Gene transfer to the central nervous system by transplantation of cerebral endothelial cells.

A cerebral endothelial immortalized cell line was used in transplantation experiments to deliver gene products to the adult rat brain. Survival of grafted cells was observed for at least 1 year, without any sign of tumor formation. When genetically modified to express bacterial beta-galactosidase and transplanted into the striatum, these cells were shown, by light and electron microscope analysis, to integrate into the host brain parenchyma and microvasculature. Following implantation into the striatum and nucleus basalis of adult rats, endothelial cells engineered to secrete mouse beta-nerve growth factor (NGF) induced the formation of a dense network of low-affinity NGF receptor-expressing fibers near the implantation sites. This biological response was observed from 3 to 8 weeks after engraftment. The present study establishes the cerebral endothelial cell as an efficient vector for gene transfer to the central nervous system.

Calcium-dependent release specificities of various cell lines loaded with different transmitters.

By loading cells in culture with acetylcholine (ACh) we have characterized a calcium-dependent release mechanism and shown that it was expressed independently of synthesis or storage of ACh. (Israël et al., 1994, Neurochemistry International 37, 1475-1483; Falk-Vairant et al., 1996a, Proc. Natl. Acad. Sci. U.S.A. 93, 5203-5207; Falk-Vairant et al., 1996b, Neuroscience 75, 353-360; Falk-Vairant et al., 1996c, Journal of Neuroscience Research 45, 195-201). The transmitter loading procedure was applied to two other transmitters, gamma-aminobutyric acid (GABA) and glutamate (Glu). We could then study the specificity of the release mechanism for the three transmitters in a variety of cell lines, including neural-derived cells. Four different calcium-dependent release phenotypes were identified: two were specific for ACh or GABA, and two co-released two transmitters ACh and GABA but not Glu, or ACh and Glu but not GABA. We conclude that release mechanisms having different specificities are expressed by the cell lines studied, they become functional after loading the cells with the relevant transmitters. These observations will help the identification of proteins controlling the specificity of release, and provide an interesting model for pharmacological studies.

Oligoclonal beta-galactoside-specific gamma immunoglobulins allow the immunocytochemical detection of cellular antigenic determinants expressed in mouse brain after surgical injury.

Histological brain sections were probed with human oligoclonal lectin-like IgGs (L-IgG) purified from normal serum. In intact brain, antigenic determinants for these IgG were restricted to some blood vessel endothelial cells. By contrast, during the inflammatory reaction following a surgical injury, these determinants were detected at the cell surface of different cell types, within and near the lesion site. The cells reacting with L-IgG consisted of endothelial cell, mature astrocytes, activated microglial and ependymal cells.

Migration pathways, differentiation and survival of macroglial cells from a xenograft implanted into the thalamus of newborn mice.

Embryonic rabbit corpus callosum transplants were grafted into thalamus of newborn shiverer mice in order to compare the fates of oligodendroglial and astroglial cells derived from the transplants. Our model allowed the identification of the two populations of macroglial cells. The thalamus was chosen as site of implantation because of its situation at a crossroad of numerous neuronal fascicles. Previous studies, where the dorsal striatum was used as site of implantation, had shown that corpus callosum was one of the favorite routes of migration for both populations of macroglial cells. In the present study special attention was given to the comparison of the migration pathways and areas of settlement of implanted astroglia and oligodendroglia. The internal capsule, the medial lemniscus, the crus cerebri and the thalamic radiations were used by both populations of transplant derived macroglial cells for their migrations through the host parenchyma. They integrated into the host tissue on these routes or further away in areas such as the putamen, the mesencephalon or the colliculi. Signs of degeneration of the implanted astroglia were often observed after 1 month post-implantation.

Localization of TNF alpha and IL-1 alpha immunoreactivities in striatal neurons after surgical injury to the hippocampus.

Since the inflammatory process develops after transplantation to the brain, we sought to determine the presence of cytokines following a surgical trauma to the brain of an adult mouse. We report the early and marked presence of tumor necrosis factor-alpha and interleukin-1 alpha in neuronal somata of the striatum following a surgical injury to the hippocampus. The expression of cytokines later extends to neuronal cells of the hippocampus, thalamus, cerebral cortex, brain stem, and cerebellum and to glial cells of the corpus callosum. By contrast, these cytokines are not expressed by neuronal cells following injury to other regions, such as the striatum, cerebellum, and cortex. This study suggests a possible role for certain neurons in the brain's early reaction to a penetrating injury.

Comparative migration and development of astroglial and oligodendroglial cell populations from a brain xenograft.

In previous studies of brain transplantation, the fate of the implanted glial cells has been investigated separately; that is, the interest has been focused either on the astroglia or on the oligodendroglia. However, the two populations of implanted glial cells may interact with each other, for example by secreting species-specific factors or by inducing reactions by the host. We have used two different models of brain transplantation: one that allows the identification of the implanted astrocytes, and another that allows the identification of the implanted oligodendroglia. The present model is a combination of both; it consists of the grafting of embryonic rabbit brain fragments into the brains of neonatal Shiverer mice. The myelin made by the implanted oligodendrocytes is identified by anti-myelin basic protein immunohistochemistry. The implanted astrocytes are identified by a monoclonal antibody that combines with rabbit but not with mouse glial fibrillary acidic protein. This study shows that although they use the same major routes of migration, both populations of glial cells tend to move differently. They demonstrate areas of common settlement but also areas where only one population of implanted glia is present. From the site of implantation in the dorsal striatum, the major routes of migration are the corpus callosum, the white matter fascicles in the striatum, and the internal capsule. After a delay of 6 weeks, no significant prevalence of one population of implanted glial cells over the other was observed.

Xenogenic transplantation into newborn rodent brain: neovascularization of the graft by the host.

Neoangiogenesis of transplants implanted into the brains of newborn rodent hosts was evaluated by immunohistochemistry for 2 weeks after the operation. The use of species-specific antibodies directed against mouse endothelial cells demonstrated the respective participation of the host and the donor in the formation of new vessels in the graft after crossed rabbit into mouse and mouse into rat transplantation experiments. We show that blood vessels made by host endothelial cells begin to penetrate the transplant 24 h after grafting, and cross it completely by 72 h. Simultaneously, host astrocytes invade the transplant.