Enzymatic digestion of the human amniotic basement membrane (HABM) using heparinase Ⅰ, heparinase Ⅱ, collagenase and chondroitinase ABC enabled us to study the supportiveness of HABM for forebrain neuronal growth. HABM-coated 24-well plates were enzymatically digested for 1 hr. at 37℃ before dissociated neurons from the forebrain of E8 chick embryo were seeded. The neurons were cultured for 20hr. in RPMI 1640 medium containing 20mmol/L HEPES and 60％ fetal calf serum before being quantified for neuronal growth by an automatic colorimetric microassay. The assay, utilizing a tetrazolium derivative named MTT [3-(4, 5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide] which can be converted by viable cells to form a formazan product, can be measured with an Elisa reader. The results revealed that heparinase Ⅰ, collagenase, and chondroitinase ABC had significantly removed from the HABM the attachment sites for neurons, and that heparinase Ⅱ had significantly increased the sites for attachment and/or enhanced the survival growth of neurons.

We have investigated the growth and interactions between dissociated cells from different visual structures obtained from neonatal albino rats, namely, retina (R), superficial gray of the tectum (T), the lateral posterior portion of the dieneephalon (G), and occipital cortex (C). Dissociated cells obtained were either cultured separately or mixed with another cell type in 24-well plates coated with laminin. After 3 days of culture surviving cells were quantified by the MTT colorimetric microassay.The optical densities obtained from mixed cultures with cells from any two of the four types (G, R, T, C) of brain tissues were 1.3 to 3 times higher than the sum of their corresponding individual cultures except for those of G+C and T+G. Among them R+T and R+G were the most active cultures, followed by R+C and T+C. Further, condition medium from tectal cells (Tc) increased the activities of R and G for 3 and 2 times, respectively, and condition medium from retinal cells (Re) increased the activities of G and C for 1.3 and 2 times, respectively. Therefore it is suggested that neuronotrophic factors may be present in Tc and Rc.

Using enzymatic digestions on human amniotic basement membrane (HABM), it was observed ultrastructurally that heparinase Ⅱ digestion resulted in formation of microvilli on the surface of HABM and thinning and vesiculation of lamina densa and lamina lucida layers. This suggested that the heparinase Ⅱ-digested HABM had increased its surface area for more efficient cell attachment. The main action of heparinase Ⅱ is to digest heparan sulphate on HABM surface, which leads to exposure of more sites of laminin for cell attachment and neurite outgrowth; therefore heparinase Ⅱ-digested HABM can increase neuronal growth to 150%. For those HABM digested by heparinase Ⅰ, collagenase Ⅰ, and chondroitinase AC, they lost most of the sites for attachment and neurite outgrowth and as a consequence, the growth of neurons decreased. This morphological study supports heparinase Ⅱ digested HABM as an ideal substratum for neuronal attachment and growth.

The expression of serotonin-like immunoreactivity in day 4 neuronal cultures from E8 chick embryo forebrains was investigated by PAP immunocytochemical method. It was found that 98% of the dissociated forebrain cells cultured on laminin substratum were neuron specific enolase immunoreactive positive; and 10.4% of these neurons were mainly multipolar or bipolar and serotonin immunoreactive positive. In order to verify whether the remaining 2% NSE negative forebrain cells were neuroglial cells, we had used anti-glial fibrillary acid protein antibody to show that only 2% of the cultured forebrain cells were composed of GFAP immunoreactive positive cells, i. e. neuroglial cells.

Using transmitted and reflected light confocal laser scan microscopic methods, we have studied the optical tomograms starting from outer layers to inner layers of an unstained human retina whole mount. The rod-structures which were located at the first superficial tomogram of the retina, had a width of 1.5 ?m and their center-to-center distance of 1-1.3 ?m. Toward the inner tomogram, there were the cone-structures which had a width of 3-3.2 ?m on their basal portion and their center-to-center distance of 5-11 ?m. The density for the rod-shape structures was 443 954 unit/mm~2 and the cone-shape structures was 20 128 unit/mm~2. We have discussed the characteristics of tomograms obtained from the laser scan microscope and its application in clinical uses.

In order to study the cause of mental deterioration and increase of plaques and tangles in the basal forebrain in Alzheimer's disease, we have used isolated neural cells from the nuclei of amygdala (A), the hippocampus(H), the nuclei of medial septum (S), and the basal nuclei of Meynert (M) of basal forebrain, and the retina (R) of newborn rats, to study the effect of cellular interactions on the growth activity of these cells. The growth activity was measured quantitatively using MTT colorimetric microassay method. The MTT results revealed that, after 3 days of culture, the most active cell growth were found in mixed cultures of A+H and H+R reaching up to 212％ and 270％ of their respective individual control cultures (p

By immunizing Balb/c mice with immunoglobulin IgG which was isolated from the D_3 monoclonal antibody specific to the retinal ganglion neuronotrophic factor (RGNTF-McAb) with a Phast System gel electrophoretic method, we have developed anti-idiotypic antibodies against the RGNTF-McAb. A MTT colorimetric microassay designed to measure the effect of conditioned media from hybridomas containing antibodies on the growth activities of retinoblastoma (Rb) cells, was used to screen for idiotypic antibodies from these hybridomas. The results showed that G_4 hybridoma produced an anti-idiotypic antibody which could bind to the D_3 RGNTF-McAb and neutralize the inhibitory function of D3 on Rb cells, and possessed the trophic activity similar to RGNTF in promoting the growth activity of Rb cells. Immunohistochemical studies with anti-idiotypic antibody revealed that Rb cells and embryonic and neonatal rat retinal ganglion cells possessed coarse immunoreactive positive granules distributed on their cell surface membrane. These granules are identified as the receptors for RGNTF. Therefore, we concluded that the G_4 monoclonal antibody is not only an anti-idiotypic antibody but also an anti-paratopic antibody, It may be very useful in studying the mechanism of action of RGNTF and has a widespread applications in future.

Monoclonal antibody specific to the retinal ganglion neuronotrophic factor (RGNTF) and anti-idiotypic monoclonal antibody specific to the receptor of RGNTF were used to localize immunohistochemically and quantify the amounts of RGNTF and its receptor in the paraffin sections of embryonic E_(16) to E_(20) rat retinae. The results revealed that RGNTF contents in the retinal ganglion cells of E_(16) to E_(20) embryonic retinae decreased lineally and very significantly (P

We have used our retinal ganglion neuronotrophic factor (RGNTF) monoclonal antibody and anti-idiotypic antibody to study the immunohistochemical localization and quantitation of RGNTF and its receptor in the human retinoblastomal (Rb) nude mice model before and after RGNTF monoclonal antibody immunotherapy. The results revealed that for those nude mice responders treated with RGNTF monoclonal antibody and with Rb grade reduction, their RGNTF and receptor contents in Rb cells were reduced significantly from 100% before therapy to 32.4% and 25% after therapy, respectively. These quantitative results of RGNTF and its receptor contents between each grades differ significantly (P

We have used our anti-MNTF monoclonal antibody (MNTF-McAb) and MNTF-anti- idiotypic monoclonal antibody (MNTF-Id-McAb) to study the changes in localization and quantities of MNTF and its receptor in tongue muscles of 30 adult rats after 4 days, 2, 3 and 5 weeks, and 2 and 5 months of post-denervation of the hypoglossal nerve. The LSM VIDAS image analysis results revealed that 4 days post-denervation, the MNTF content in tongue muscles of the denervated side had reduced to 75%-80%as compared to the control intact side 100%, however, the reduction of MNTF receptor was insignificant; 2-3 weeks post- denervation, the contents of MNTF and its receptor in the denervated side were significantly reduced to 65%-70% and 50%-55%, respectively; 5 weeks post- denervation, the contents of MNTF and its receptor had started to increase especially for the receptor; and gradually the contents of both MNTF and its receptor in denervated side had recovered and reached to a similar levels as in the control side at 2 months and 5 months post-denervation. These results indicated that MNTF and its receptor are synthesized by the tongue muscle cells and their syntheses are regulated by the hypoglossal nerve; and that there is a close relationship between MNTF and its receptor with the regeneration of hypoglossal nerve.