CULTIVATION OF SUPERIOR CERVICAL GANGLION OF RAT / 解剖学报

ABSTRACT

The discovery of the nerve growth factor(NGF),a specific protein material,by Rita Levi-Montalcini and her colleagues(1954)led to the observation that ifNGF is added to the culture medium,sympathetic and sensory neurons grow well inculture,for it is a potent and specific maintenance factor for these two types of neu-rons.Therefore,we chose the superior cervical ganglion(SCG)as a model systemfor nerve tissue culture in order to study the development,differentiation and rege-neration of nervous tissue in vitro.This report presents what we have observed onthe cultures of SCG that have been successfully established in our laboratory bymeans of explant technique.SCG from newborn rats were explanted onto collagen or plasma-coated coverslipsand maintained in Maximow depression slide assemblies at 37℃.The fluid me-dium was replaced twice a week and consisted of 1/3 calf serum,1/3 synthetic medium199 and 1/3 Hanks' BSS,or of equal parts of calf serum and Hanks' BSS,supplementedwith 600 mg% glucose.NGF(a crude extract of mouse salivary gland)was added tothe medium in a proportion of 1:20;for control,no NGF was added to the medium in some cultures.The cultures were examined microscopically every day,Nissl's stai-ned,Bodian's protargol and ammonium silver impregnated preparations were madeperiodically.Time lapse microcinematographic records were also made.We have planted altogether 209 SCG from newborn rats.All of them grew suc-cessfully in culture with NGF or without NGF,but the SCG with NGF grew muchmore rapidly than those without NGF.NGF strongly stimulates the outgrowth ofneurites and maintains the survival of the sympathetic neurons.Neurites grew outindividually or,more frequently,as bundles extending radially toward the peripheryor forming a meshwork.The tips of elongating neurites expanded to form growthcones from which are projected long slender microspikes(filopodia).These microspi-kes continually waved about,extending,and retracting as the growth cone movedover a substratum.The behavior of the growing tip of neurite is best analyzed bytime lapse microcinematography.The Schwann cells emerged from the explant andproliferate to accompany the neurites.These cells,fusiform or filiform in shape,were usually arranged in alignment along the neurites.They could be easily distingui-shed from fihroblasts.Mitosis of Schwann cells had been observed and recorded bytime lapse microcinematography.The bodies of the sympathetic neurons did not mig-rate away from the explant.