Sex-specific regional brain bilirubin content in hyperbilirubinemic Gunn rat pups.

BACKGROUND: The hyperbilirubinemic j/j Gunn rat is frequently used to study the effects of neonatal hyperbilirubinemia on the developing central nervous system (CNS). Despite evidence that the cerebellar region and males are predisposed to bilirubin-induced brain injury in this animal model, there are limited regional and no sex-specific brain bilirubin content data. OBJECTIVE/METHODS: To characterize and contrast the regional (cortex, brainstem, cerebellum) and sex-specific CNS bilirubin contents of hyperbilirubinemic j/j Gunn rat pups and their age-matched (15-19 days) nonjaundiced J/j counterparts. Pups were injected 24 h prior to sacrifice with sulfadimethoxine (200 mg/kg i.p.) to enhance the CNS bilirubin content. RESULTS: The CNS bilirubin contents in each region and total serum bilirubin levels were significantly greater in jaundiced j/j pups versus nonjaundiced J/j pups. Within the sulfadimethoxine-treated male j/j cohort, the mean brain bilirubin content was highest in the cerebellum (18.9 +/- 7.8 microg/g), intermediate in the brainstem (10.7 +/- 8.0 microg/g), and lowest in the cortex (4.7 +/- 3.0 microg/g) (F = 11.31, p < 0.001 by ANOVA), and the cerebellar bilirubin level was significantly higher than in the littermate-matched sulfadimthoxine-treated j/j female pups (p < 0.02). The serum albumin levels were not different between j/j male and j/j female pups. CONCLUSIONS: We conclude that the brain bilirubin content of hyperbilirubinemic j/j Gunn rat pups is greater than in nonjaundiced J/j pups and varies as a function of CNS region and sex. We speculate that the higher cerebellar bilirubin content may preferentially predispose male j/j Gunn rat pups to bilirubin-induced neurotoxicity.

Cryopreserved microencapsulated hepatocytes--transplantation studies in Gunn rats.

Hepatocyte transplantation has been shown to provide significant metabolic support in several animal models of liver diseases. However, for it to be a viable alternative for supplementation of liver function in disease, large quantities of isolated hepatocytes would be necessary. At the present time there are no inexpensive routine methods for cryopreservation of hepatocytes. Existing procedures are cumbersome and require expensive programmable freezers. Hepatocyte cultures are sensitive and easily damaged in handling. By utilizing techniques of microencapsulation and cryopreservation we have attempted to overcome these problems. We have developed a simple, convenient, and inexpensive technique for the long-term storage of hepatocytes. Biological activity of the nonfrozen isolated encapsulated hepatocytes (IEH) and cryopreserved IEH (cIEH) was assessed both in tissue culture and by transplantation in Gunn rats. Significant urea and protein syntheses were detectable during the 10-day culture period even in the 30-day cIEH. Additionally, transplanted IEH and cIEH significantly reduced hyperbilirubinemia in Gunn rats for up to 30 days posttransplantation. Control (empty) microcapsules did not lower serum bilirubin levels. Thus we conclude: (1) cryopreservation of IEH is a convenient and cost-effective method for preserving and storing hepatocytes; (2) cryopreserved IEH function as well as nonfrozen IEH both in vitro and in vivo; (3) microencapsulation may protect hepatocytes from the adverse effects of cryopreservation.

Jaundiced Gunn rats have increased synaptic delays in the ventral cochlear nucleus.

Recordings were made in vitro from cochlear nuclei of Gunn rats, a strain with a recessive mutation that predisposes rats to hyperbilirubinemia at birth. Delays between shocks to the auditory nerve and earliest synaptic responses of the cochlear nuclear neurons were on average longer in Gunn rats than in heterozygotes. Injections of sulfonamide further increased average synaptic delays in jaundiced rats. Responses to injected current in rats were like those in mice.

Purkinje cell abnormalities and synaptogenesis in genetically jaundiced rats (Gunn rats).

The course of cytological abnormalities and synaptogenesis of Purkinje cells were investigated in the culmen of cerebella from homozygous Gunn rats with hereditary hyperbilirubinemia from postnatal day 7 to adulthood (5-10 months old). The affected Purkinje cells were abundant at day 7. A large number of Purkinje cells reached the fully advanced stage of degeneration during the ensuring 16 days and disappeared between days 12 and 30. The Purkinje cells remaining at day 30 were less affected and recovered by the adult stage. Various abnormalities in Purkinje cell synaptogenesis with the parallel fibers, climbing fibers, and basket and stellate cell axons were observed. Primitive junctions between parallel fibers and Purkinje dendritic shafts were often found in adulthood. The parallel fiber boutons lacking postsynaptic partners and facing astrocytic processes were often noted from day 18 to adulthood. The persistence of such presynaptic elements suggests some mechanisms for stabilizing the synaptic elements once they have been formed. Many of the parallel fiber synaptic boutons with or without their postsynaptic partners were enlarged and were assumed to be transsynaptically affected by Purkinje cell damage. A number of climbing fiber synapses with perisomatic process of Purkinje cells, which are transient in normal synaptogenesis, were present at day 30 and a few of them were still found even in adulthood. Basket and stellate cell synapses were often found in abundance on the remaining Purkinje cells in adulthood, though they were not frequently encountered during the development period.

Biliary excretion in Sprague-Dawley and Gunn rats during manganese-bilirubin-induced cholestasis.

We previously showed that alterations of the bile canalicular membrane are likely to occur following a cholestatic regimen composed sequentially of manganese and bilirubin. The present study was designed primarily to investigate the biliary excretion of organic bile constituents following administration of the manganese-bilirubin combination. Experiments in hyperbilirubinemic Gunn rats were also performed to determine whether the unconjugated or the conjugated form of bilirubin is involved in this cholestatic interaction. Male Sprague-Dawley rats and male homozygous Gunn rats were given the following (i.v.): (a) manganese (4.5 mg per kg); (b) unconjugated bilirubin (25 mg per kg); (c) bilirubin ditaurate (38 mg per kg); (d) manganese-unconjugated bilirubin, or (e) manganese-bilirubin ditaurate. Bile flow was measured and bile was analyzed for manganese, total bilirubin, bile salts, cholesterol and phospholipid content. The results show that: (i) manganese-unconjugated bilirubin treatment caused about a 50% reduction in bile flow in Sprague-Dawley rats, whereas in Gunn rats the manganese-bilirubin ditaurate treatment resulted in about a 75% reduction, and (ii) in both strains, bile salt excretion was not appreciably modified during the cholestatic phase, as biliary bile salt concentration increased. The results suggest that although important differences regarding the form of bilirubin apparently exist, unconjugated bilirubin could be implicated in the cholestatic interaction in both strains of rats. Manganese-bilirubin-induced cholestasis is not related to a defect in bile salt excretion. The latter supports our contention that diminished canalicular membrane permeability to water is likely to be a key factor in this form of experimental cholestasis.

Salicylate nephropathy in the Gunn rat: potential role of prostaglandins.

We examined the potential role of prostaglandins in the development of analgesic nephropathy in the Gunn strain of rat. The homozygous Gunn rats have unconjugated hyperbilirubinemia due to the absence of glucuronyl transferase, leading to marked bilirubin deposition in renal medulla and papilla. These rats are also highly susceptible to develop papillary necrosis with analgesic administration. We used homozygous (jj) and phenotypically normal heterozygous (jJ) animals. Four groups of rats (n = 7) were studied: jj and jJ rats treated either with aspirin 300 mg/kg every other day or sham-treated. After one week, slices of cortex, outer and inner medulla from one kidney were incubated in buffer and prostaglandin synthesis was determined by radioimmunoassay. The other kidney was examined histologically. A marked corticomedullary gradient of prostaglandin synthesis was observed in all groups. PGE2 synthesis was significantly higher in outer medulla, but not cortex or inner medulla, of jj (38 +/- 6 ng/mg prot) than jJ rats (15 +/- 3) (p less than 0.01). Aspirin treatment reduced PGE2 synthesis in all regions, but outer medullary PGE2 remained higher in jj (18 +/- 3) than jJ rats (9 +/- 2) (p less than 0.05). PGF2 alpha was also significantly higher in the outer medulla of jj rats with and without aspirin administration (p less than 0.05). The changes in renal prostaglandin synthesis were accompanied by evidence of renal damage in aspirin-treated jj but not jJ rats as evidenced by: increased incidence and severity of hematuria (p less than 0.01); increased serum creatinine (p less than 0.05); and increase in outer medullary histopathologic lesions (p less than 0.005 compared to either sham-treated jj or aspirin-treated jJ). These results suggest that enhanced prostaglandin synthesis contributes to maintenance of renal function and morphological integrity, and that inhibition of prostaglandin synthesis may lead to pathological renal medullary lesions and deterioration of renal function.