The Effect Of Interleukin-2 On Survival And Morphology In Rat Primary Cortical Neurons

INTRODUCTION:Interleukin-2 (IL-2) is not only an important immunoregulatory molecule, it is also an important neuroregulatory molecule in the CNS. Duing the past ten years, IL-2-like bioactivity, IL-2-like immunoreactivity, IL-2-like mRNA, IL-2 binding sites, IL-2 receptor and IL-2 receptor(IL-2R) immunoreactivity have been reported. They mainly locate on neurons and glias. Investigations in vivo and in vitro have elucidated the following: IL-2 and IL-2R have been detected in the brain; IL-2 promotes survival and neurite extension of cultured neurons, stimulates oligodendrocyte proliferation and maturation, affects the hypothalamic-pituitary function and produces behavioral and electrocorticogram spectrum changes. In addition, It has reported that IL-2 has an analgesic effect in the CNS and stimulating the proliferation and differentiation of T lymphocytes.Concerning about the effect of IL-2 on CNS, investigations in some laboratories have demonstrated that IL-2 showed survival-promoting effects on hippocampus in high cell density cultures but did not show any effect on the neuronal survival in low cell density culture. Furthermore, IL-2 promoted the extension of processes of hippocampal neurons independent on density. These results suggest that IL-2 supportsneuronal survival indirectly and promotes extension by directly acting on brain neurons. So the effects of IL-2 on neuronal survival and morphology are mediated by different mechanism.However, others have reported that IL-2 may function as an inhibitory factor. After three days of exposure to IL-2, neurite retractions, cellular swelling, vacuolations and cell rapture were evident. So the effect of IL-2 on neurons was still disputed. Furthermore little is known about the possible role of IL-2 on morphology in cortical neurons. In this thesis we investigate the effects of IL-2 on cortical neurons.OBJECTIVES:1. To investigate the effect of IL-2 on survival of cortical neurons.2. To test the effect of IL-2 is time-dependent or density-dependent.3. To clarify the effect of IL-2 on morphology of cortical neurons.METHODS:1. The effect of IL-2 on survival: Primary cortical neurons were prepared from brains of newborn rats. The neurons at DIV(days in vitro) 10 were exposed to IL-2 for 24 hours, and the survival was evaluated by MTT assay.2. The effect of IL-2 on morphology: the neurons at DIV 4 were transfected with GFP-actin gene, and then the morphology was investigated after 48 hours, Data were analyzed by Excel 2003 and Metamorph software.RESULTS:1. Addition of recombinant human IL-2 significantly supported the survival of cortical neurons in high cell density culture. As the result of MTT assay, OD value changes from 0.46 ± 0.04 to 0.61 ± 0.03 (P<0.05), And the effect is concentration-dependent. But it did not show any effect on the neuronal survival inlow cell density culture, OD value changes from 0.30±0.02 to 0.31±0. 05 (P> 0.05).2. The effect of IL-2 on cortical neurons is time-dependent and concentration -dependent. When exposed to IL-2 for 12 hours, there is no apparently effect, but it is effective after 24 hours, and the higher in concentration, the stronger in function.3. The effect of IL-2 on cortical neurons morphology: Addition of IL-2 promoted extension for primary neurons in low cell density cultures, but it did not function on protrusion of cell body and branch of primary neurite.CONCLUSIONS:1. IL-2 significantly promoted the survival of cortical neurons in high cell density culture, but did not show any effect on the neuronal survival in low density culture. In high cell density culture condition, neuron-neuron and neuron-glia is maximized. These results suggest that IL-2 probably supports neurons survival indirectly. It is possible that IL-2 stimulates secretion of some neurotrophic factor from neuron or glia to support survival.2. Addition of IL-2 promoted the extension of processes of cortical neurons in low cell density cultures. Since neuronal-glias and neuronal-neuronal interaction was strongly restricted in low density culture, IL-2 probably affects neuronal morphology by directly acting on the neuronal cells. The different effects of IL-2 on branch and extension suggest that neurite extension and branching are independently regulated by different mechanisms.