Neuroprotective action of erythropoietin against cell death induced by hypoxia in SH-SY5Y cells

WENKER SHIRLEY; CHAMORRO MARÍA EUGENIA; VOTA DAIANA; VITTORI DANIELA; NESSE, ALCIRA

Buzios

Congreso; First Congress IBRO-LARC of Neurosciences of Latin America, the Caribbean and the Iberian Peninsula; 2008

Sociedad Brasilera de Neurociencias y Comportamiento, Sociedad Argentina de Investigación en Neurociencias, Sociedad Chilena de Neurociencia, Sociedad de Neurociencia del Uruguay

OBJECTIVES: On  exposure  to  hypoxia,  brain  tissue  becomes  sensitive  to  cell  injury and death, which could cause neurodegenerative outcomes. Erythropoietin  (Epo) has emerged as a multifunctional  factor  that could play a significant role  in  tissues outside the  hematopoietic  system. We  decided  to  investigate whether  Epo might  be  able  to prevent  deleterious  effects  of  hypoxia. METHODS: Human  neuroblastoma SH-SY5Y cells exposed to hypoxia were used. Treatments with Epo were made before and after hypoxia  injure.  Morphological  alterations  were  observed  by  scanning  electron microscopy  (SEM).  Cell  viability  was  determined  by  MTT  assay.  Apoptosis  was detected  by  DNA  laddering  and  by  nuclear  fluorescence  microscopy  (Hoechst). Expression of Bcl-2 members was analyzed by RT-PCR. RESULTS: Hypoxia exposure for  16  or  24  h  caused  morphological  changes  like  lost  of  neuritogenesis  and  cell detachment. Under this condition, a significantly reduced cell viability dependent on the exposure-length (H16 71±9%, H24 28±15%; P<0.05 with respect to controls; n=4) and a significant increase in cell apoptosis were detected (H16 35±2% vs. controls P<0.05; n=5). These results are in accordance with cell morphology alterations and membrane blebbing  observed  by  SEM.  The  effects  triggered  by  hypoxia  in  this  undifferentiated neuroblastoma  cell  line were  totally  prevented  by  a  previous  treatment with Epo  (25 U/ml,  9  h)  as  shown  by  data  of  cell  viability: H16  71±7%, E9/H16  100±4%; P<0.05; n=5, and percentage of apoptotic cells: H16 28±1%, E9/H16 7±2%; P<0.001; n=5. A post treatment with Epo up to 48 h after 16 h of hypoxia did not overcome the injures. Cell exposure  to hypoxia showed downregulation of RNAm  levels of  the antiapoptotic member Bcl-xL (RT-PCR) to near 45±12% of the control levels (P<0.05; n=3), while the previous treatment with Epo has the opposite effect (Bcl-xL RNAm: 147±12%; P<0.05). CONCLUSIONS:  these  results demonstrate  that  short  treatments with Epo  could not reverse  the  damages  caused  by SH-SY5Y  cell  cultures  under  low  oxygen  condition. However,  Epo  prevented  the  apoptosis  induced  by  hypoxia,  suggesting  a neuroprotective action partially dependent on the modulation of Bcl-xL.