The influence of donor age on the survival of solid and suspension intraparenchymal human embryonic nigral grafts.

In many species, graft survival and graft-derived behavioral recovery are affected by the embryonic donor age. We compared the ability of solid and suspension grafts of human embryonic mesencephalic dopaminergic (DA) neurons at different embryonic stages to survive intraparenchymal transplantation into 6-OHDA lesioned immunosuppressed rats. Suspension grafts survived best when donor age was between postconception (PC) days 34 and 56. Transplants displayed numerous healthy tyrosine hydroxylase immunoreactive (TH-IR) neurons which sent extensive neuritic processes into the host striatum. Suspension grafts survived poorly when donor age was greater than 65 days. Solid implants displayed comparable viability of TH-IR neurons when donor age was between 44 and 65 days. No solid grafts contained TH-IR cells when donor tissue was older than 72 days. The suspension and solid methods of transplantation resulted in comparable survival of robust grafts, but solid grafts resulted in more intergraft variability than suspension grafts, particularly among the more marginal implants. Our results demonstrate that the upper limit for survival of human embryonic DA suspension grafts correlates well with the period of development of the human nigrostriatal pathway. The "window" for donor age of solid human embryonic DA grafts appears to be extended by about 9 days in comparison to suspension grafts. These data suggest that the upper age limit for grafting human mesencephalic DA neurons should be PC day 56 for suspension grafts, and PC day 65 for solid implants. Older donors are likely to produce grafts with fewer surviving DA neurons.

A model three-dimensional culture system for mammalian dopaminergic precursor cells: application for functional intracerebral transplantation.

Grafts of fetal neural tissue, rich in dopamine (DA) neurons, have previously been shown to improve the symptoms of parkinsonism, both in humans and in animal models. In order to circumvent some of the problems associated with cell transplant therapy, such as the limited availability of transplant tissue, we have established a reaggregate (three-dimensional) tissue culture system that can be used to proliferate normal mammalian neuronal precursors. We demonstrate the in vitro growth of DA-neuronal precursors derived from embryonic porcine ventral mesencephalon, Carnegie stages 15-18. Cultures of DA-neuronal precursors were maintained in F12 medium supplemented with Chang C Supplement for 5 days and switched to serum-free N2 medium for an additional 10 days. Cultures labeled with tritiated thymidine on Days 5-7 in vitro revealed that 43.5% of the DA neurons had incorporated the label, indicative of cell division. Histological examination of the cultured cells demonstrated rosette-like structures, similar to developing neuroepithelium in vivo. Neuronal maturation in vitro was stimulated by dibutyryl cyclic AMP (dbcAMP). Exposure to 5 mM dbcAMP for 7 days stimulated tyrosine hydroxylase (TH), neuron-specific enolase, and 200-kDa neurofilament accumulation three- to sixfold above control levels. After 15 days in vitro, cultured cells reversed amphetamine-induced rotation when grafted into the striata of hemiparkinsonian rats. Successful transplants of cultured neurons were dependent upon a minimum density of DA neurons within the graft (greater than 100 DA neurons/mm3 of graft volume). Data suggest that the percentage of TH neurons can be increased about threefold by culturing the aggregates in tyrosine-free medium, which selects for TH-positive cells. The ability to cultivate mammalian neuronal precursor cells in vitro may eventually make graft therapy a more practical approach to treatment of neurological diseases.

Development of dopaminergic neurons in the human substantia nigra.

A series of 29 human embryonic brains were examined in order to characterize the ontogeny of dopaminergic neurons within the developing substantia nigra. Embryos from Postconception Weeks 4.0 to 11.2 (last menstrual period 6.0-13.2) were studied. Immunohistochemical staining was performed using a polyclonal antibody to tyrosine hydroxylase. Tyrosine hydroxylase-like immunoreactivity was first seen in cells of the ventral mesencephalon at 6.5 weeks adjacent to the ventricular zone. Ventral migration of TH-positive cells began at 6.7 weeks. Neural process extension was first identified in tyrosine hydroxylase-positive neurons at 8.0 weeks. The ascending nigrostriatal bundle was also first demonstrated at 8.0 weeks. Tyrosine hydroxylase containing neurites were seen initially in the developing putamen at 9.0 weeks. Only a few tyrosine hydroxylase-positive cells remained adjacent to the ventricular zone at Week 10.0 and all had disappeared from the ventricular zone by 11.2 weeks. At this latter stage, a large number of dopaminergic cells had elaborated neural processes. The sequence of developmental events of human mesencephalic dopaminergic neurons is similar to the equivalent period of ontogeny in other mammals. The duration of the developmental period is, however, significantly protracted.

Xenografting of fetal pig ventral mesencephalon corrects motor asymmetry in the rat model of Parkinson's disease.

A suspension of cells from embryonic day 21 fetal pig ventral mesencephalon was transplanted into the striatum of 20 immunosuppressed rats with 6-hydroxydopamine-induced lesions of the nigrostriatal dopamine pathway. Of these rats, 15 showed reduction of amphetamine-induced ipsilateral rotation by 9 weeks and complete reversal of rotation by 14-17 weeks. Animals maintained stable reversal of rotations (contralateral direction) until cessation of Cyclosporin A (CyA) treatment at 15-20 weeks. Within 4-9 weeks after CyA removal, these rats showed exclusively ipsilateral rotations during behavioral testing which were comparable to pre-transplant levels, suggesting that the grafts were rejected upon cessation of CyA treatment. Rats were sacrificed and tyrosine hydroxylase (TH) immunohistochemistry was performed at several time points, both on and off CyA, to examine a possible correlation between the degree of rotational behavior and the number of TH-positive surviving grafted cells. Staining showed large numbers (230-12,329) of TH-positive surviving cells in animals displaying a high degree of rotational correction (1.6 to -9.6 net ipsilateral rotations/min) after cessation of CyA treatment. Two control groups, those transplanted with non-neuronal cells from the pig ventral mesencephalon (n = 5) and those receiving only daily CyA injections (n = 4) showed no significant reduction of net ipsilateral rotations throughout the experiment. No TH-positive surviving cells were seen in the one non-neuronal transplant analyzed. This data demonstrates long-term retention of xenografted tissue with immunosuppression and its concomitant restoration of normal motor behavior in the rat model of Parkinson's disease.

The survival of dentate gyrus neurons in dissociated culture.

Using a technique for dissociating cells from the area dentata of postnatal rats, we have been able to routinely establish low density cultures of dentate granule neurons that can be grown in the presence or absence of serum. Non-granule neurons from the hilar region and glial cells (both astrocytes and oligodendrocytes) are also present, but can be readily distinguished from the granule cells in these cultures. Unlike dissociated hippocampal pyramidal cells, which frequently resemble their in vivo morphology, dissociated dentate granule cells bear little resemblance to their normal in vivo counterparts, but are very similar in appearance to the ectopic granule cells seen in the reeler mouse. This suggests that extrinsic factors are the principal determinants of the mature form which granule neurons assume in vivo. On the other hand, the dissociated granule cells are able to express certain other aspects of their in vivo phenotype including the synthesis and transport of an antigen which is characteristically found in mossy fibers. Certain neuropeptide-containing non-granule neurons found in these cultures are also capable of maintaining aspects of their in vivo phenotype.

On the numbers of neurons in fields CA1 and CA3 of the hippocampus of Sprague-Dawley and Wistar rats.

In a previous study it was found that there are significant differences in the numbers of granule cells in the dentate gyrus of adult Sprague-Dawley and Wistar rats and also that the continued postnatal addition of new cells to the dentate gyrus has quite different consequences in the two strains. We have now extended these observations to the two major cytoarchitectonic fields of the hippocampus (the regio superior or field CA1; and the regio inferior or field CA3). The mean number of pyramidal neurons in field CA1 of 1-month-old Sprague-Dawley rats is 420,000 (+/- 60,000 S.E.), while Wistar rats at the same age have 320,000 (+/- 20,000). The numbers of neurons in field CA3 in the two strains are: 330,000 (+/- 30,000) and 210,000 (+/- 20,000), respectively. Whether these strain differences reflect specific differences in the neural organization of the hippocampal formation in the two strains, or are related to more general differences in total body weight or brain weight, is unknown. Since during the first two days postnatally we estimate that there are between 358,000 and 491,000 cells in field CA1 of Sprague-Dawley rats, it would seem that there is no significant naturally-occurring neuronal death in this hippocampal field. This may be due to the extensive collateral projections of the hippocampal pyramidal neurons.

On the number of neurons in the dentate gyrus of the rat.

We have estimated the number of dentate granule cells in Sprague-Dawley and Wistar rats at 1, 4 and 12 months of age. In Sprague-Dawley rats the number of granule cells is relatively constant throughout this period at about 1 million. In Wistar rats, on the other hand, there is a progressive increase in the number from about 700,000 at 1 month to 1 million at 4 months; thereafter the number declines to about 800,000 at 1 year. Estimates of the numbers of cells in the polymorphic zone that can be stained immunohistochemically for somatostatin, cholecystokinin, vasoactive-intestinal peptide, and glutamic acid decarboxylase show no appreciable differences in the two strains.

An improved in vitro immunization procedure for the production of monoclonal antibodies against neural and other antigens.

A simplified in vitro immunization procedure has been developed for the production of monoclonal antibodies against a variety of neural antigens. The procedure, which can be applied to both soluble and membrane-bound antigens, involves the addition of an adjuvant peptide, N-acetylmuramyl-L-alanyl-D-isoglutamine, and has been found to improve all fusion parameters.

Production and characterization of monoclonal antibodies against the "brain-specific" proteins 14-3-2 and S-100.

We have raised mouse hybridomas that secrete monoclonal antibodies against bovine brain-specific proteins 14-3-2 and S-100, and we have characterized the antibodies by immunoperoxidase and immunofluorescence methods in sections and in tissue cultures of rat brain. One monoclonal antibody to 14-3-2 (E8.F9) has been found to react strongly with bovine 14-3-2 and with rat neuron-specific enolase in an enzyme-linked immunosorbent assay (ELISA) and to react weakly with rat nonneuronal enolase. This pattern of specificity is reflected in strong neuronal labeling and occasional weak glial labeling in immunocytochemical preparations. After appropriate tissue fixation, E8.F9 could be shown to be localized primarily to the cytoplasm of neurons; with less adequate fixation nuclear labeling was also seen. A monoclonal antibody to the calcium binding protein S-100 (G12.B8) reacted strongly with bovine S-100 in an ELISA and with the major protein bands in electrophoretically separated S-100. In immunocytochemical preparations G12.B8 labeled the cytoplasm of astrocytes. Both antibodies are of the IgG1 subclass. Because of its specificity, the antibody against the S-100 protein promises to be useful as an immunological marker for astrocytes in the adult animal and in mature tissue cultures of brain cells. Although it has been thought that the generally low levels and relatively late appearance of S-100 during ontogeny may restrict its usefulness as a marker for developing astrocytes, preliminary immunocytochemical evidence indicates that G12.B8 selectively labels radial glial cells and astrocytes or astrocyte precursors as early as, or even earlier than, antibodies against the glial fibrillary acidic protein. The antibody against neuron-specific enolase is likely to be of limited use as a neuronal marker because of its crossreactivity with nonneuronal enolase.

Prostaglandins E1 and E2 interact with prostaglandin F2alpha to regulate initiation of DNA replication and cell division in swiss 3T3 cells.

Prostaglandin (PG) E1 or E2 added at 2-1,000 ng/ml to quiescent cultures of Swiss 3T3 cells synergistically enhanced the rate of initiation of DNA replication stimulated by PGF2 alpha alone or with insulin. Neither PGD2 nor PGF1 alpha had any effect with PGF2 alpha. An increase in the rate of entry into S phase also occurred when PGE1 or PGE2 was added 8 or 15 hr after addition of PGF2 alpha. However, adding PGE1 and PGE2 together with PGF2 alpha did not further enhance the synergistic effect observed with PGE1 or PGE2 separately. The synergistic effect was also observed in stimulation of 2-deoxyglucose uptake but not in early changes of intracellular levels of cAMP. These results may be relevant in understanding the control of fibroblastic proliferation in wound healing and may provide an alternative mechanism for oncogenic transformation.