Is bilirubin able to affect the cell cycle in Gunn rat brain? - An in vivo and in vitro study -

The hyperbilirubinemic jj Gunn rat is a well established animal model for Crigler-Najjar type I Syndrome and neonatal jaundice. Similarly to humans, they present neurological deficits and what is more a marked cerebellar hypoplasia with a prominent loss and degeneration of Purkinje cells and granule neurons. Since high levels of bilirubin have been proven to arrest the cell cycle progression, we addressed the question if the cerebellar hypoplasia observed in the hyperbilirubinemic Gunn rat could be somehow linked to a cell cycle arrest, and if this cell cycle arrest was affecting selectively primary cultures of astrocytes and cerebellar granule neurons. In the in vivo study we report that the high levels of bilirubin present in the cerebellum of hyperbilirubinemic Gunn rat cause a cell cycle arrest in the late G0/G1 phase, characterized by a decrease in the protein expression of Cyclin D1, Cyclin A, Cyclin A1 and most importantly Cdk2. Meanwhile an increase in protein expression of total Cyclin E, due to a rose in the levels of low molecular weight Cyclin E forms (a supposed attempt to bypass the cell cycle arrest), was in vain. Furthermore, we observed an increment in the 18 kDa fragment of Cyclin E (implicated in the amplification of the apoptotic pathway) suggesting us the presence of an increased apoptosis. Consistent with this speculation, the levels of the cleaved form of Poly (ADP-ribose) Polymerase (PARP-1) were increased. In the in vitro study we support the selectivity of bilirubin to damage specific cells as cerebellar granule neurons. Cerebellar granule cells viability was more affected respect to astrocytes in the same treatment conditions. The cell cycle was affected by high concentration of bilirubin only in cerebellar granule cells. We hypothesised that the characteristic cerebellar hypoplasia of hyperbilirubinemic Gunn rat may be due to the conjunction between cell cycle arrest and apoptosis, and that these two processes are intimately connected. Furthermore, only granule neurons cell cycle was affected. Cryopreservation has been used routinely in prolonged storage of many mammalian tissues. The cryopreservation of neural cells/ tissue started to be interesting after the successful transplantation of such tissues, mainly for research. Several studies have been performed to achieve cryopreservation of granule cells, however, for these cell types there is no defined protocol for cryopreservation with sufficient success to enable it to be incorporated into routine clinical practice. As we were thinking to perform cerebellar granule cells transplantation as a way to treat Gunn rats cerebellar hypoplasia, we started to set up a protocol for cerebellar granule cells cryopreservation using the slow-freezing methodology. Cerebellar granule cells were successfully cryopreserved with a protocol that involves the use of 10 % of DMSO as cryoprotective agent, a freezing rate of 2.1°C/min, and a fast (154.4°C/ min) rewarming at 39°C. The cells cryopreserved in this way had a good cell viability and were kept in culture for 7 days. More experiments have to be made to standardize this protocol.