BILIRUBIN LEVELS IN RAT BRAIN 23 1

17. Roda A, Cappelleri G. Aldini R. Roda E. Barbara L 1982 Quantitative aspects cytes isolated from fetal and sucking rats. Gastroenterology 78: 1324 (abstr) of the interaction of bile acids with human serum albumin. J Lipid Res 24. Suchy FJ, Balistreri WF 1982 Uptake of taurocholate of hepatocytes isolated 23:490-495 from developing rats. Pediatr Res 16:282-285 18. Roda A, Roda E, Aldini R, Festi D, Mazzella G, Sama C, Barbara L 1977 25. Suchy FJ, Balistreri WF, Heubi JE, Searchy JE, Levin RS 1981 Physiologic Development. validation and application of a single-tube radioimmunoassay cholestasis: elevation of the primary serum bile acid concentrations in normal for cholic and chenodeoxycholic conjugated bile acids in human serum. Clin infants. Gastroenterology 80: 1037- 104 1 Chem 23:2107-2113 26. Suchy FJ, Heubi JE, Balistreri WF, Belknap WM 1981 The enterohepatic 19. Roda A, Roda E, Festi D. Aldini R, Mazzella G, Sama C, Barbara L 1978 A circulation of bile acids in suckling and weanling rats. Gastroenterology radioimmunoassay for lithocholic acid conjugates in human serum and liver 80: 135 1 (abstr) tissue. Steroids 32: 13- 19 27. Tikanoja T, Tikanoja S, Simell 0 1981 Plasma conjugated cholic acid in 20. Sewell RB. Hardy KJ. Smallwood RA. Hoffman NE 1980 Fetal bile salt premature and term newborns and young infants. Acta Pediatr Scand metabolism: placental transfer of taurocholate in sheep. Am J Physiol 70:491-495 239:G354-357 28. Vlahcevik ZR, Schwartz CC, Gustafsson J, Halloran LG, Danielsson H, Swell 21. Sewell RB, Hardy KJ. Smallwood RA. Hoffman NE 1982 Fetal bile salt L 1980 Biosynthesis of bile acids in man. Multiple pathways to cholic and metabolism: placental transfer of dihydroxy bile salts in sheep. Am J Physiol chenodeoxycholic acid. J Biol Chem 255:2925-2933 6:G172-175 29. Watkins JB, Goldstein E, Coryer R, Brown ER, Eraklis A 1979 Sulfation of 22. Strange RC. Hume R, Eadington DW. Nisumo IA 1981 Distribution of bile acids in the fetus. In: Presig R, Bircher J (eds) The Liver Quantitative glycocholate in blood from human fetuses and adults. Pediatr Res 15:1425- Aspects of Structure and Function. Editio Cantor, Aulendorf, pp 249-254 1428 30. Watkins JB 1983 Placental transport: bile acid conjugation and sulfation in 23. Suchy FJ. Balistreri WF 1980 Maturation of bile acid conjugation in hepato- the fetus. J Pediatr Gastroenterol Nutr 2:365-373

003 1-3998/85/1902-023 1$02.00/0 PEDIATRIC RESEARCH Vol. 19, No. 2, 1985 Copyright O 1985 International Pediatric Research Foundation, Inc. Printed in (I.S. A.

Effect of Postnatal Anoxia on Bilirubin Levels in Rat Brain

FEDERICO MAYOR, JR., MONSERRAT PAGES, JAVIER D~EZ-GUERRA, FERNANDO VALDIVIESO, AND FEDERICO MAYOR Departamento de Bioquimica y Biologia Molecular, Centro de Biologia Molecular, Facultad de Ciencias Universidad Autonoma, Madrid, Spain

ABSTRACT. The effects of a period of anoxia 18-24 h Despite the current development of neonatology and the use after birth on bilirubin levels in rat brain were investigated of exchange transfusion and phototherapy, kernicterus continues during anoxia, recovery, and development. Postnatal an- to occur, specially in sick, premature, or low birth weight babies, oxia induces a significant, temporary increase (up to 200% as has been recently pointed out by different reports (20, 3 1, 36). with respect to control values) in newborn rat brain bili- Despite the various diagnostic and therapeutic measures intended rubin levels during anoxia and short-term recovery. Pre- to prevent bilirubin encephalopathy, at present no blood chem- treatment of the newborn rats with a single dose of the istry value (including bilirubin concentration) or clinical factors drug sulfixosazole markedly enhances bilirubin accumula- can completely predict the risk of kernicterus. tion in the brain of the anoxic rats. A second rise in brain Perinatal asphyxia, together with prematurity, acidosis, respi- bilirubin concentration is detected in a group of the new- ratory distress syndrome, and the use of certain drugs have been born rats 3-6 days after oxygen deprivation. Autoradi- reported to be the major predisposing risk factors for the occur- ographic localization of radiolabeled bilirubin following in rence of bilirubin encephalopathy in newborn infants with low vivo experiments suggests that this substance is preferen- levels of serum bilirubin (l,6, 15, 20,43). These clinical reports, tially accumulated in some areas of the newborn rat brain together with the fact that the neurological damage associated as a consequence of postnatal anoxia, and indicates, to- with perinatal asphyxia or anoxia shows brain regional distribu- gether with the effect of sulfixosazole, that as a result of tion and clinical manifestations somewhat similar to those ob- anoxia, a displacement of unbound bilirubin from blood to served in bilirubin encephalopathy (9, 27, 29), have prompted the nervous tissue occurs. Our results confirm the impor- us to investigate the relationship between episodes of lack of tance of anoxia as a risk factor for the develo~mentof oxygen in the postnatal period and the accumulation of bilirubin bilirubin-induced encephalopathy. The possible ielevance in the brain. of intracerebral hemorrhages caused by perinatal asphyxia producing delayed bilirubin accumulation in the newborn rat brain is suggested. (Pediatr Res 19: 231-236, 1985) MATERIALS AND METHODS Chemicals. I', 2, 3', 4, 5, 6', 7', 8 [14C]bilirubin(62.5 mCi/ Received May 16, 1984; accepted July 1 1, 1984. mmol) was obtained from The Radiochemical Centre, Amer- Correspondence Prof. F. Mayor, Departmento de Bioquimica y Biologia Molec- sham, Bucks, UK. Sulfixosazole and bilirubin were purchased ular, Centro de Biologia Molecular, Facultad de Ciencias, Universidad Autonoma de Madrid, Madrid-34, Spain. from Sigma Chemical Co., St. Louis, MO. All other reagents This work was supported by a grant from the Comision Asesora de Investigaci6n used were of the highest purity available. Cientifica y Tbcnica (Ministry of Science and Education of Spain). Animals. Dated pregnant rats of the Wistar strain weighing MAYOR ET AL. 2% g were individually caged and fed on standard laboratory computer-generated densitometric maps were obtained by meas- food and water ad libitum. The time of birth was noted within 4 uring the optical density of the radioactively exposed film with h. Pups were born vaginally and reared with their dams until the aid of a microdensitometer (Photomation System P-1700, 18-24 h after birth. Optronics International Inc., Chelmsford, MA) connected by Anoxia. The experimental animals (18-24 h of postnatal age) means of an Assembler language to a PDP 11/45 computer were rendered anoxic for periods up to 20 min, essentially as (Digital Equipment Corp., Maynard, MA) with a Fortran IV previously described (37). Groups of four to six newborn rats language. The image was digitalized, the optical densities coded were placed in 500-ml glass jars. The jars were stoppered except into 9 gray tones, and the densitometric map printed by the for small intake and outlet taps, and partially immersed in a 37" computer using a Varian printer-plotter. A similar method of C water bath to maintain a constant thermal environment. image analysis has been recently published (14). For the in vitro Anoxia was achieved by bubbling 100% nitrogen at a rate of 4- autoradiographic studies, the brain sections were prepared and 6 literlmin through water at 37" C and delivering the gas into incubated for 30 rnin with 0.4 pM radioactive bilirubin in 120 the 500-ml jars. In this experimental model, the oxygen tension mM-KC1122 mM-potassium phosphate buffer, pH 7.2, as de- of the effluent gas was less than 0.03 mm Hg, and total anoxemia scribed. The tissue sections were developed after an exposure of is obtained within 2 rnin after exposure to nitrogen (1 I). The 20-30 days, and the slides viewed by both bright-field and dark- control newborns were exposed, under the same conditions, to field microscopy. Under these conditions, the autoradiographic humidified air. Littermates were removed after 5, 10, or 20 min technique provides quantitative data because autoradiographic of anoxia and then immediately frozen in liquid N,. Other grain densities are proportional to time of exposure and tissue animals rendered anoxic for 20 rnin were allowed to recover in content of radioactivity (44). air at 37" C. In the short-term recovery experiments, the surviving newborns (approximately 80%) were kept at 37" C for different RESULTS intervals prior to freezing in liquid N2. In the long-term experi- ments the survivors, after a I-h period of recovery at 37" C, were The effect of postnatal (18-24 h of age) exposure to nitrogen returned to their mothers, whose litters were normalized to 8- atmosphere on rat brain bilirubin levels during anoxia and the 10 pups, and kept with them for 1-7 days. When required, the first 6 h of recovery is shown in Figure I. The brain bilirubin pups were sacrificed by immersion in liquid N2. From the time levels in the control littermates remained constant throughout of freezing until use, animal carcasses were stored at -80" C. the experimental period and are similar to those previously In some experiments, newborn rats were pretreated 30 min reported (34). During the first 5 rnin of anoxia, brain bilirubin before the onset of the anoxic episode either with 600 pg/g levels increased abruptly and reached a maximum at 35-50 min sulfixosazole administered subcutaneously or with 9 pg/g, bili- (15-30 rnin of recovery). As recovery continued, a gradual rubin injected intraperitoneally with a 100-j~1Hamilton syringe decrease in brain bilirubin levels was observed in the anoxic equipped with a '/3-mm gauge needle (Hamilton Co., Reno, NV). newborns. Control animals received the same volume of 0.9% (w/v) NaCl. In order to investigate whether postnatal anoxia (on the 1st Bilirubin solutions were prepared by dissolving crystalline bili- day of life) has any long-term effect on rat brain bilirubin levels, rubin in a small volume of 0.1 M NaOH, diluting with saline, groups of rats that survived a 20-min anoxic episode were re- and adjusting the pH to 8 (25). turned to their dams and kept with them for up to 8 days of life Determination of brain bilirubin levels. Whole-brain bilirubin (i.e. for up to 7 days after exposure to anoxia). Brain bilirubin levels in individual control and anoxic newborn rats were deter- concentrations were determined at different ages and compared mined as previously described (34). Brain tissue was homoge- to those of control littermates. The results are shown in Table 1. nized with 3 volumes of 0.25 M, sucrose solution. The extraction No significative changes in brain bilirubin levels were observed mixture containing 1 ml tissue homogenate, 4 ml distilled water, in the control rats during the developmental period studied. A 5 ml chloroform, and 12 ml methanol was homogenized. After significant increase in brain bilirubin concentration in the anoxic adding 8 ml distilled water the whole mixture was centrifuged rats was detected at 4-7 days of age (3-6 days after the anoxic for 30 rnin at 10000 x g. The procedure was camed out in dim episode). By day 8, the brain bilirubin levels in the treated group light below 4" C. Bilirubin in the chloroform phase was deter- were restored to control values. mined directly from the difference between the absorbance at It should be mentioned that this rise in brain bilirubin concen- 452 and 490 nm on a Pye-Unicam SP-1700 double-beam spec- tration shows an interesting difference with respect to that ob- trophotometer, using commercial bilirubin as a standard. For served during anoxia and short-term recovery (see Fig. 1). In the the determination of brain bilirubin levels in I-day-old rats, the latter case, all newborns subjected to anoxia experienced an forebrains of two newborn rats were pooled. increase in their brain bilirubin content, with narrow differences Analysis of the data. Statistical analysis was performed by the between individual values for a given period of anoxia or recov- Student's t test for nonpaired samples and comparison was made ery. On the other hand, the individual brain bilirubin levels of with the control group. p values of 0.05 or less were taken as the anoxic group at 4-7 days of age differ considerably. Whereas significant, and the results are expressed as the mean + SEM. some animals show values indistinguishable from those of con- Autoradiography. One-day-old rats were used. Immediately trol rats, others have a higher accumulation of bilirubin in brain, after 20-min exposure to 100% nitrogen, 2 ,uCi of ['4C]bilirubin with a wide range of levels. For instance, at 5 days of life, when were administered intraperitoneally into the anoxic and control the mean brain bilirubin level for the anoxic group (I 8 animals) rats in 100 p1 of 0.9% saline. Thirty minutes later, the animals was 0.355 k 0.052 (significantly higher than control rats), seven were sacrificed by decapitation and the brains rapidly removed treated rats showed values similar to those of controls, whereas and fixed in 10% formalin. Four- to five-millimeter slices of 1 l (60%) exhibit a wide range (up to 0.9 pg bilirubinlg wet appropriate regions were cut. Following repeated washings in weight) of brain bilirubin concentrations more than 0.25 pg/g ethanol and xylol the tissue was embedded in paraffin and wet weight, a value never reached by control rats throughout the multiple serial sections, 3-4 pm in thickness, were obtained on experimental period. an microtome and mounted onto subbed slides. The paraffin In order to understand the mechanism by which postnatal was removed by washing with xylol (two 10-min rinses). Finally, anoxia induces an increase in brain bilirubin levels more clearly, the slides were covered with AR-10 autoradiographic stripping we studied the effect of different pretreatments of newborn rats film (Eastman Kodak, Rochester, NY), air dried, and placed in on brain bilirubin concentrations during the first period of light-tight boxes. The tissue sections were developed after an recovery from 20-min postnatal anoxia (see Table 2). Pretreat- exposure of 10-12 wk at 4" C and examined microscopically ment with a single dose of sulfixosazole-a drug known to under dark-field and bright-field illumination. In some cases, displace serum albumin-bound bilirubin, increasing the blood BILIRUBIN LEVELS IN RAT BRAIN

0 0 10 20 50 80 200 I 380 , 'Tzr Recovery Time (minl Fig. 1. Brain bilirubin levels during anoxia and recovery. Whole-brain bilirubin levels, expressed as pg/g brain wet weight were determined in the anoxic (0)and control (0)neonatal rats. The brains of two animals were pooled for each determination. The results are the means + SEM of the number of determinations shown in parentheses. The dashed area denotes mean control values & SEM ***p < 0.001 for comparison of anoxic and control newborns. Table 1. Effect of postnatal anoxia on bilirubin levels in rat Table 2. Effect of dzfferent treatments on brain bilirubin levels in brain during development newborn rats Brain bilirubin levels (pg/g wet wt) Treatment Control rats Anoxic rats Exposure Recovery to anoxia from anoxia Brain bilirubin level Pretreatment (min) (min) (~g/gwet wt) Control 0.212 + 0.015 (17) None 20 30 0.446 & 0.0 16 (4)** None 20 60 0.334 + 0.021 (7)** None 20 180 0.234 + 0.0 19 (4) Saline? 0.197 + 0.037 (4) The newborn rats were rendered anoxic for 20 min. The surviving Sulfixosazolet 0.358 a 0.030 (4)** anoxic rats and the control littermates were reared for UD to 8 davs of Sulfixosazole$ 20 30 0.622 + 0.057 (1 I)** life with their dams. The results are the means + SEM of the number of Bilirubin$ 20 60 0.356 0.021 (14)** animals shown in parentheses. * p < 0.05, ** p < 0.01 for comparison of Bilirubin$ 20 180 0.289 -t 0.0 19 (7)* anoxic and control neonates of the same age. Brains of two animals were pooled for each determination. The results are the means + SEM of the number of determinations shown in levels of unbound, "free" bilirubin (3)-by itself promotes a parentheses. * p < 0.0 1 ** p < 0.00 1, for comparison of treatment and significant rise (p< 0.00 1) in brain bilirubin levels in comparison control nwborns. with control rats, although the bilirubin concentration reached t Brain bilirubin levels were determined 90 rnin after the injection. is less than that detected after a period of 20 min anoxia (see $ Injected 30 min before the onset of the anoxic episode. Fig. 1). On the other hand, pretreatment with sulfixosazole significantly enhanced (p < 0.05) the increase in brain bilirubin levels observed after 30 rnin of recovery from a 20-min anoxic rats, whereas no significant differences with respect to controls episode. The bilirubin accumulation rose to 0.622 + 0.057 pg/g were detected at the same time of recovery in the animals of brain wet weight, approximately three times the control values, subjected to 20-min anoxia without prior injection of bilirubin. and was also significantly higher (p < 0.05) than those detected In another set of experiments, radiolabelled bilirubin was in animals injected with sulfixosazole, but not subjected to injected to control and 20-min anoxic rats, and the animals anoxia. sacrified 30 rnin later, when the maximum level of bilirubin Unlike sulfixosazole, pretreatment with a single dose of bili- accumulation is reached in the brain of the anoxic animals (see rubin prior to anoxia did not significantly enhance the increase Fig. 1). We have used autoradiographic techniques, detailed in in brain bilirubin levels induced by postnatal anoxia under our the "Methods and Materials" section, to localize bilirubin distri- experimental conditions. However, bilirubin pretreatment seems bution in nervous tissue sections examined with the light micro- to slow down the gradual decrease in brain bilirubin observed in scope. No noticeable accumulation of radioactive bilirubin was the anoxic rats after the first 30 min of recovery. Thus, brain detected in the brain of control rats, with the radioactive grains bilirubin values at 180 rnin of recovery in the bilirubin-pretreated located mainly in the brain blood vessels (results not shown). On group were significantly higher (p < 0.0 1) than those of control the other hand, tissue sections from the brain of the anoxic 234 MAYOR ET AL.

Fig. 2. Autoradiography of in vivo bilirubin distribution in brain areas from anoxic newborn rats. Densitometric maps were printed by a PDP 1 1/45 computer after image analysis of autoradiograms of ['4C]bilirubin distribution. The brain section shown in A includes the hippocampus (H), the dentate gyms (GD), and the lateral nucleus of the thalamus (LT),whereas the densitometric maps B and C show bilirubin distribution at the level of the choroid plexus of the walls of the lateral ventricle and in a cerebral cortex area, respectively. Bar, 150 pm. The white spots correspond to high densities of ['4C]bilirubin autoradiographic grains. newborn animals showed a remarkable presence of autoradi- The regional distribution of bilirubin in brain sections has alsc ographic grains, indicating a displacement of bilirubin from the been studied by autoradiography after experiments in vitro blood to the nervous tissue as a result of postnatal anoxia. where tissue sections from control and anoxic rats were incubated Furthermore, in examining different areas of the brain, regions in the presence of 0.4 pM [14C]bilirubin(Table 3). Under oul with a high density of grains were observed; these include hip- experimental conditions, bilirubin binds to the cellular struc- pocampus, thalamus, some areas of the cerebral cortex, and the tures, with no significant differences between the brain area: choroid plexus of the walls of the lateral ventricle (see Fig. 2). In examined in the control rats, except the cerebellum, where 2 the two latter regions, radiolabelled bilirubin appeared concen- slightly higher grain density was found. In the anoxia-treatec trated in intense, local radioactive spots (Fig. 2 B and C). These rats, higher autoradiographic grain densities than in control rat: results suggest that when bilirubin is accumulated by the newborn and than other brain regions of the anoxic rats were detected ir rat brain as a consequence of postnatal anoxia, a relatively the cerebellum, hippocampus, and striatum. However, no de. specific or preferential distribution of the pigment in the nervous fined brain areas for bilirubin accumulation could be detectec tissue occurs. by this method. BILIRUBIN LEVELS IN RAT BRAIN 235 Table 3. Regional distribution of radioactive bilirubin in brain local bilirubin concentrations and of the mechanisms of bilirubin slices as determined by autoradiography* neurotoxicity. Treatment of the newborn Lucey et al., (24), in monkeys, and Chen et al. (7,8), in rabbits, rats have reported that neonatal anoxia causes deposition of bilirubin in the brain (detected by histological techniques), only when Brain area Control Anoxia superimposed to experimentally induced severe hyperbilirubi- Cerebellum 150 + 10 202 + 6 nemia. Our findings indicate that postnatal anoxia, without Hippocampus loo+ 15 180 & 10 concomitant hyperbilirubinemia, is able to promote bilirubin Stnatum 110+ 17 170+ 17 accumulation in the newborn brain. Thalamus 80+ 15 107 -c 21 The remarkable, transient rise in brain bilirubin levels ob- Hypothalamus 90 + 7 98 k 18 served during anoxia and the early recovery phase might be Neocortex 85 f 12 76 f 14 explained by the acidosis induced by anoxia. Blood pH declines by approximately 0.6 pH units in newborn rats subjected to 20- * Brain slices from control and 20-min anoxic rats were incubated in the min anoxia (37). A decreased pH has been shown to enhance presence of 0.4 pm [I4C] bilirubin and processed for autoradiography. the in vitro interaction of bilirubin with liposomes (12) and The results are expressed as number of radioactive grains per 10,000 pm2 human erythrocyte membranes (33), as well as bilirubin incor- and are the means + SEM for at least 15 serial sections. poration into culture cells (28) and brain slices (18). In agreement with these data, we have found that bilirubin interaction with rat brain synaptosomal membranes is markedly augmented as pH decreases (unpublished work). A decline in blood pH, increasing DISCUSSION the interaction of bilirubin with the brain cellular membranes, The experimental model we have selected to render the new- could displace bilirubin from its binding to albumin, allowing born rats anoxic has the advantage of having been previously pigment accumulation in the brain. This process should be used for studying the alterations in brain energy metabolism and favored, as it has been confirmed in our experiments, in the blood parameters (pH, pcoz, po2, glucose, lactate, etc.) promoted presence of drugs such as sulfixosazole (known to displace bili- by a 20-min anoxic episode (1 1,37-39). Furthermore, the period rubin from albumin) as well as in neonatal hyperbilirubinemia, of 20-min anoxia, although producing considerable biochemical in which the free pigment concentration in blood is augmented and circulatory changes, has a high survival index (1I), thus (6). In addition, Diamond and Schmid (10) have reported that allowing the study of the brain bilirubin levels during recovery acidosis induced by infusion of diluted HCl increases brain from anoxia and the neonatal development period. bilirubin levels in guinea pigs. Neonatal acidosis has been put The results reported herein indicate that 1) Postnatal anoxia forward as an important risk factor for the development of induces a significant, temporary increase (up to 200% with bilirubin encephalopathy (4, 43), and appears frequently in the respect to control values) in whole-brain bilirubin levels during clinical reports of newborns diagnosed with kernicterus (19). anoxia and short-term recovery in all the newborn rats exposed Furthermore, comparing the changes in blood pH of 1-day-old to nitrogen atmosphere at 18-24 h postpartum (Fig. 1). Treat- rats during 20-min anoxia and the following recovery (37) with ment of the newborn rats with a single dose of sulfixosazole those of brain bilirubin levels depicted in Figure 1, a clear inverse before subjecting them to 20-min anoxia markedly enhances relationship between these parameters appears. Wennberg and bilirubin accumulation by the brain of the anoxic rats, whereas Ahlfors (42), discussing the possible role of acidosis in inducing an injection of bilirubin slows down the decrease in brain bili- kernicterus, have pointed out that arterial blood pH might not rubin concentrations observed after the first 30 min of recovery reflect the actual tissue acidosis adequately and that in as- (Table 2). These data, together with the autoradiographic locali- phyxiated newborns the pH gradient between different brain zation of radioactive bilirubin in tissue sections from the brain regions and central arterial blood might be large. In fact, a better of anoxia-treated rats (Fig. 2), strongly suggest that, as a result of inverse relationship (p < 0.01 and < 0.05, respectively) than with anoxia, a displacement of unbound bilirubin from blood to the blood pH is obtained when comparing brain bilirubin levels with nervous tissue occurs. 2) A second rise in brain bilirubin levels lactate concentrations in brain or in cerebrospinal fluid during was detected in a group of the newborn rats 3- to 6 days after 20-min anoxia and the early phase of recovery (37). exposure to anoxia (Table l), when all other biochemical param- Although all these data are consistent with a role for acidosis eters altered by anoxia had already been restored to control in promoting accumulation of unbound bilirubin in the brain, it values (1 1, 37, 39). A relatively wide range of brain bilirubin is not possible to disregard an accumulation of albumin-bound concentrations was observed in the rats affected. 3) Autoradi- bilirubin in the nervous tissue as a result of either a reversible ographic localization of radiolabeled bilirubin following in vivo opening of the blood-brain bamer induced by anoxia (17) or the experiments suggests that bilirubin is preferentially accumulated occurrence of a vasogenic brain edema (26). Levine et al. (21) in some areas of the newborn rat brain as a consequence of postnatal anoxia (Fig. 2). This, in turn, suggests that the brain have recently reported that albumin-bound bilirubin is able to bilirubin concentrations determined in the whole brain of the penetrate the brain when the blood-brain bamer is reversibly anoxic rats could be an underestimate of the actual levels of the opened by means of an hyperosmolar infusion. pigment in local regions of the nervous tissue. Furthermore, the On the other hand, the increase in brain bilirubin levels that brain areas where bilirubin is accumulated preferentially, coin- takes place 3-6 days after the anoxic episode (Table 1) might cide roughly with those more frequently stained in bilirubin reflect the occurrence of intracerebral hemorrhages of different encephalopathy (19) and with those more severely damaged as a size and location during the period of anoxia and/or recovery, result of perinatal asphyxia (38). On the other hand, the brain as a result of in situ catabolism up to bilirubin of the heme group regions where local, intense spots of radioactive bilirubin appear of the hemoglobin of the extravasated blood. (i.e. choroid plexus, cerebral cortex) are related to the areas of Although this conclusion remains speculative and needs to be higher incidence of intracerebral hemorrhages during the peri- confirmed, a number of data are consistent with this explanation. natal period (20, 35). First, it seems to be established that perinatal asphyxia gives rise The brain bilirubin levels determined in the brain of control to intracerebral bleeding in human neonates (especially in pre- rats are similar to those previously described and probably reflect matures) probably as a consequence of alterations in the cerebral the albumin-bound bilirubin present in the blood vessels (34). blood flow (22, 40). Recently, Zhukova and Hallman (45) have The possible pathological relevance of brain bilirubin levels reported that experimental perinatal asphyxia in rats promotes remains to be established when we have a better knowledge of intracerebral bleeding, most of all in the periventricular areas, 236 MAYOR ET AL. the thalamus, and the choroid plexus. In the latter area, we have in perinatal rat brain: relation to survival in anoxia. J Neurochem 24:27 1 12. Eriksen EF, Danielsen H, Brodersen R 198 1 Bilirubin-lipsome interaction. J detected, in the in vivo autoradiographic experiments, intense, Biol Chem 256:4269 local spots of radioactive bilirubin (see Fig. 2B) that could be an 13. Fishman RA 1980 Cerebrospinal fluid in disease of the nervous system WB indication of the occurrence of an hemorrhage, as extravasated Saunders Company, ~hiladelphia blood contains bilirubin, free or bound to albumin. Bada et al. 14. Fuxe K, Calza L, Benfenati F, Zini I, Agnati LF 1983 Quantitative autoradi- omhic localization of 'H-imi~raminebindine sites in the brain of the rat: - z - (2) and Goddard et al. (16) have also detected a high incidence relationship to ascending 5-hydroxytryptamine neuron systems. Proc Natl of bleedings in the choroid plexus. The intraventricular and Acad Sci USA 80:3836 periventricular hemorrhages are the most frequent in human 15. Gartner LM, Snyder RN, Chabon RS, Bernstein J 1970 Kernicterus: high newborns, particularly in preterm neonates (20, 33, although incidence in premature infants with low serum bilirubin concentrations. Pediatrics 45:906 bleeding occurs as well in the region of the cerebral cortex (4 l), 16. Goddard J, Lewis RM, Armstrong DL, Zeller RS 1980 Moderate, rapidly where we have also found local accumulation of radiolabeled induced hypertension as a cause of intraventricular hemorrhage in the bilirubin following anoxia (Fig. 2C). newborn beagle model. J Pediatr 96:1057 Second, the presence of macrophages in areas of brain hem- 17. Johansson B 1974 Blood-brain bamer dysfunction in acute arterial hyperten- sion after papaveine-induced vasodilatation. Acta Neurol Scand 50:573-580 orrhages has been observed (5, 45). Macrophages have the en- 18. Kashiwamata S, Suzuki F, Semba RK 1980 Affinity of young rat cerebral slices zymes required to catabolize the heme group up to bilirubin (30). for bilirubin and some factors influencing its transfer to the slices. Japan. J Roost et al. (32) have reported the occurrence of heme oxygenase Exp Med 50:303 activity in cerebral cortex, choroid plexus, and arachnoid follow- 19. Kim MH, Yoon JJ, Sher J, Brown AK 1980 Lack of predictive indices in ~ernicterus:a comparison of clinical and pathologic factors in infants with ing experimental subarachnoid bleeding. The changes in the or without kernicterus. Pediatrics 662352 composition of the cerebrospinal fluid after such hemorrhages 20. Leblanc R, O'Gorman A 1980 Neonatal intracranial hemorrhage-a clinical have been studied in human adults (13). The hemoglobin level and serial computerized tomographic study. J Neurosurg 53:642 reaches a maximum 36 h after bleeding and then disavvears 2 1. Levine RL, Fredericks WR, Rapport SI 1982 Entry of bilirubin into the brain due to opening of the blood-brain bamer. Pediatrics 69:255-259 gradually, whereas bilirubin reaches its maximum conceniiation 22. Lou HC 1980 Perinatal hypoxic-ischemic brain damage and intraventricular 3-4 days after the subarachnoid hemorrhage. This delay is similar hemorrhage-a pathogenetic model. Arch Neurol 37585 to that observed in our experiments. Furthermore, a relationship 23. Lucey JF 1982 Bilirubin and brain damage-a real mess. Pediatrics 69:38 1 between the occurrence of intracerebral bleeding (detected by 24. Lucey JF, Hibbard E, Behrman RE, Esquivel de Gallardo FO, Windle WF 1964 Kerniecterus in asphyxiated newborn Rhesus monkeys. Exp Neurol computer-assisted tomography), and a xantochromathic cerebro- 9:43 spinal fluid has been reported in human neonates (20). Recently, 25. Mustafi MG, King TE 1970 Binding of bilirubin with lipid. J Biol Chem Lucey (23) has suggested a role for intracerebral bleeding in the 245:1084 pathogenesis of kernicterus. 26. Myers RE 1977 In: Gluck L (ed) "Intrautercina Asphyxia and the Developing Brain." Year Book Medical Publishers, Inc., Chicago, pp 37-97 Finally, the preferential accumulation of bilirubin in different 27. Myers RE 1979 A unitary theory of causation of anoxia and hypoxia brain areas of the brain (see Table 3 and Fig. 2), as well as the relatively pathology. In: Fahn C, Davis JN, Rowland LP (eds) Cerebral Hypoxia and wide range of brain bilirubin levels detected 3-6 days after the its Consequences. Advances in Neurology. vol26. Raven Press, New York, anoxic episode (Table l), might be interesting factors in an pp 195-213 28. Nelson T, Jacobsen J, Wennberg RP 1974 Effect of pH on the interaction of attempt to explain the varying degrees of neurological damage bilirubin with albumin and tissue culture cells. 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