Neurotrophic effects of FPF-1070 (Cerebrolysin®) on cultured neurons from chicken embryo dorsal root ganglia, ciliary ganglia, and sympathetic trunks

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We examined the effect of FPF-1070 (Cerebrolysin®) on neurite outgrowth in explant cultures of dorsal root ganglia (DRG), sympathetic trunks (ST), and ciliary ganglia (CG) from 10- to 11-day chicken embryos. FPF-1070 significantly promoted neurite

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  J Neural Transm (2000) 107: 1253–1262 Neurotrophic effects of FPF-1070 (Cerebrolysin®) on culturedneurons from chicken embryo dorsal root ganglia, ciliary ganglia,and sympathetic trunks T. Satou 1 , T. Itoh 1,2 , Y. Tamai 3 , H. Ohde 2 , A. J. Anderson 4 , and S. Hashimoto 1 1 2nd Department of Pathology, Kinki University School of Medicine, 2 Fujimoto Pharmaceutical Corp., Research Institute, and 3 3rd Department of Internal Medicine, Kinki University School of Medicine,Osaka, Japan 4 Institute for Brain Aging and Dementia, University of California, Irvine, CA, U.S.A.Received October 20, 1999; accepted April 17, 2000 Summary.  We examined the effect of FPF-1070 (Cerebrolysin®) on neuriteoutgrowth in explant cultures of dorsal root ganglia (DRG), sympathetictrunks (ST), and ciliary ganglia (CG) from 10- to 11-day chicken embryos.FPF-1070 significantly promoted neurite outgrowth in DRG and ST neuronsat all concentrations examined, in comparison with phosphate buffered saline-treated negative controls; however, this effect on neurite outgrowth was not assignificant as that observed for nerve growth factor-treated positive controlson DRG and ST neurons. Additionally, FPF-1070 exhibited an inverted Urelationship between concentration and effectiveness in DRG and ST neurons.In contrast, FPF-1070 did not affect neurite outgrowth in CG neurons althoughciliary neurotrophic factor-treated positive controls showed striking neuriteoutgrowth. Our results demonstrate that FPF-1070 has different neurotrophiceffects depending on the subpopulation of neurons. This study clarifies a rolefor neurotrophic activity in the mechanism of action of FPF-1070. Keywords:  Cerebrolysin®, neurotrophic factor, culture, chicken, neuron,FPF-1070. Introduction FPF-1070 is a commercially available drug (distributed under the trade nameCerebrolysin) that is prepared by standardized enzymatic breakdown of lipid-free pig brain proteins and consists of 85% free amino acids and 15% biologi-cally active small peptides (10   Kd) (Gschanes et al., 1997). While FPF-1070has been employed in the clinical treatment of cognitive deficits in dementia  1254T. Satou et al. and stroke sequelae for more than 40 years (Gschanes et al., 1997), themechanisms of action of this nootropic drug remain to be elucidated. Positiveeffects of FPF-1070 on learning and memory have been reported in animalmodels (Francis-Turner and Valouˇ sková, 1996; Hutter-Paier et al., 1996) andclinical trials (Kofler et al., 1990; Rüther et al., 1994). Several potential mecha-nisms of action for these effects of FPF-1070 on neuronal tissue have beensuggested. In particular, it has been reported that FPF-1070 provides meta-bolic stabilization of neurons via protein synthesis modulation (Piswanger etal., 1990), prevention of lactose acidosis (Windisch and Piswanger, 1985),prevention of free radical formation (Sugita et al., 1993), and interaction withadenosine (Xiong et al., 1995) or GABAb receptors (Xiong et al., 1996).The role and function of neurotrophic factors, including nerve growthfactor (NGF), in the morphogenesis and functional maintenance of the ner-vous system has been defined and expanded in recent years (Vantini, 1992;Yuen et al., 1996). FPF-1070 has been reported to exhibit similar effects toNGF in vivo, in particular, FPF-1070 ameliorates septal neuron loss afterfimbria-fornix transection (Akai et al., 1992). Additionally, we have previ-ously reported that FPF-1070 has effects on nerve fiber elongation similar tothose of NGF on cultured chick embryo dorsal root ganglia (DRG) neurons(Satou et al., 1994). In this study, we examined the effects of FPF-1070 oncultured neurons derived from sympathetic trunks (ST) and ciliary ganglia(CG), in comparison with DRG explants, in order to clarify the effects of FPF-1070 on various aspects of the peripheral nervous system. Methods Cell culture was performed according to the procedures described by Barde et al. (1980).These are summarized as follows: Thirty chicken embryos aged 10 to 11 days old weresacrificed to obtain approximately 300 pieces of neural tissue from the dorsal root ganglia(DRG), 30 pieces from the sympathetic trunks (ST) and 50 pieces from the ciliary ganglia(CG). These different neuronal tissues were separately collected in dishes (Falcon) filledwith 0.1M phosphate buffered saline (PBS) (pH of 7.4). After being washed with PBSonce, the samples were transferred into centrifuge tubes, spun at 1,500rpm for 5 minutes,and the supernates completely removed. 30ml of a 0.02% EDTE (Nakarai Co. Ltd.) plus0.05% trypsin (GIBCO) solution was added to the tubes containing DRG cells and 12mlof this solution was added to the tubes containing ST and CG cells. After gently pipetting,the samples were incubated in a 5%-CO 2  incubator at 37°C for 40 minutes. Followingincubation, the tubes were centrifuged at 1,500rpm for 5 minutes, and the supernatescarefully removed. 24ml of Eagle-MEM culture media (Nissui Co. Ltd.) supplementedwith 10% fetal calf serum (FCS) (GIBCO) was added to the tubes containing DRG cellsand 12ml of this supplemented media was added to the tubes containing ST and CG cells.Neuronal tissues were then resuspended by gently pipetting several times, the suspensionsfiltered through nylon mesh and recollected in 6cm-diameterdishes (Falcon) at a volumeof 12ml per dish, and the dishes incubated in a 5%-CO 2  incubator at 37°C for 130 minutes.After incubation, a fraction of the media containing floating cells was carefully collectedin the tubes. Floating cells in the collected media were counted by a counting chamberand each media were diluted with Eagle-MEM/10% FCS up to 3   10 3  cells/ml to preparecell suspensions. 1ml per well of these cell suspensions was plated in each well of 24-wellpolystyrene culture plates (Falcon) coated with 1% poly-d-lysine (SIGMA). Immediatelyafter plating, a series of test samples was prepared by adding 5, 10, 20, 40 or 80 µ l of FPF-1070 (provided by E BEWE  A RZNEIMITTEL Ges.m.b.H. Pharmaceutical Laborato-  Neurotrophic effects of FPF-1070 on cultured neurons1255ries) per culture well. Negative controls were prepared using identical volumes of PBS.For DRG and ST cultures, which are known to be NGF responsive, positive controls wereprepared by adding 5 or 10 µ l of NGF (TAKARA Co. Ltd.) solution which was preparedby dissolving 1 µ g NGF in 1ml PBS including 0.04% bovine serum albumin. As a result,final concentration of NGF was 5 or 10ng/ml in the culture medium. For CG cultures,which are known to be CNTF responsive, positive controls were prepared by addingCNTF (MBL Co. Ltd.) to a final concentration of 2ng/ml in the culture medium. Addi-tionally, the effect of 5 or 10ng/ml NGF on CG cultures was tested as described above forDRG and ST cultures.FPF-1070-treated cultures, negative control cultures, and positive control cultureswere maintained in a 5%-CO 2  incubator at 37°C for 4 days, followed by evaluation forneurite outgrowth. For DRG cultures, the observed groups were as follows: FPF-1070-treated, n   40 wells for each drug concentration; PBS-treated negative controls, n   12wells; 5 µ l NGF-treated positive controls, n   12 wells; 10 µ l NGF-treated positive con-trols, n   12 wells. For ST cultures, the observed groups were as follows: FPF-1070-treated, n   40 wells for each drug concentration; PBS-treated negative controls, n   16wells; 5 µ l NGF-treated positive controls, n   24 wells; 10 µ l NGF-treated positive con-trols, n   29 wells. For CG cultures, the observed groups were as follows: FPF-1070-treated, n   4 wells for each drug concentration; PBS-treated negative controls, n   9wells; 5 µ l NGF-treated positive controls, n   4 wells; 10 µ l NGF-treated positive controls,n   4 wells; CNTF-treated positive controls, n   4 wells. Nerve fiber outgrowth wasevaluated by observing neurons that formed nerve fibers twice or more as long as theirown cytoplasm under a phase-contrast microscope, and quantifying outgrowth accordingto a 3 point scale. Evaluation and quantification was as follows: score 1, when elongatingnerve fibers from a single neuron were observed; score 2, when elongating nerve fibersfrom a pair of neurons were observed; score 3, when nerve fibers extended from a clustercomprising more than three neurons. Scores obtained from all of the neurons observed ineach well were summed to derive a total nerve fiber outgrowth score for that well, andanalyzed for statistical significance by one way ANOVA followed by post-hoc t-tests. Results PBS-treated negative controls showed little or no nerve fiber outgrowth inDRG, ST, or CG cells (Figs. 1a, 2a and 3a). In contrast, NGF-treated positivecontrols for DRG and ST, and CNTF-treated positive controls for CG,showed obvious nerve fiber elongation (Figs. 1b, 2b and 3b). FPF-1070 in-duced nerve fiber outgrowth in DRG and ST cells (Figs. 1c and 2c), but did notinduce nerve fiber outgrowth in CG cells (Fig. 3c). As expected, NGF did notinduce nerve fiber outgrowth from CG neurons (Fig. 3d).The nerve fiber elongation scores of the FPF-1070-treated groups forDRG and ST cultures, which were significantly higher than those of the PBS-treated negative controls (Figs. 4 and 5), increased in a dose-dependentmanner at concentrations between 5 and 20 µ l/ml of FPF-1070. However, FPF-1070-induced elongation scores in DRG and ST cultures declined at concen-trations above 20 µ l/ml of FPF-1070, and did not significantly facilitate nervefiber outgrowth in ST at a concentration of 80 µ l/ml in comparison with PBS-treated negative controls (Figs. 4 and 5). In addition, FPF-1070, even at themaximally effective concentration of 10–20 µ l/ml, was not as effective as eitherconcentration of NGF in promoting nerve fiber elongation in DRG and STcultures (Figs. 4 and 5). Finally, as described qualitatively above, CNTF-treated positive CG culture controls exhibited a significant increase inquantified nerve fiber elongation scores in comparison with either FPF-1070-  1256T. Satou et al. treated or PBS-treated negative control cultures, and no significant differencein CG neurite outgrowth was observed between FPF-1070-treated and PBS-treated negative control cultures (Fig. 6). Discussion Research into the clinical applicability of neurotrophic factors for the treat-ment of various neurological disorders, such as neurodegenerative diseases(Hughes and O’Leary, 1996), dementia (Hefti et al., 1996), and peripheral Fig. 1. Cultured neurons from dorsal root ganglia (DRG) under a contrast phase micro-scope. a  DRG neurons do not exhibit neurite elongation in the PBS-treated negativecontrol condition (3.3   10) b  DRG neurons exhibit clump formation and multiple pointsof nerve fiber outgrowth and neurite elongation in the NGF-treated positive controlcondition (3.3   20) c  Scattered DRG neurons exhibit neurite elongation in the FPF-1070treated condition (3.3   20)  Neurotrophic effects of FPF-1070 on cultured neurons1257 neuropathy (Apfel and Kessler, 1996), has been an active area of investiga-tion. However, several problems for the clinical use of neurotrophic factorsremain to be addressed, including the route of administration, because thesefactors do not pass through the blood-brain barrier (Lindsay et al., 1993). Thepresent study demonstrates that FPF-1070 has an effect analogous to that of neurotrophic factors such as NGF, as assessed by the induction of nerve fiberoutgrowth on DRG and ST cells. Thus, FPF-1070, which can be administeredintravenously, appears to be a useful clinical agent that may exert itsnootropic (cognition enhancing) effects in part via neurotrophic activity. Fig. 2. Cultured neurons from sympathetic trunks (ST) under a contrast phase micro-scope. a  ST neurons do not exhibit neurite elongation in the PBS-treated negative controlcondition (3.3   10) b  ST neurons exhibit neurite elongation and clump formation in theNGF-treated positive control condition (3.3   10) c  Scattered ST neurons exhibit neuriteelongation in the FPF-1070-treated condition (3.3   10)
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