Functional central nervous system myelin repair in an adult mouse model of demyelination caused by proteolipid protein overexpression.

Two types of interventions to remyelinate the adult demyelinated central nervous system were investigated in heterozygous transgenic mice overexpressing the proteolipid protein gene. 1) A cocktail of trophic factors, "TS1," was directed toward the activation of the endogenous pool of neural progenitors to increase the number of myelinating oligodendrocytes (OL) in the brain. 2) A combinatorial approach in which OL progenitors were coinjected with TS1 into the corpus callosum of wild-type and He4e transgenic mice that displayed hindlimb paralysis. The levels of locomotor ability in these mice were evaluated after a single treatment. The data showed that a single administration of either one of the interventions had similar therapeutic effects, alleviating the symptoms of demyelination and leading to the recovery of hindlimb function. Histological and immunofluorescent examination of brain sections showed extensive remyelination that was sufficient to reverse hindlimb paralysis in transgenic mice. When the interventions were administered prior to hindlimb paralysis, He4e mice were able to walk up to 1 year of age without paralysis.

Neonatal chimerization with human glial progenitor cells can both remyelinate and rescue the otherwise lethally hypomyelinated shiverer mouse.

Congenitally hypomyelinated shiverer mice fail to generate compact myelin and die by 18-21 weeks of age. Using multifocal anterior and posterior fossa delivery of sorted fetal human glial progenitor cells into neonatal shiverer x rag2(-/-) mice, we achieved whole neuraxis myelination of the engrafted hosts, which in a significant fraction of cases rescued this otherwise lethal phenotype. The transplanted mice exhibited greatly prolonged survival with progressive resolution of their neurological deficits. Substantial myelination in multiple regions was accompanied by the acquisition of normal nodes of Ranvier and transcallosal conduction velocities, ultrastructurally normal and complete myelination of most axons, and a restoration of a substantially normal neurological phenotype. Notably, the resultant mice were cerebral chimeras, with murine gray matter but a predominantly human white matter glial composition. These data demonstrate that the neonatal transplantation of human glial progenitor cells can effectively treat disorders of congenital and perinatal hypomyelination.

Radial glial cell line C6-R integrates preferentially in adult white matter and facilitates migration of coimplanted neurons in vivo.

C6-R is a cell line derived from C6 glioma cells that exhibits key properties of radial glia including the ability to support neuronal migration in culture. To explore its potential use in promoting neuronal migration in vivo, we analyzed the behavior of C6-R cells in the intact and injured adult rat CNS. At 6-11 days postimplantation at the splenium of the corpus callosum, green fluorescent protein-labeled C6-R cells were observed primarily in either the corpus callosum or the hippocampus in the brain, and in the spinal cord they migrated more extensively in the white matter than in the grey matter. To determine whether C6-R cells retain their ability to promote neuronal migration in vivo, they were coinjected with labeled neurons into adult brain. When rat embryonic neurons were coimplanted with C6-R cells, the neurons and C6-R cells comigrated through a much larger volume than neurons alone or neurons coimplanted with fibroblasts. In brains preinjured with ibotenic acid, C6-R cells as well as coimplanted neurons distributed widely within the lesion site and migrated into adjacent brain tissue, while transplants with neurons alone were restricted primarily to the lesion site. The results suggest that radial glial cell lines can serve as a scaffold for neuronal migration that may facilitate development of experimental models for neural transplantation and regeneration.

Characterisation of the immune response in a neural xenograft rejection paradigm.

We have looked at both donor and host MHC expression in a neural xenograft rejection paradigm. Grafts of either mouse corpus callosum or an SV40 large T transformed astrocytic cell line were placed in the mid-brain of neonatal rats. Three weeks later graft rejection was induced by the application of a skin graft of the same donor origin. MHC expression in the neural graft and the host brain was examined histologically four and ten days after the animals had received a skin graft. Donor MHC expression was detected in the corpus callosal grafts at both time points and preceded host MHC expression and the lymphocytic infiltrate. The grafts of the transformed cell line could not be induced to express MHC antigens under the experimental protocol used nor were they rejected. The migratory patterns of the transformed cells were compared to the well characterised migration patterns of astrocytes from the corpus callosal grafts.

Effects of the age of donor or host tissue on astrocyte migration from intracerebral xenografts of corpus callosum.

Our previous studies in which neural tissue was transplanted into neonatal rat brains have demonstrated that the patterns of astrocyte migration are stereotypic and are mainly influenced by local factors in the host brain. Here we examine how these patterns are affected by the relative maturity of donor cells and recipient brains. In one group of animals, adult mouse corpus callosum was transplanted into the cortex or midbrain of neonatal rat brains; in a second group, corpus callosum from 2- to 3-day-old mice was placed in adult rats. The location of the grafts and the distribution of donor astrocytes were defined by using a monoclonal antibody (anti-M2) specific for mouse astrocytes. In marked contrast to previous studies involving transplantation into neonatal hosts, very few neonatal astrocytes placed into adult brains invaded the gray matter and these never migrated more than 250 microns from the graft. The migration in adult brains also failed to show targeted migration to regions such as the substantia nigra. In the second group, astrocytes from adult grafts implanted in neonatal hosts migrated in both gray and white matter of brains in patterns similar to those of neonatal cells placed in neonatal brains. These observations suggest that the astrocyte migration in the white and gray matter is guided by different cues and that the maturation of host brain environment, but not the age of donor cells, influences the pattern of cell migration.

Migration of astrocytes transplanted to the midbrain of neonatal rats.

Previous studies have indicated that transplanted astrocytes are able to survive, express glial fibrillary acidic protein (GFAP), and migrate in the host brain, and that the pattern and speed of astrocyte migration is largely determined by the location of the graft. We examine here the pattern of astrocyte migration in the midbrain by transplanting CD-1 mouse corpus callosum (P2-3) into the midbrain of neonatal rats. The location of the grafts and the distribution of donor astrocytes were assessed by using a monoclonal antibody (anti-M2) specific for mouse astrocytes. A characteristic donor astrocyte distribution was seen. The highest density of cells was in the region of the substantia nigra (SN); lower numbers were found in the medial geniculate nucleus (MGN). Donor astrocytes were also found in the superior colliculus (SC) and central gray region, but only when the body of a graft was located nearby. [3H]thymidine studies showed that the concentrations of donor astrocytes in the SN were not the result of high levels of mitotic activity: all indications were that the proportion of dividing donor cells closely matched that of host glia. The pattern of astrocyte migration in the midbrain did not follow the course established by radial glia and was not influenced by axonal degeneration in the SC after removal of eyes. Moreover, donor cells failed to migrate along the course of axonal outgrowth from co-grafted retinae. Reciprocally, axonal elongation from retinal grafts did not follow the pathway of astrocyte migration, thus suggesting that astrocyte migration and neuronal outgrowth follow different cues.

Long-term survival of mouse corpus callosum grafts in neonatal rat recipients, and the effect of host sensitization.

Previous studies have suggested that the incidence of spontaneous rejection among immunogenetically mismatched neural transplants in neonatal recipients varies significantly depending on the cellular composition of the graft material. For example, neuron-rich grafts of embryonic mouse retina generally survive for extended periods without showing signs of rejection after implantation into neonatal rats, whereas cortical xenografts, which contain abundant glial and endothelial cells as well as neurons, typically undergo rejection 4-6 weeks after implantation. To determine whether the presence of donor glia is responsible for this high incidence of spontaneous rejection, we examined the fate of a non-neuronal graft material composed predominantly of xenogeneic glial cells (post-natal day 3, PD3, CD-1 mouse corpus callosum) implanted into the mesencephalon of PD1 Sprague-Dawley rats. The distribution and survival of donor astrocytes were assessed using a monoclonal antibody specific for a mouse astrocyte surface antigen, M2. Thirteen of 16 animals sacrificed within 2 months of implantation had detectable transplants. In these animals, M2-positive cells frequently migrated well away from body of the graft, clustering in large numbers in several characteristic regions of the host brain. Unlike cortical grafts of similar age, the vast majority (93%) of callosal transplants showed no histological signs of rejection or major histocompatibility complex antigen expression in and around the transplant-derived cells. As previously noted in the neonatal retinal transplant paradigm, however, well-integrated 1-month-old corpus callosum grafts could be induced to reject by appropriate sensitization of the host immune system, implying that the host was not immunologically tolerant to the foreign neural graft. With longer survival times in unsensitized hosts, a progressively smaller percentage of animals had detectable donor astrocytes (5 of 10 animals at 3 months postimplantation and 4 of 16 animals at 4 months); in those 9 animals with surviving grafts, only small numbers of M2-positive cells were seen within the graft bed and surrounding host brain. However, only 2 of the 26 "long-term" animals showed evidence of graft rejection. These results indicate that mouse astrocytes show characteristic patterns of migration into the host brain when implanted into neonatal rats; however, these xenogeneic cells have a limited duration of survival. The infrequency with which even subtle signs of spontaneous rejection were detected in animals that had received corpus callosum xenografts suggests that an immune-mediated process is unlikely to be responsible for the time-dependent elimination of the donor astrocytes.(ABSTRACT TRUNCATED AT 400 WORDS)

Cultured epithelioid astrocytes migrate after transplantation into the adult rat brain.

A highly purified population of dividing epithelioid astrocytes has been prepared from postnatal rat corpus callosum. These cells were labelled in culture by incorporation of either [3H]thymidine or fluorescent microspheres and transplanted in a fibrin clot into the hippocampi of adult syngeneic rats. Transplanted cells divided in vivo and progressively migrated into the host brain from the site of implantation up to distances of about 1 mm. After a 1-week survival, transplant cells stained strongly for glial fibrillary acidic protein and had the thick sinuous processes characteristic of stellate astrocytes. Artefactual transfer of radiolabel to host cells was ruled out by control experiments in which either the proportion of transplant cells that were radiolabelled was varied or radiolabelled transplant cells were killed prior to implantation. Astrocyte migration over the first days after implantation was determined to occur at a rate of approximately 100 microns per day. Transplant cells moved into both grey and white matter areas of the host brain and over the migratory period were commonly observed to be associated with blood vessels. Some transplant cells were directly juxtaposed against neuronal perikarya and dendrites. Many labelled astrocytes were located in areas that were apparently completely free of damage caused by implantation. These results define a class of mature astrocytic cells that have the ability to migrate through the adult brain. The existence of pathways for cell movement in the adult CNS has implications for the mechanisms of tissue remodelling after injury and transplantation, for regenerative repair of the CNS, and for the dynamics of cell-cell contacts in the normal adult mammalian brain.

Disappearance of xenogenic astrocytes transplanted into newborn mice is associated with a T-cell response.

Following transplantation of fragments of embryonic rabbit brain into the brains of newborn mice, the proportion of mice bearing detectable xenogenic astrocytes increases to over 80% in the first 3-4 weeks. Recent studies have demonstrated that the host response at this time was dominated by non-specific elements of host defense: macrophages, microglia and astrocytes. In the second phase, the proportion of mice bearing xenogenic astrocytes declines rapidly after 4 weeks and reaches zero by 10 weeks. In the present experiments, designed to characterize the host defense during this period, a dramatic increase in the proportion of mice displaying T-cells in the brain in the fourth and fifth weeks after transplantation was found. This corresponded with a marked decline of xenogenic astrocytes. Both subsets of T-cell, helper-inducer (L3T4) and cytotoxic-suppressor (Lyt2), were found, with L3T4 more numerous in many samples. T-cells were found at the site of transplantation and at sites of migration. The division of the host-defense response in this model into a phase of antigen non-specific cells followed by a period when T-cells appear and transplanted astrocytes disappear, should facilitate kinetic studies into the mechanisms of brain-graft rejection.

Migration of xenogenic astrocytes in myelinated tracts: a novel probe for immune responses in white matter.

Experimental brain transplantation allows the study of the development of the immune response against brain antigens within the brain itself. This laboratory has developed a transplantation model in which rabbit embryo brain fragments are placed in the brains of newborn mice. The migration of xenogenic astrocytes is traced by a monoclonal antibody which combines with donor but not host glial fibrillary acidic protein. In the first 4 weeks after transplantation, the donor astrocytes successfully migrate, often within myelinated tracts. Following this period, T cells make their appearance and xenogenic astrocytes disappear by 10 weeks. The propensity for clearly identified foreign astrocytes to migrate in myelinated tracts coupled with a well-defined time course of host-vs-graft interaction suggested that the model could be used to study the immune response in white matter. The studies reported here provide sequential examples of the relationship between migration by foreign astrocytes in myelinated tracts and the development of the host immune response. Extensive migration in white matter tracts was first observed in the absence of any T cell response. Subsequently T cells were found at the transplantation site. Finally Ia was found to be expressed on blood vessels and microglia were strongly reactive in white matter that contained T cells but no foreign astrocytes. These observations support the suggestion that the model can be used to more precisely define cellular immune events that occur within white matter.

Timing and patterns of astrocyte migration from xenogeneic transplants of the cortex and corpus callosum.

The timing, pattern, and pathway of astrocyte migration were investigated in vivo by transplantation of CD-1 mouse cerebral cortex (E13-14) or corpus callosum (P2-3) into neonatal rat cortex. A monoclonal antibody specific for a mouse astrocyte surface antigen (M2) was used to identify the location of the grafts and the migrated donor astrocytes. Within the host cortex, astrocytes from cortical grafts began migration at post-transplantation day (PTD) 7. Over the next 4 days, the most distant displaced donor cells were found progressively further away from the grafts, migrating at a rate of about 220 microns/day. After PTD 11, the migration rate for the farthest displaced donor cells slowed to 25 microns/day, and the cells appeared to stop at about PTD 16 at a distance of 1,100 microns from the edge of the graft. Astrocytes had a faster migration speed in the white matter and covered a longer distance (5 mm) than those in the gray matter, extending on occasion into the contralateral hemisphere. The patterns of astrocyte migration differed depending on local cues around the transplant. Donor astrocytes that had been implanted into the host cortex migrated toward the host cortical surface, sometimes in several radial lines. Astrocytes from grafts, especially callosal grafts, placed in the subcortical white matter migrated along the host fiber tracts. Many astrocytes transplanted into the hippocampus formed laminar patterns close to the hippocampal neuronal layers. These results suggest that the direction, pattern, and speed of astrocyte migration are influenced by local substrates in the host brain.

Short-term post-grafting morphological alterations of glia from an adult brain transplant.

Fragments of corpus callosum from adult rabbit have been implanted into the brain of newborn mice. Previous studies had shown that under such conditions transplant-derived astroglial cells differentiate in the host and survive for at least 2 months. The present study was devised to clarify the fate of the differentiated astrocytes present in the adult transplant by using combined ultrastructural and immunohistochemical approaches. These mature cells are shown to degenerate and die within 2 days after the implantation. Therefore, we suggest that stem cells present in adult tissue would account for the surviving population of transplant-derived glial cells.

Interspecies identification of astrocytes after intracerebral transplantation.

Species-specificity of the Tp-GFAP 1 (glial fibrillary acidic protein) monoclonal antibodies raised against calf GFAP was established by means of immunochemical techniques. Since it was shown to combine with rabbit GFAP but not with mouse GFAP it allows the characterization of a new experimental model potentially useful in the study of the fate of implanted astrocytes after intracerebral graft of CNS fragments. Preliminary observations indicate that embryonic and newborn rabbit astrocytes are able to survive, express GFAP and migrate when implanted into newborn mouse brain.