Liposome‐Bilirubin Interaction: A Novel Strategy to Eliminate Bilirubin from Systemic Circulation

In the present study, we established the role of liposomes in removal of bilirubin from systemic circulation of the hyperbilirubinemic rats. Bilirubin has been demonstrated to possess inherent tendency to interact with liposomes through ionic as well as hydrophobic interactions. The size as well as lamellarity of the liposomes does not seem to affect their binding with bilirubin. However, the charge on the surface of liposomes plays an important role in bilirubin‐liposome interaction, e.g., bilirubin binds more extensively with positively charged liposomes as compared to the neutral or negatively charged liposomes. The present study further demonstrates that liposomes were effective in reducing the increased plasma bilirubin level in hyperbilirubinemic model animals as well. The results of the study suggest that positively charged liposome‐mediated selective homing of excess plasma bilirubin to the hepatocytes seems to offer an important strategy in management of hyperbilirubinemic conditions.     

Liposome-bilirubin interaction: a novel strategy to eliminate bilirubin from systemic circulation

In the present study, we established the role of liposomes in removal of bilirubin from systemic circulation of the hyperbilirubinemic rats. Bilirubin has been demonstrated to possess inherent tendency to interact with liposomes through ionic as well as hydrophobic interactions. The size as well as lamellarity of the liposomes does not seem to affect their binding with bilirubin. However, the charge on the surface of liposomes plays an important role in bilirubin-liposome interaction, e.g., bilirubin binds more extensively with positively charged liposomes as compared to the neutral or negatively charged liposomes. The present study further demonstrates that liposomes were effective in reducing the increased plasma bilirubin level in hyperbilirubinemic model animals as well. The results of the study suggest that positively charged liposome-mediated selective homing of excess plasma bilirubin to the hepatocytes seems to offer an important strategy in management of hyperbilirubinemic conditions.     

Binding of bilirubin with albumin-coupled liposomes: implications in the treatment of jaundice

In the present study, we demonstrated the suitability of liposomes as a method of removing plasma bilirubin in hyperbilirubinemic rats. The liposomes have innate tendency to bind with bilirubin through hydrophobic interaction. Among different types of liposomes, the positively charged liposomes were found to have maximum affinity to free bilirubin. However, the entrapment or coupling of serum albumin on the surface of egg phosphatidylcholine liposomes can render a several-fold increase in their bilirubin binding capacity. The proteoliposomes were able to preferentially bind with bilirubin even in the presence of erythrocytes. Interestingly, these liposomes were found to displace bilirubin bound on the surface of erythrocytes as well. The results of the present study further demonstrate that albumin-bearing liposomes were equally effective in removing plasma bilirubin in experimental jaundiced animals. These observations indicate that liposome-mediated selective homing of excess plasma bilirubin to the liver cells (cf. hepatocytes) may help in the development of safer strategy for the treatment of hyperbilirubinemic conditions in the model animals.     

Binding of bilirubin with albumin-coupled liposomes: implications in the treatment of jaundice

In the present study, we demonstrated the suitability of liposomes as a method of removing plasma bilirubin in hyperbilirubinemic rats. The liposomes have innate tendency to bind with bilirubin through hydrophobic interaction. Among different types of liposomes, the positively charged liposomes were found to have maximum affinity to free bilirubin. However, the entrapment or coupling of serum albumin on the surface of egg phosphatidylcholine liposomes can render a several-fold increase in their bilirubin binding capacity. The proteoliposomes were able to preferentially bind with bilirubin even in the presence of erythrocytes. Interestingly, these liposomes were found to displace bilirubin bound on the surface of erythrocytes as well. The results of the present study further demonstrate that albumin-bearing liposomes were equally effective in removing plasma bilirubin in experimental jaundiced animals. These observations indicate that liposome-mediated selective homing of excess plasma bilirubin to the liver cells (cf. hepatocytes) may help in the development of safer strategy for the treatment of hyperbilirubinemic conditions in the model animals.     

Degradation of plasma bilirubin by a bilirubin oxidase derivative which has a relatively long half-life in the circulation

To enhance degradation of unconjugated bilirubin in hyperbilirubinemic subjects, we synthesized a bilirubin oxidase (EC 1.3.3.5) (BO) derivative (PEGBO) by covalently linking (2,4-bis[O-methoxy(polyethyleneglycol)]-6-chloro-s-triazine) (PEG) to the enzyme. Intravenously injected BO in rats disappeared from the circulation with a half-life of 2.5 min; the half-life of PEGBO was 190 min. Intravenously injected BO minimally and transiently decreased plasma bilirubin levels in jaundiced Gunn rats and in bile-duct-ligated jaundiced rats. In contrast, PEGBO rapidly and substantially decreased plasma bilirubin levels and the effect persisted for longer than 3 h. Renal dysfunction often occurs in patients with liver diseases. To study the role of bilirubin toxicity for the kidney, functions of transtubular transport for organic anions was measured in bile-duct-ligated jaundiced animals before and after treatment with PEGBO. Bile duct ligation decreased urinary excretion of phenolsulfophthalein (PSP), an organic anion used for renal function test. Treatment of the jaundiced animals with PEGBO increased the rate of PSP disappearance from the circulation and normalized its urinary excretion. Thus, PEGBO might be useful for the study of bilirubin toxicity in jaundiced animals.     

Targeted suppression of heme oxygenase-1 by small interference RNAs inhibits the production of bilirubin in neonatal rat with hyperbilirubinemia

Background  

Excessive accumulation of bilirubin contributes to neonatal hyperbilirubinemia in rats. Heme oxygenase (HO) is one of the rate-limiting enzymes in catabolizing heme to bilirubin. In the present study, we investigated whether suppression of rat HO-1 (rHO-1) expression by small interference RNAs (siRNAs) reduces bilirubin levels in hyperbilirubinemic rats.     

Impaired fertility in the jaundiced female (Gunn) rat

The hyperbilirubinemic female Gunn rat has been reported to have impaired fertility. A total of 267 jaundiced (j/j) and 91 nonjaundiced (+/j) female Gunn rats were used in a series of experiments to characterize the nature of this reduced fertility. Sixteen percent of the jaundiced females mated and delivered litters which were characteristically small in number (4.5 pups). A comparison of the gross observations at necropsy of jaundiced and nonjaundiced pregnant rats indicated that the number of implantation sites and live fetuses were significantly lower in the jaundiced females. The number of fetal resorptions in these rats were significantly higher; whereas, the number of corpra lutea was similar for both genotypes. The significantly lower plasma bilirubin levels in the pregnant jaundiced rats compared to the nonpregnant suggested that the observed effect on fertility was related to the concentration of plasma bilirubin.     

An analysis of the DOCA-salt model of hypertension in HO-1-/- mice and the Gunn rat

Heme oxygenase-1 (HO-1) is induced in the vasculature in the DOCA-salt model of hypertension in rats. Whereas the HO system and its products may exert vasodilator effects, recent studies have suggested that the HO system may predispose to hypertension. The present study examined the effects of selected components of the HO system, specifically, the HO-1 isozyme and the product bilirubin in the DOCA-salt model of systemic hypertension; the experimental approach employed mutant rodent models, namely, the HO-1(-/-) mouse and the hyperbilirubinemic Gunn rat. DOCA-salt induced HO-1 protein in the aorta in HO-1(+/+) mice and provoked a significant rise in systolic arterial pressure in HO-1(-/-) mice but not in HO-1(+/+) mice; this effect could not be ascribed to impaired urinary sodium excretion or impaired glomerular filtration rate in the DOCA-salt-treated HO-1(-/-) mice. The administration of DOCA salt to uninephrectomized rats significantly increased systolic arterial pressure in wild-type rats, an effect that was attenuated in the mutant Gunn rat; this reduction in systemic hypertension in the DOCA-salt-treated Gunn rat was not due to a greater induction of HO-1 in the vasculature or to a more avid urinary sodium excretion. DOCA-salt impaired endothelium-dependent and endothelium-independent vasorelaxation in wild-type rats but not in Gunn rats; prior exposure to bilirubin repaired the defect in endothelium-dependent vasorelaxation in aortic rings in DOCA-salt-treated rats. DOCA salt stimulated vascular production of superoxide anion in wild-type but not in Gunn rats. We suggest that HO-1 and the product bilirubin may exert a countervailing effect in the DOCA-salt model of systemic hypertension.     

Involvement of multidrug resistance-associated protein 2 (ABCC2/Mrp2) in biliary excretion of micafungin in rats

The drug transporter, multidrug resistance-associated protein 2 (ABCC2/Mrp2), is known to play important roles in excretion of various drugs. In the present study, we investigated whether Mrp2 is involved in the transport of micafungin, a newly developed antifungal agent. When Sprague-Dawley rats received an intravenous injection of micafungin (1 mg/kg) in combination with cyclosporine, the cyclosporine significantly delayed the disappearance of micafungin from plasma and decreased the systemic clearance and volume of distribution at steady-state of micafungin to 54% and 65% of the corresponding control values, respectively. When Sprague-Dawley rats received a constant-rate infusion of micafungin, cyclosporine significantly decreased the steady-state biliary clearance of micafungin (~80%). A significant decrease in the biliary clearance of micafungin (~60%) was observed in Eisai hyperbilirubinemic rats, which have a hereditary deficiency in Mrp2. The present findings at least suggest that Mrp2 is involved mainly in the hepatobiliary excretion of micafungin in rats.     

Role of glucagon in the splanchnic and systemic hemodynamic changes induced by portal-systemic blood shunting

Portal-systemic blood shunting is often accompanied by hyperglucagonemia and hemodynamic changes. To determine this causal relation, splanchnic and systemic hemodynamics (radioactive microspheres) and plasma glucagon levels (radioimmunoassay) were assessed in conditions of total portal-systemic shunting in portacaval-shunted (PCS) rats and in sham-operated (SO) normal rats. To compare these results, another hemodynamic study was undertaken basally and during glucagon infusion in nonoperated normal rats. PCS rats showed a threefold greater plasma glucagon concentration than SO animals (924 +/- 134 vs. 309 +/- 18 pg/ml, p less than 0.01), and they developed a hyperdynamic splanchnic circulation with higher portal venous inflow than SO rats (8.29 +/- 1.1 vs. 5.09 +/- 0.4 ml/min/100 g, p less than 0.05). Infusion of a pharmacological dose of glucagon in normal rats increased portal venous inflow (from 4.92 +/- 0.33 to 6.24 +/- 0.48 ml/min/100 g, p less than 0.05) so as to imply this hormone in the development of the hyperdynamic splanchnic circulation in conditions of portal-systemic shunting. However, the discrepancies in systemic hemodynamics between PCS and glucagon-infused rats may be a result of the different plasma glucagon levels reached in the two groups.     

Galanin influences vasopressin and oxytocin release from the hypothalamo-neurohypophysial system of salt loaded rats.

Galanin is a peptide present in the nervous system and peripheral tissues which exerts a broad range of physiological functions. The influence of centrally administered galanin (Gal; 100 pM i.c.v.) on arginine vasopressin (AVP) and oxytocin (OT) content in the hypothalamus and neurohypophysis as well as on their blood plasma concentration was estimated in male Wistar rats drinking ad libitum 2% solution of natrium chloride per 48 hours. In euhydrated rats and subsequently applied i.c.v. with Gal a significant fall in the hypothalamic and neurohypophysial content of OT but not AVP was observed, however, without simultaneous changes in these neurohormones blood plasma concentration. On the contrary, i.c.v. injection of Gal to salt-loaded rats caused a marked raise in AVP and OT level in the hypothalamus and neurohypophysis with subsequent diminution of both neurohormones concentration in blood plasma. These results suggest that in euhydrated rats Gal has an inhibitory influence on the biosynthesis as well as axonal transport of OT, but not AVP. On the contrary, in salt-loaded rats galanin restricts secretion of both neurohormones into the systemic circulation.     

Systemic leakage of 15 micron radioactive microspheres over a period of 24 hours, in awake late-pregnant guinea pigs

The present study was designed to determine the reliability of regional blood flow measurements with 15 microns radioactive microspheres, when 24 h have elapsed between reference sampling and sacrifice. The study was performed in 12 chronically-instrumented late-pregnant guinea pigs. The fraction of microspheres recovered from the lungs was consistently higher, by about 2% of the cardiac output after such a 24-h period, as compared to microsphere experiments performed immediately prior to sacrifice. This finding suggests dislodgement of radioactive particles from the systemic circulation between the time of completion of reference sampling and the subsequent 24 h. No correlation could be demonstrated between the change in lung fraction and the change in any organ fraction in particular. Therefore, it is highly unlikely that the microspheres accumulated in the lungs over this period originate from the placenta or any other specific organ. It is concluded that in the awake late-pregnant guinea pig, the 2% loss of microspheres from the systemic circulation during the 24 h following reference sampling, does not invalidate the cardiac output distribution derived.     

Characterization of inducible nature of MRP3 in rat liver

We found previously that expression of multidrug resistance-associated protein (MRP) 3 is induced in a mutant rat strain (Eisai hyperbilirubinemic rats) whose canalicular multispecific organic anion transporter (cMOAT/MRP2) function is hereditarily defective and in normal Sprague-Dawley (SD) rats after ligation of the common bile duct. In the present study, the inducible nature of MRP3 was examined, using Northern and Western blot analyses, in comparison with that of other secondary active [Na(+)-taurocholic acid cotransporting polypeptide (Ntcp), organic anion transporting polypeptide 1 (oatp1), and organic cation transporter (OCT1)] and primary active [P-glycoprotein (P-gp), cMOAT/MRP2, and MRP6] transporters. alpha-Naphthylisothiocyanate treatment and common bile duct ligation induced expression of P-gp and MRP3, whereas expression of Ntcp, oatp1, and OCT1 was reduced by the same treatment. Although expression of MRP3 was also induced by administration of phenobarbital, that of cMOAT/MRP2, MRP1, and MRP6 was not affected by any of these treatments. Moreover, the mRNA level of MRP3, but not that of P-gp, was increased in SD rats after administration of bilirubin and in Gunn rats whose hepatic bilirubin concentration is elevated because of a defect in the expression of UDP-glucuronosyl transferase. However, the MRP3 protein level was not affected by bilirubin administration. Although the increased MRP3 mRNA level was associated with the increased concentration of bilirubin and/or its glucuronides in mutant rats and in SD rats that had undergone common bile duct ligation or alpha-naphthylisothiocyanate treatment, we must assume that factor(s) other than these physiological substances are also involved in the increased protein level of MRP3.     

Effect of obstructive jaundice on the fate of a nephrophilic organic anion in the rat

Renal transtubular transport of phenolsulfophthalein (PSP), a nephrophilic organic anion that circulates bound to albumin, was studied in normal and bile-duct-ligated rats. Intravenously injected PSP disappeared from the circulation more rapidly in bile-duct-ligated jaundiced rats than in intact animals. However, urinary excretion of PSP was significantly lower in the former than in the latter. Kinetic analysis revealed that binding of PSP to plasma protein(s) was significantly lower with jaundiced rats than with intact animals. Addition of albumin to plasma samples from bile-duct-ligated rats markedly increased PSP binding. The decreased PSP binding returned to normal levels after treating the jaundiced plasma with bilirubin oxidase, an enzyme that degrades amphiphilic bilirubin to water soluble metabolites. These results suggest that bilirubin might be the major metabolite that occupied the PSP binding site(s) on albumin in jaundiced rats. When PSP was injected bound to equimolar amount of albumin, the rate of PSP disappearance from the circulation decreased and urinary excretion of the ligand increased markedly; urinary excretion of PSP was significantly larger in bile-duct-ligated rats than in intact animals. These results suggest that the renal transport capacity for amphiphilic organic anions, such as PSP, might be increased compensatively in bile-duct-ligated animals, and that the apparent decrease in renal secretory transport for PSP might result from, at least in part, random distribution of the ligand to extrarenal tissues due to decrease in the binding activity of albumin.     

The role of albumin in the hepatic transport of bilirubin: studies in mutant analbuminemic rats

Bilirubin and other cholephilic organic anions are bound to albumin in the circulation; their hepatic uptake involves a carrier-mediated process. To investigate the possible role of serum albumin in the transhepatic transport of a cholephilic ligand, plasma clearance of radioactive bilirubin and its biliary excretion as well as its interaction with plasma proteins were compared between normal and mutant analbuminemic rats (NAR). With a tracer amount of 3H-labeled bilirubin, its plasma clearance and biliary excretion were comparable in both animal groups. However, the plasma clearance of a loading dose of the ligand was significantly increased and its biliary recovery was low in NAR as compared with normal animals. In accord with these findings in vivo, gel permeation chromatographic analysis revealed that the bilirubin binding capacity of serum proteins was significantly lower in NAR than in control animals. When bilirubin was administered to NAR as a mixture with equimolar albumin, its plasma disappearance was considerably decreased and its biliary recovery was increased. Similar effects were observed when albumin was replaced by an equimolar amount of glutathione S-transferases (ligandins). These observations indicate that, although ligand-protein interaction in the circulation is important for directing bilirubin to the plasma membranes of the hepatocyte, this mechanism is not specific for albumin.     

Bolus administration of obestatin does not change glucose and insulin levels neither in the systemic nor in the portal circulation of the rat

Obestatin is a second peptide derived from the preproghrelin polypeptide. It was originally thought to have anorexigenic effects, thereby functioning as an antagonist of ghrelin. However, this has been a subject of debate ever since. Since acylated ghrelin strongly induces insulin resistance, it could be hypothesized that obestatin plays a role in glucose homeostasis as well. In the present study we evaluated the effect of obestatin on glucose and insulin metabolism in the systemic and portal circulation. Obestatin 200 nmol/kg was administered systemically as a single intravenous bolus injection to fasted pentobarbital anesthetized adult male Wistar rats. Up to 50 min after administration, blood samples were taken to measure glucose and insulin concentrations, both in the portal and in the systemic circulation. The effect of obestatin was evaluated in fasted and in glucose-stimulated conditions (IVGTT) and compared to control groups treated with saline or IVGTT, respectively. Intravenous administration of obestatin did not have any effect on glucose and insulin concentrations, neither systemic nor portal, when compared to the control groups. Only the glucose peak 1 min after administration of IVGTT was slightly higher in the obestatin treated rats: 605.8 ± 106.3% vs. 522.2 ± 47.1% in the portal circulation, respectively (NS), and 800.7 ± 78.7% vs. 549.6 ± 37.0% in the systemic circulation, respectively (P < 0.02), but it can be debated whether this has any clinical relevance. In the present study, we demonstrated that intravenously administered obestatin does not influence glucose and insulin concentrations, neither in the portal nor in the systemic circulation.     

Selective transport of cytokine-induced neutrophil chemoattractant from the lung to the blood facilitates pulmonary neutrophil recruitment

The CXC chemokines cytokine-induced neutrophil chemoattractant (CINC) and macrophage inflammatory protein-2 (MIP-2) are potent neutrophil chemoattractants in rats. We have previously shown that CINC, unlike MIP-2 and most other proinflammatory cytokines, is elevated in the systemic circulation in response to an intratracheal (IT) challenge. Therefore, we hypothesized that CINC generated within the lung selectively enters the vascular compartment to facilitate pulmonary neutrophil recruitment. Rats were administered IT LPS, and plasma CINC and MIP-2 levels were measured 90 min and 4 h after injection, along with mRNA expression in lung, spleen, liver, and kidney. Ninety minutes and 4 h after IT LPS, CINC and MIP-2 mRNA expression were largely confined to lung homogenate, but of the two chemokines, only CINC was present in plasma. In separate experiments, rats received IT injections of recombinant CINC and/or MIP-2. Here, plasma levels of CINC, but not MIP-2, were significantly increased throughout the 4-h observation period. This finding was verified by individually administering (125)I-labeled forms of each chemokine. Instillation of recombinant MIP-2 or CINC into the lung increased the number of neutrophils recovered in bronchoalveolar lavage fluid at 4 h, and this effect was enhanced when both chemokines were administered together. In addition, intravenous (IV) CINC, but not IV MIP-2, increased pulmonary neutrophil recruitment in response to IT MIP-2. Our results show that CINC, in contrast to MIP-2, is selectively transported from the lung to the systemic circulation, where it promotes neutrophil migration into the lung in response to a chemotactic stimulus.     

New sorbent for bilirubin removal from human plasma: Cibacron Blue F3GA-immobilized poly(EGDMA–HEMA) microbeads

Cibacron Blue F3GA-immobilized poly(EGDMA–HEMA) microbeads were investigated as a specific sorbent for bilirubin removal from human plasma. The poly(EGDMA–HEMA) microbeads were prepared by a modified suspension copolymerization technique. Cibacron Blue F3GA was covalently coupled to the poly(EGDMA–HEMA) microbeads via the nucleophilic reaction between the chloride of its triazine ring and the hydroxyl groups of the HEMA molecule, under alkaline conditions. Bilirubin adsorption was investigated from hyperbilirubinemic human plasma on the poly(EGDMA–HEMA) microbeads containing different amounts of immobilized Cibacron Blue F3GA, (between 5.0–16.5 μmol/g). The non-specific bilirubin adsorption on the unmodified poly(EGDMA–HEMA) microbeads were 0.32 mg/g from human plasma. Higher bilirubin adsorption values, up to 14.8 mg/g, were obtained with the Cibacron Blue F3GA-immobilized microbeads. Bilirubin molecules interacted with these sorbents directly. Contribution of albumin adsorption on the bilirubin adsorption was pronounced. Bilirubin adsorption increased with increasing temperature.     

Reduced circulating oxidized LDL is associated with hypocholesterolemia and enhanced thiol status in Gilbert syndrome

A protective association between bilirubin and atherosclerosis/ischemic heart disease clearly exists in vivo. However, the relationship between bilirubin and in vivo oxidative stress parameters in a clinical population remains poorly described. The aim of this study was to assess whether persons expressing Gilbert syndrome (GS; i.e., unconjugated hyperbilirubinemia) are protected from thiol oxidation and to determine if this, in addition to their improved lipoprotein profile, could explain reduced oxidized low-density lipoprotein (oxLDL) status in them. Forty-four matched GS and control subjects were recruited and blood was prepared for the analysis of lipid profile and multiple plasma antioxidants and measures of oxidative stress. GS subjects possessed elevated plasma reduced thiol (8.03±1.09 versus 6.75±1.39 nmol/mg protein; P<0.01) and glutathione concentrations (12.7±2.39 versus 9.44±2.45 μM; P<0.001). Oxidative stress status (reduced:oxidized glutathione; GSH:GSSG) was significantly improved in GS (0.49±0.16 versus 0.32±0.12; P<0.001). Protein carbonyl concentrations were negatively associated with bilirubin concentrations and were significantly lower in persons with >40 μM bilirubin versus controls (<17.1 μmol/L; P<0.05). Furthermore, absolute oxLDL concentrations were significantly lower in GS subjects (P<0.05). Forward stepwise regression analysis revealed that bilirubin was associated with increased GSH:GSSG ratio and reduced thiol concentrations, which, in addition to reduced circulating LDL, probably decreased oxLDL concentrations within the cohort. In addition, a marked reduction in total cholesterol concentrations in hyperbilirubinemic Gunn rats is presented (Gunn 0.57±0.09 versus control 1.69±0.40 mmol/L; P<0.001), arguing for a novel role for bilirubin in modulating lipid status in vivo. These findings implicate the physiological importance of bilirubin in protecting from atherosclerosis by reducing thiol and subsequent lipoprotein oxidation, in addition to reducing circulating LDL concentrations.     

Alterations in the Cell Cycle in the Cerebellum of Hyperbilirubinemic Gunn Rat: A Possible Link with Apoptosis?

Severe hyperbilirubinemia causes neurological damage both in humans and rodents. The hyperbilirubinemic Gunn rat shows a marked cerebellar hypoplasia. More recently bilirubin ability to arrest the cell cycle progression in vascular smooth muscle, tumour cells, and, more importantly, cultured neurons has been demonstrated. However, the involvement of cell cycle perturbation in the development of cerebellar hypoplasia was never investigated before. We explored the effect of sustained spontaneous hyperbilirubinemia on cell cycle progression and apoptosis in whole cerebella dissected from 9 day old Gunn rat by Real Time PCR, Western blot and FACS analysis. The cerebellum of the hyperbilirubinemic Gunn rats exhibits an increased cell cycle arrest in the late G0/G1 phase (p < 0.001), characterized by a decrease in the protein expression of cyclin D1 (15%, p < 0.05), cyclin A/A1 (20 and 30%, p < 0.05 and 0.01, respectively) and cyclin dependent kinases2 (25%, p < 0.001). This was associated with a marked increase in the 18 kDa fragment of cyclin E (67%, p < 0.001) which amplifies the apoptotic pathway. In line with this was the increase of the cleaved form of Poly (ADP-ribose) polymerase (54%, p < 0.01) and active Caspase3 (two fold, p < 0.01). These data indicate that the characteristic cerebellar alteration in this developing brain structure of the hyperbilirubinemic Gunn rat may be partly due to cell cycle perturbation and apoptosis related to the high bilirubin concentration in cerebellar tissue mainly affecting granular cells. These two phenomena might be intimately connected.