The Influence Of Ammonium Chloride On Rat Hippocampal Neurons

Objective:To observe the effects of the morphology and the influence on cytoskeleton on ammonia intoxication in hippocampal neurons, to elucidate the mechanism of CNS dysfunction in patients with hepatic encephalopathy.Methods:1. Extract the rat hippocampus, and cultivate the rat hippocampal neurons.At the seventh day,to join different concentrations of NH₄ CL in hippocampal neurons.After24 hours,we adopt inverted microscope to observe neurons morphology, according to the change of neurons, choosing the appropriate ammonia concentration toestablish the ammonia poisoning model,and to simulate the hepatic encephalopathy;2. At the seventh day, we join 0.5ul NH₄CL（1mol / L）to hippocampal neurons. After24 hours,the neurons were stained with calcein-AM, we adopt fluorescence microscopy to observe the neurons,and to calculate the cellular density;3. At the ninth day, we join 0.5ul NH₄CL（1mol / L）to hippocampal neurons. After24 hours,neurons were stained with rhodamine-phalloidin,we adopt fluorescence microscopy and laser scanning confocal microscopy changes to observe the F-actin of neurons, and to count the F- actin fluorescence intensity,and Image-J were applied for statistical treatment of neurite.Results:1. Normal control group of neurons grew well, cell body plump, neurite profile clear,forming a network structure; no significant difference between the low concentration NH₄CL（0.125mmol/L） hippocampal neurons with normal control group, with the increasing concentration of nerve,cell bodies appear black particles,intracellular nucleus shrinkage, cracking, neurite varicose stiff, broken, or even disappear, in the high concentrations（5.0mmol/L） group,a large number of neuronal apoptosis;The neurite length decreases with the increase of concentration of NH₄ CL, and the neurite length of the 5 mmol/L group compared with the control group are statistical significant（P < 0.05）, 0.125, 0.5, 1.0, tendency compared with the control group of neurons length don’t have statistical significance（P > 0.05）;2.We identify the survival of hippocampal neurons with calcein-AM staining, that morphology of hippocampal are consistent compared with the control group, neurons grew well, staining, the fluorescence intensity of the hippocampal neurons（5mmol/L）stained by calcein are weaker,the cellular density are lower（P<0.05）;3.At the control group of neurons,F-actin bundles neatly into the main distribution in the cell bodies and projections. Ammonia poisoning group, F-actin disorganized,structural damage, cell apoptosis tended.Conclusions:1.Hippocampal neurons were cultured in vitro;2.The growth of hippocampal neurons are inhibited as concentration of ammonia increase;The ammonia induce neuronal apoptosis;3.Ammonia poisoning can lead to the depolymerization of F-actin,the number of neurite reduce in the ammonia poisoning group, suggests that ammonia poisoning may have inhibition in the process of neuron morphological development..