n n rats f r o m birth to 72 hours. T h e s e r u m a l b u m i n levels are also shown. T h e s e r u m bilirubin a n d s e r u m a l b u m i n levels are so a d j u s t e d on the scale as to s h o w their m o l a r equivalents.
28
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Fig. 2. Serum bilirubin and albumin levels in jaundiced rats from birth to adult life. Int e r u p t e d lines on the g r a p h r e p r e s e n t one s t a n d a r d deviation.
Volume 75 Number 6, part 1
to r e a c h a p e a k of 10 to 13 rag. p e r cent at 14 to 16 days, after which it declines, reaching the m e a n a d u l t level of 4 to 6 rag. per cent by 30 days. G u n n rats born of h o m o zygous m o t h e r s ( j j / j j matings) h a v e higher serum bilirubin levels at birth (1 to 2 rag. per cent) than those born of non j a u n d i c e d (Jj) m o t h e r s (0.5 rag. p e r cent). F r e q u e n t l y such animals die 4 to 5 days after birth (Fig.
1). T h e animals treated with sulfadimethoxine, regardless of age or initial serum bilirubin level, h a d serum bilirubin concentrations 18 hours after injection of 1.5 mg. p e r cent or less, except in 2 instances. These exceptions were in the 2 oldest animals, treated at age 33 days, which h a d bilirubin levels of 2.5 a n d 2.0 mg., respectively, 18 hours after the administration of sulfadimethoxine (Table I). At autopsy the fat and small intestines of the s u l f o n a m i d e - t r e a t e d rats were b r i g h t yellow, strikingly different from those of their j a u n d i c e d littermate controls, whose bilirubin h a d r e m a i n e d p r i m a r i l y in the bloodstream. S y m p t o m a t o l o g y . I n healthy litters, 70 to 75 p e r cent of u n t r e a t e d G u n n rats develop signs of central nervous system disease during the first 18 days of life. A b n o r m a l neurologic signs usually a p p e a r between 8 and 12 days after birth, when m o t o r function is sufficiently developed to allow recognition of ataxia, generally the first sign of central nervous system dysfunction. I n animals younger t h a n 8 days, hypotonia is the first indication of bilirubin e n c e p h a l o p a t h y , followed by lethargy, feeding difficulty, a n d weight loss. I n severely affected o l d e r animals a t a x i a is followed by hypotonia, after which l e t h a r g y a n d feeding difficulties occur. Finally, the animals display erratic hyperactivity, increasing lethargy, opisthotonus, convulsions, a n d death. I n an optimally h e a l t h y colony none of the homozygous animals die, b u t 40 to 50 per cent have various degrees of a t a x i a in a d u l t life. Thus, in 20 to 30 p e r cent the ataxia is transient a n d confined to the first 3 or 4 weeks of life (see also J o h n s o n a n d associatesS).
Sulfadimethoxine
10 73
T a b l e I. A g e of animals at the time of injection, a n d severity of brain d a m a g e (0 to 6+) before a n d after injection. All animals were killed 18 to 24 hours after t r e a t m e n t (Rx) with 150 rag. p e r k i l o g r a m of s u l f a d i m e t h o x i n e by subcutaneous injection. L i t t e r m a t e s given d u m m y injections served as controls. A n i m a l s m a r k e d G A were perfused with g l u t a r a l d e h y d e , discoloration of the b r a i n due to bilirubin could not be assessed in them. N o t e that severity of sulfonamideinduced d a m a g e was directly r e l a t e d to preinjection serum bilirubin level Abnormal CNS Age in signs days at _ _ ___ in)ec- Be[ore Alter lion Rx I Rx 18 hr. 0 4+ 1 1 1 1
I '] Brain
discolUn- Treated I oration treated + 6.0 ~1.0
0 0 0 0
3+ 3+ 4+ 3+
+ + + +
2 2 4 5 7 8 9 I0 10 10
0 0 0 0 0 0 1+ 0 0 0
3+ 6+ 4+ 3+ 3+ 5+ 3+ 4+ 6+ 2+
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+ + + GA ++ +++ ++ + GA GA ++ GA GA +++ +++ +++ +++ +++
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13 13 14 15 15 15 15 16 16 21 22 22 28 28 33 33
0 0 2+ 1+ 3+
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Serum bilirubin in rag. %
+++ +++ ++ +++ ++ GA + + + + +
6.5 6.5 6.5 5.0
5.5 7.5 7.0 7.5 6.0 7.0 6.0 9.0 9.0 5.5 5.5 5.5 6.5 6.5 6.5 11.5 11.5 11.5 11.5 12.0 10.0 9.0 12.0 8.5 7.0 7.5 7.5 5.0 5.0 6.0 6.0
~1.0 ~1.0 ~1.0 ~1.0
~1.0 ~ 1.0 ~ 1.0 ~ 1.0 ~ 1.0 ~ 1.0 ~ 1.0 ~1.0 ~1.0 1.5 ~ 1.0 ~ 1.0
~1.0 ~1.0 % 1.0
<1.0 ~1.0 ~1.0 <1.0 < 1.0 <1.0 < 1.0 ~1.0 ~1.0 ~1.0 <1.0 ~1.0 ~1.0 ~1.0 2.0 2.5
1 0 74
Schutta and Johnson
The Journal o[ Pediatrics December 1969
Fig. 3. Cerebellar cortex from a jaundiced rat, aged 16 days. The Purkinje cells marked P~ and P, are normal. Those marked P,, P,, and P~ show osmiophilic inclusions. There are no abnormalities in the molecular or granule cell layers (Original magnification •
In this experiment, a striking increase in brain damage followed treatment with sulfadimethoxine. Animals treated at age 18 to 48 hours, when serum bilirubin levels of littermates ranged between 5.0 and 6.5 rag. per cent at death 18 hours later, developed more severe signs of damage (3+ to 5+) than older treated animals with serum bilirubin levels in the same range (2+ to 3+). Otherwise the severity of damage related closely to the pretreatment serum bilirubin concentration (Table I ) . Histologic observations. Until 10 days after birth the Purkinje cells in the rat are immature and at times difficult to identify, but thereafter they can be recognized with ease, and accurate counts of normal and abnormal Purkinje cells can be made (Fig. 3). In untreated animals, between days 11 and 13 the number of abnormal Purkinje cells varied from 54 to 71 per cent (10 animals) ; at 14 to 16 days the proportion of abnormal Purkinje cells varied from 52 to 82 per cent (19 animals); thereafter the counts varied between 60 and 80 per cent up to day 22 (11 animals). From day 22 to 33 th~ counts varied considerably, from between 27 and
75 per cent (10 animals). The explanation for the variability is the resorption of many necrotic cells, which decreases the number of abnormal cells. T h a t there is loss of Purkinje cells and other neurons in the cerebellar folia is evident from the considerable atrophy which is present in many animals, particularly those which have ataxia. Asymptomatic animals examined between 11 and 21 days tended to have less than 70 per cent abnormal Purkinje cells, whereas those with ataxia had 70 to 82 per cent. There was thus a considerable variation in the abnormal cell counts from different litters, but the counts among littermates varied not more than 5 per cent, provided that the symptomatology prior to death was similar. Typical abnormalities were found in all animals, including those which had no clinical signs of central nervous system damage at any time. In all instances animals treated with sulfadimethoxine had more abnormal Purkinje cells in the vermis than untreated littermates with identical clinical status before treatment. One animal treated at 21 days had 1+ ataxia before and 3+ ataxia 24 hours after treatment. When the cell count of that ani-
Volume 75 Number 6, part 1
SuI[adimethoxine
%
of
abnormal
Purkinje
1075
cells
I
Jaund i c e d "
+
sulfa
,o % 80
70
60
50
40 30 20 10
11
II
12 age
13 -
-
16
21
22
28
33
33
days
Fig. 4. Histograms showing the proportions of abnormal cells in treated and untreated animals. The animal treated at day 21 was compared with an untreated littermate which had 2 to 3+ ataxia; this explains why the counts are similar.
m a l was c o m p a r e d with that of an uninjected l i t t e r m a t e with 2-3+ ataxia, the ceil counts were almost identical. H e r e the discrepancy in the symptomatology before t r e a t m e n t explains the similar p r o p o r t i o n of a b n o r m a l cells in both animals (Fig. 4 ) . I n rats which were treated at an age when Purkinje cell counts were impossible to accomplish accurately, the degree of d a m a g e was j u d g e d by the presence or absence of abn o r m a l P u r k i n j e cells in the plastic-embedd e d sections. I n the past a n d in this study we were u n a b l e to see any abnormalities in animals y o u n g e r t h a n 64 hours. T r e a t e d rats which were killed before t h a t age h a d m a n y a b n o r m a l Purkinje cells. I n the animals given sulfadimethoxine 18 to 24 hours after birth, necrotic Purkinje cells were also present. Necrotic cells do not occur in u n t r e a t e d rats until 10 to 11 days. I n treated animals which d i d not convulse, the n a t u r e of the morphologic changes as observed in the light microscope were identical to those f o u n d in u n t r e a t e d animals; however, the a b n o r m a l i ties were m o r e p r o n o u n c e d a n d necrosis of
Purkinje cells was more common. I n the cerebral cortex of u n t r e a t e d rats affected neurons occur very rarely, whereas in sulfad i m e t h o x i n e - t r e a t e d G u n n rats cortical neurons with intracytoplasmic inclusions were relatively common. I n the colliculi a n d tile cerebellar nuclei, neurons with osmiophilic cytoplasmic inclusions were also more comm o n in the treated animals. Evidence of acute cell necrosis was not present in these areas unless the animals were opisthotonic or h a d convulsed, when petechial h e m o r rhages were also present throughout the brains and m a n y areas showed swelling of gIial cells a n d neuronal shrinkage or swelling. W e believe that these changes are more likely the expression of terminal respiratory failure than acute bilirubin intoxication. Brain discoloration. I n u n t r e a t e d j a u n diced animals m i l d diffuse staining of the brain was obvious 4 to 8 hours after b i r t h ; staining r e m a i n e d diffuse u p to age 36 hours. Thereafter, the basal ganglia, the cerebellar cortex, the cerebellar nuclei, the colliculi, the eighth nerve nuclei, a n d also the gray
10 7 6
Schutta and Johnson
matter of the spinal cord were more deeply stained than the rest of the brain. In the cerebellum the vermis was distinctly more stained than the hemispheres. In cerebellar slices the yellow discoloration was visible only in the Purkinje cell and granule cell layers, the molecular and external granular layers appearing unstained. From 16 days on the intensity of staining progressively diminished; by 21 days no staining was apparent in untreated animals. It must be stressed that mild degrees of staining are very difficult to appreciate in fresh brains or those preserved by immersion fixation and may be overlooked unless the remaining blood, which obscures the yellow color of bilirubin, is washed out. In the animals treated with sulfadimethoxine from 18 to 24 hours after birth, diffuse yellow staining of the brains was more apparent than in untreated rats. Beyond that age all animals which were given sulfadimethoxine showed distinctly more marked localized yellow staining of the brain as compared with the untreated jaundiced rats. The increase in yellow staining was most marked in animals which were treated 12 to 16 days after birth. In animals treated as late as 21 to 33 days after birth, moderate staining of the vermis, the colliculi, and the cerebellar nuclei was noted (Table I ) . DISCUSSION
The deleterious effects of sulfonamides in jaundiced babies have been well documented? 3 This syndrome has been reproduced in Gunn rats, which upon administration of certain sulfonamides become sick and have increased bilirubin staining of their brains.14, 15 In the present study all animals treated with sulfadimethoxine developed acute signs referable to the central nervous system and had more yellow staining of the brains than untreated littermates. Sulfadimethoxine efficiently displaced bilirubin from its albumin binding sites, thereby allowing the unbound bilirubin to diffuse freely into the tissues, including the brain. T h e less marked reduction of serum bilirubin by sulfadimethoxine in the 33-day-old"animals is probably the result of the higher serum
The ]ournal o[ Pediatrics December 1969
albumin concentration of older animals and the greater capacity of older rats to excrete sulfonamides. The capacity of albumin to bind bilirubin has been known for some time, 16 but its importance in protecting the central nervous system from bilirubin toxicity was not suggested until 1954 by Lathe 17' is and subsequently by Brown and associates? 9 It was experimentally demonstrated in 1959 by Odell,20, ~1 whose observations have been repeatedly confirmed by others? 4' is, 22-24 The natural course of the encephalopathy in Gunn rats provides further confirmation of the importance of bilirubin binding to albumin. Central nervous system damage occurs during the first 2 to 2 ~ weeks of life when the bilirubin-albumin molar ratio is close to unity. T h e percentage of abnormal Purkinje cells in untreated animals was found to be at a maximum at day 16 (52 to 82 per cent), after which time there was a gradual decline. We believe that this decline is due to the death of severely injured cells and absence of further injury beyond that age in healthy animals. Additional neurologic damage can be induced by displacement of bilirubin from its binding sites. In Gunn rats which were treated with sulfadimethoxine between 4 and 22 days after birth, the severity of acute bilirubin encephalopathy was related to the serum bilirubin concentration present before treatment, suggesting a relationship between the amount of available bilirubin and central nervous system signs. Clinical signs of acute central nervous system intoxication occurred in animals treated up to 33 days after birth, at which time the animals are quite mature (21 days is the usual weaning time for laboratory rats). This suggests that unbound bilirubin has access to the central nervous system beyond the newborn period, which is in accord with previous observations: Johnson and associates s have shown that adult Gunn rats injected with bilirubin and sulfonamides develop ataxia; Diamond and Schmid 2~ demonstrated that 14C-labeled bilirubin enters the central nervous system in adult Gunn rats treated with sulfisoxazole. The extreme toxicity of sulfonamides to
Volume 75 Number 6, part 1
the jaundiced rat in the first two days of life and the relatively mild symptomatology in animals treated at 28 and 33 days deserve further comment. The total serum protein concentration of the rat on day 1 is approximately one quarter that at age 4 weeks. Furthermore, the excretory capacity of the newborn animal for drugs is low, so the levels of drugs in the body after a standard close are almost certainly higher in younger animals. These factors would bring about a more complete displacement of bilirubin from ali extracellular binding sites in the very young rats. Also, the volume of tissue other than brain capable of taking up bilirubin must be very much smaller in the immature rats than in older animals, which means that relatively more bilirubin is available to enter the brain in the young animals. p H homeostasis is also more precarious in very immature rats, probably resulting in an earlier onset of acidosis during the course of bilirubin intoxication. Acidosis is profound in kernicteric animals as they approach death (central nervous system gradings of 4+ to 6+). We believe that these factors account for the differences in the severity of symptomatotogy between the very young and somewhat older Gunn rats treated with sulfadimethoxine. It has been observed that serum bilirubin levels in premature babies may remain relatively low and yet severe bilirubin encephalopathy may develop. This is especially true in babies with respiratory distress. ~s, 26 The adult human brain does not stain with bilirubin even in the presence of severe jaundice, except in areas of necrosis. In some experimental models it is impossible to produce yellow staining of the brain by administering bilirubin intravenously in the absence of anoxia. Therefore, the occurrence of kernicterus has been explained by postulating a blood-brain barrier to bilirubin, which was thought to be either imperfectly developed in the young or damaged by anoxia. 27"32 However, recent studies of the blood-brain barrier to protein have failed to demonstrate differences between mature and immature brains, and the notion that the blood-brain barrier systems are more
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permeable in young brains is probably not correct. 3a A blood-brain barrier to free bilirubin (i.e., unbound to protein) has never been demonstrated. Our results suggest that bilirubin enters the central nervous system of quite mature animals after it has been displaced from its albumin binding sites. The low serum bilirubin levels in association with severe central nervous system disturbance can be explained by low serum albumin levels or the dissociation of bilirubin from available albumin binding sites by acidosis a n d / o r competing anions.S, 2o, a7 Even in the presence of relatively high serum bilirubin concentrations, unbound bilirubin can exist in appreciable quantities only in the presence of acidosis or competing anions such as free fatty acids, sulfonamides, or salicylates when there as an abundance of serum protein, as is tile case in the older healthy human baby and in tile mature Gunn rat. The fact that bilirubin discoloration in adult brains occurs only in the presence of pre-existing damage has little relevance to the problems of bilirubin encephalopathy. In necrotic areas the bloodbrain barrier to protein is destroyed and the bilirubin albumin complex has no more difficulty in entering the brain than the Evans blue-albumin complex or other markers used to demonstrate the state of the bloodbrain barrier to protein. '~~ These considerations and the results of the present study suggest that it is superfluous to postulate a blood-braln barrier for bilirubin to explain the incidence of kernicterus, and that the brain is protected from the entry of bilirubin by binding of bilirubin to protein. Petechial hemorrhages, neuronal swelling or shrinkage, and swelling of glial cells occurred only in animals which had convulsed or which were opisthotonic. Such animals were anoxic and acidotic, as judged by the strikingly dark appearance of their blood at the time of death. (In animals whose symptomatology did not exceed 3+, the blood always appeared bright red.) There can be no doubt that the cytotoxicity of bilirubin can be enhanced by anoxia, through its direct action on the neurons and also by
10 78
Schutta and Johnson
virtue of the c o n c o m i t a n t acidosis, since the binding of bilirubin to protein is less efficient at a lower p H ? s I t seems reasonable to assume that a neuron d a m a g e d by a n o x i a will be m o r e severely i n j u r e d a n d have less chance of recovering after exposure to bilirubin than an otherwise healthy cell. F u r thermore, acidosis in itself a p p e a r s to enhance the toxicity of bilirubin, as j u d g e d by its effects on cell cultures of mouse fibroblasts ~6 a n d organ cultures of cerebellumW T h e m a r k e d yellow discoloration of basal ganglia associated with h y p e r b i l i r u b i n e m i a gave rise to the term kernicterus? s I n our animals the cerebral cortex was v e r y definitely stained, p a r t i c u l a r l y a f t e r t r e a t m e n t with sulfadimethoxine, although the intensity of the discoloration was less t h a n in the basal ganglia, the cerebellar cortex, the cerebellar nuclei, the eighth nerve complex, o r the colliculi. I t has been suggested t h a t the differential staining occurs because of preexisting bilirubin d a m a g e s9-41 in n e u r o n a l groups which are exceedingly vulnerable to bilirubin. 22 However, we know of no convincing evidence that a d a m a g e d n e u r o n will take u p m o r e bilirubin t h a n u n d a m a g e d cells. T h e r e is no d o u b t that the Purkinje cells of the young G u n n rat are more easily d a m a g e d by bilirubin t h a n most o t h e r n e u r o n s / T h e colliculi a n d cerebellar nuclei, which show relatively little evidence of d a m age in the G u n n rat, are the most intensely discolored areas. Yellow areas in kernicterus are those which have high regional blood flow; a u t o r a d i o g r a p h s p r e p a r e d for the est i m a t i o n of regional blood flow 42, 43 show a striking resemblance to the p a t t e r n of discoloration f o u n d in kernicterus. I t is therefore possible that the intensity of bilirubin staining in the central nervous system is principally related to regional differences in cerebral blood flow, which m u s t p l a y an i m p o r t a n t role in d e t e r m i n i n g the q u a n t i t y of bilirubin deposited in various areas. T h e m e c h a n i s m of the high vulnerability of some neurons to bilirubin remains to be d e t e r m i n e d . However, since it is suspected t h a t regional blood flow parallels regional m e t a b o l i c rates, 42 it m a y well be tha~ vuln e r a b i l i t y to bilirubin d e p e n d s on the m e t -
The lournal o[ Pediatrics December 1969
abolic rate. T h i s also seems to be the case in e x p e r i m e n t a l n e o n a t a l asphyxia, in which the distribution of d a m a g e 44 closely resembles t h a t of bilirubin e n c e p h a l o p a t h y . This suggestion is f u r t h e r s u p p o r t e d by in vitro studies which show that bilirubin affects respiratory enzyme systems 2' as, 42, 4s and uncouples oxidative phosphorylation, s' 46 H o w ever, the possibility of inhibition by bilirubin of a specific enzyme system in vivo, u p o n which certain neurons m a y d e p e n d m o r e t h a n other cells, is w o r t h further study, because some deeply stained areas do not app e a r to be severely d a m a g e d . We wish to thank Marlene Catalde and Dennis Martell for help in preparing tissues for morphological study, Sandra Valenta, Anthony Mignano, and Edward Smith for help with bilirubin and serum protein estimations, and Muriel Schwartz and Sharon Oaks for secretarial assistance.
REFERENCES 1. Claireaux, A. E , Cole, P. G., and Lathe, G. H.: Icterus of the brain in the newborn, Lancet 2: 1226, 1953. 2. Day, R. L.: Inhibition of brain respiration in vitro by bilirubin reversal of inhibition by various means, Proc. Soc. Exper. Biol. & Med. 85: 261, 1954. 3. Kortsak, A. S., editor: Kernicterus, Toronto, 1961, University of Toronto Press. 4. Gunn, C. K.: Hereditary acholuric jaundice in a new mutant strain of rats, J. Hered. 29: 137, 1938. 5. Sehmid, R., Axelrod, J., Hammaker, L., and Swarm, R. L.: Congenital jaundice in rats due to a defect in bilirubln glucuronide formation, J. Clin. Invest. 37: 1123, 1958. 6. Crigler, J. F., and Najjar, V. A.: Congenital familial nonhemolytic jaundice with kernicterus, Pediatrics 10: 169, 1952. 7. Schutta, H. S., and Johnson, L.: Bilirubin eneephalopathy in the Gunn rat, J. Neuropath. & Exper. Neurol. 26: 377, 1967. 8. Johnson, L., Garcia, M. L., Figueroa, E., and Sarmiento, F.: Kernicterus in rats lacking glucuronyI transferase. II. Factors which alter bilirubin concentration and frequency of kernicterus, Am. J. Dis. Child. 101: 322, 1961. 9. Malloy, H. T., and Evelyn, K. A.: The determination of bilirubin with the photoelectric colorimeter, J. Biol. Chem. 119: 481, 1937. 10. Karnovsky, M. J.: A formaldehyde-glutaraldehyde fixative of high osmolarity for use in electron microscopy, J. Cell. Biol, 27: 270, 1965.
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11. Luft, J. H.: Improvement in epoxy resin embedding methods, J. Biochem. & Biophys. Cytol. 9: 409, 1961. 12. Guyer, S.: Personal communication, 1966. 13. Silverman, W. A.: The effects in premature infants of sulfisoxazole in the neonatal period, in Kortsak, A. S., editor: Kernicterus, Toronto, 1964, University of Toronto Press, p. 196. 14. Johnson, L., Sarmento, F., Blanc, W. A., and Day, R.: Kernicterus in rats with an inherited deficiency of glucuronyl transferase, Am. J. Dis. Child. 97: 591, 1959. 15. Blanc, W. A., and Johnson, L.: Studies in kernicterus. Relationship with sulfonamide intoxication. Report on kernicterus in rats with glucuronyl transferase deficiency and review of pathogenesis, J. Neuropath. & Exper. Neurol. 18" 165, 1959. 16. Martin, N. H.: Preparation and properties of serum and plasma proteins. XXI. Interactions with bilirubin, J. Am. Chem. Soc. 71: 1230, 1949. 17. Lathe, G. H.: The chemical pathology of bile pigments, Biochem. Soc. Symp. 12: 34, 1954. 18. Lathe, G. H.: Exchange transfusion as a means of removing bilirubin in haemolytic disease of the newborn, Brit. M. J. 1: 192, 1955. 19. Brown, A. K., Zuelzer, W. W., and Robinson, M. S.: Studies in hyperbilirubinemia, Am. J. Dis. Child. 93: 274, 1957. 20. Odell, G. B.: Studies in kernieterus. I. The protein binding of bilirubin, J. Clin. Invest. 38: 823, 1959. 21. Odell, G. B.: The dissociation of bilirubin from albumin and its clinical implications, J. PEDIAT. 55: 268, 1959. 22. Diamond, I., and Schmid, R.: Experimental bilirubin encephalopathy. The mode of entry of bilirubin 14C into the nervous system, J. Clin. Invest. 45: 678, 1966. 23. Menken, M., Barrett, P. V. D., Swarm, R. L., and Berlin, N. I.: Kernicterus development of an experimental model using bilirubin 14C, Arch. Neurol. 15: 68, 1966. 24. Ostrow, J. D., and Schmid, R.: The protein binding of 14C bilirubin in human and murine serum, J. Clin. Invest. 42: 1286, 1963. 25. Harris, R. C., Lucey, J. F., and MacLean, J. R.: Kernicterus in premature infants associated with low concentration of bilirubin in plasma, Pediatrics 21: 875, 1958. 26. Stern, L., and Denton, R. L.: Kernicterus in small premature infants, Pediatrics 35: 483, 1965. 27. Rozdilsky, B., and Olszewski, J.: Experimental study of the toxicity of bilirubin in newborn animals, J. Neuropath. & Exper. Neurol. 20: 193, 1957. 28. Chen, H., Lien, I. N., and Lu, T. C.: Kernicterus in newborn rabbits, Am. J. Path. 46: 331, 1965. 29. Swinyard, C. A., editor: Kernicterus and its importance in cerebral palsy, Springfield, Ill., 1961, Charles C Thomas, Publisher.
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30. Waters, W. J., and Bowen, W. R.: Bilirubin encephalopathy. Preliminary studies related to production, Pediatrics 15: 45, 1955. 31. Zuelzer, W. W.: Bilirubin and mental retardation, A. Res. Nerv. & M e n t . Dis. 3 9 : 1 8 3 1962. 32. Ernster, L., Herlin, L., and Zetterstrom, R.: Experimental studies on the pathogenesis of kernicterus, Pediatrics 20: 647, 1957. 33. Dobbing, J.: The development of the blood brain barrier, in Lajtha, A., editor: Brain barrier systems, Amsterdam, 1968, Elsevier Publishing Company, p. 417. 34. Bakay, L.. In Lajtha, A., editor: The blood brain barrier, Springfield, Ill., 1968, Charles C Thomas, Publisher, p. 78. 35. Odell, G. B., and Cohen, S.: The effect of pH on the protein binding of bilirubin, Am. J. Dis. Child. 100: 525, 1960. 36. Cowger, M. L., Igo, R. P., and Labbe, R. F.: The mechanism of bilirubin toxicity studied with purified respiratory enzyme and tissue culture systems, Biochemistry 4: 2763, 1965. 37. Silberberg, D., and Johnson, L.: Personal communication. 38. Schmorl, G.: Zur Kenntniss Des Ikterus Neonatorum Insbesondere Derbei Autretender Gehirn Veranderungen, Verhandl. deutsch. path. Gesellsch. 6: 109, 1903. 39. Lucey, J. F., Hibbard, E., Behrman, R. E., de Gallardo, E., and Windle, W. F.: Kernicterus in asphyxiated newborn rhesus monkeys, Exper. Neurol. 9: 43, 1964. 40. Day, R. L.: Kernicterus: Further observations on toxicity of bile pigments, Pediatrics 17: 925, 1956. 41. Haymaker, W., Margoles, C., Pentschew, A., Jacob, H., Lindenberg, R., Arroyo, L. S., Stochdorph, O., and Stowens, D.: Pathology of kernicterus and posticteric encephalopathy, in Swinyard, C. A., editor: Kernicterus and its importance in cerebral palsy, Springfield, Ill., 1961, Charles C Thomas, Publisher, p. 21. 42. Sokoloff, L.: Local cerebral circulation at rest and during altered cerebral activity induced by anaesthesia and visual stimulation, in Ketty, S. S., and Elkes, J., eds.: The regional chemistry, physiology and pharmacology of the nervous system, Oxford, 1961, Pergamon Press, p. 107. 43. Reivich, M., Isaachks, G., Evarets, E., and Ketty, S. S.: The effect of slow wave sleep and R E M sleep on regional cerebral blood flow in cats, .J Neurochem. 15: 301, 1968. 44. Rank, J. B., and Windle, W. F.: Brain damage in the monkey Macaca Mulatta by asphyxia neonatorum, Exper. Neurol. 1: 130, 1959. 45. Waters, W. J., and Bowen, W. R.: Bilirubin encephalopathy: Studies related to cellular respiration, Am. J. Dis. Child. 90: 603, 1955. 46. Zetterstrom, R., and Ernster, L.: Bilirubin, an uncoupler of oxidative phosphorylation in isolated mitochondria, Nature 178: 1335. 1956.