Microglia, MHC expression and graft rejection.
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Biomedical subjects
R D Lund
Publications and source records attributed to R D Lund.
Optic nerve degeneration induces the expression of MHC antigens in the rat visual system.
The brain has long been considered to be an immunologically privileged site. However, privilege is not absolute, as has been shown by the inability of foreign tissue grafts to survive indefinitely in the brain. The rejection of this tissue is accompanied by the upregulation of major histocompatibility complex (MHC) antigen expression. Therefore it is essential to define conditions that influence the expression of these antigens in the brain, especially since such a definition may further the understanding of disease processes that lead to the autoimmune destruction of the central nervous system. Here we show that both MHC class I and class II antigens are expressed within 1 or 2 days of eye removal by cells showing the morphological characteristics of microglia. Expression is seen along the optic pathway and within the brainstem centers to which optic axons project. In the early stages of the reaction, MHC class I antigen expression is seen throughout the optic pathway, including the terminal distribution areas of the subcortical visual centers, while MHC cells class II are localised mainly to degenerating myelinated fiber systems. These changes are not accompanied by any alteration in the integrity of the blood-brain barrier. During the second week postlesion, class I positive cells are found beyond the confines of the degenerating pathways, while class II positive cells are seen within regions such as the stratum griseum superficiale of the superior colliculus, where few myelinated axons are present. There is subsequent diminution of MHC positive cells, but a small number of cells are still seen 60 days post-lesion. Focal lesions within the eye show that at early survival times, while class I MHC positive cells are distributed throughout the nerve, class II positive cells are largely absent from the unmyelinated segment of the nerve. Retrograde changes in the retina after nerve section are accompanied only by MHC class I antigen expression. These observations show that neural degeneration is accompanied by a rigid sequence of events involving expression of MHC antigens by microglia. If foreign antigens were present in the brain while these events were taking place, it is possible that such antigens would be recognised and destroyed by the host immune system.
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Transient outgrowth from retinae implanted in the neonatal rat cerebral cortex.
Retinae placed in the cortex of neonatal rodents show substantial outgrowth, both along the subpial margin and within the cortical white matter, which is not sustained beyond 6 days post-transplantation. These results, in combination with previous studies, suggest that optic axon outgrowth is sustained for a finite period, after which it requires target-derived influences for its long-term maintenance.
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Retinal transplants in congenitally blind mice: patterns of projection and synaptic connectivity.
Embryonic retinae were transplanted onto the midbrain of neonatal congenitally anophthalmic mice and neonatal mice from which both eyes had been removed. When donor mice of the AKR strain were used, the detailed patterns of the transplant projections to the host brain were demonstrated with an antibody to Thy-1.1, which specifically stains neural tissue derived from AKR donors. Many of the subcortical visual centers were innervated, and only small differences were encountered between anophthalmic and eye-enucleated mice. The terminal arbors of transplant-derived axons could not be classified as in normal animals, although several distinct arbor types were seen. In the superior colliculus, the laminar arrangements that characterize normal retinal arbors were disrupted. Despite this, the synaptic patterns formed by transplant-derived axons in the superior colliculus of anophthalmic mice compared very closely with those of retinal axons in normal, sighted animals. These observations indicate that the ability of a retinal transplant to innervate the host brain and to form the synaptic arrays characteristic of optic terminals are not dependent on prior innervation, nor do they appear to be influenced by the events that follow eye removal.
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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.
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Anatomical and functional consequences of induced rejection of intracranial retinal transplants.
Retinae from embryonic rats transplanted over the midbrain of newborn host rats establish connections with visual centres of the host brain, which mediate a pupilloconstrictor response in the host eye when the transplant is stimulated by light. The changes in the size of the host pupil can be measured accurately with a pupillometry system. We have taken advantage of the additional observation that while grafts between rat strains, as between Long Evans and Sprague-Dawley strains, may survive indefinitely, they can be induced to reject by skin grafting from the strain providing the donor retinal tissue. Combining pupillometry with skin grafting provides a useful way of examining correlated anatomical and behavioural changes associated with graft rejection from its earliest stage to the point of overt destruction. Even within three days of skin grafting, the amplitude and speed of constriction as well as the response latency all showed significant enhancement from normal, and this was sustained for a further week or more. Response deterioration followed during the second week post-skin grafting, but the exact timing varied considerably among animals. Anatomical observations of the process of retinal rejection showed the first invasion of lymphocytes to occur between days 5 and 7 and total degeneration of the retinal transplant and its projections to occur by two to three weeks post-skin grafting. The lymphocytic infiltration was preceded by upregulation of microglia, which expressed both class I and II major histocompatibility antigens and by activation of astrocytes identified by their expression of glial fibrillary acidic protein. Within the target region of retinal transplant axons, major histocompatibility antigen expression and astrocytic responses preceded degeneration of transplant derived axons (demonstrated by the Fink-Heimer stain) and there was no evidence for any lymphocytic lymphocytic infiltration during transplant rejection. These observations show that the earliest stages of microglial activation are accompanied by an enhancement of response parameters, but that the functional failure finally occurs only at an advanced stage of graft destruction. The absence of lymphocytic infiltration into areas receiving terminals from axons of transplant origin, even though these contain significant numbers of reactive microglia, suggests that the terminal axonal processes are not a primary target for the immune response.
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The migration of astrocytes after implantation to immature brains.
Using a species-specific marker, we have found that astrocytes, taken from donors of varying ages from fetal to adult, migrate in highly stereotypic patterns in immature host brains. Migration is primarily within and towards cell layers, although some cells are seen to migrate along fibre bundles. This contrasts with studies using the same approach in mature hosts, where migration is predominantly within fibre layers, largely excluding cellular regions.
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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.
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Neonatal host astrocyte migration into xenogeneic cerebral cortical grafts.
Migration of host astrocytes into grafts was investigated by transplantation of rat cortex (E16) into the cortex or midbrain of neonatal mice. Host astrocytes, visualized by the mouse astrocyte-specific antibody, began to invade the grafted cortex during the first week post-transplantation and sequentially migrated substantial distances throughout the graft. Host cells in the grafts which were undergoing immune rejection became hypertrophic. These results have important implications when assessing interactions between host and graft cells.
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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)
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A role for microglia in the maintenance of photoreceptors in retinal transplants lacking pigment epithelium.
Studies on intact retina have pointed to a necessary role for retinal pigment epithelium in the maintenance of photoreceptor outer segments and for regeneration of visual pigment. However, it has been shown that when embryonic retinae are separated from the pigment epithelium and transplanted into the brain of neonatal rats, the transplanted photoreceptors develop outer segments and the retina responds to light in the apparent absence of pigment epithelial cells. We confirm that there are no retinal pigment epithelium cells associated with transplanted retinae in the present series of experiments and show that a row of cells, composed predominantly of microglia of host origin, border the graft. These cells can be seen to contain engulfed outer segments when they are apposed to the outer retina, suggesting that the microglia have assumed, at the least, the phagocytic function normally associated with retinal pigment epithelium. Microglial cells and their processes are also found within the transplant, but these cells are typically devoid of phagosomes, indicating an absence of phagocytic activity. The close physical association of these resting microglia with the transplant may facilitate their role in antigen presentation under specific conditions of immune provocation.
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cFos labeling in rat superior colliculus: activation by normal retinal pathways and pathways from intracranial retinal transplants.
Previous studies in this laboratory have shown that intracranial retinal transplants can establish both anatomical and functional connections with the host brain. Embryonic rat retinae transplanted intracranially into neonatal host brains are capable of evoking appropriate physiological and behavioral responses when illuminated. The present study employs the specific expression of the cFos protein to identify brain regions in which immediate-early gene activation can be recognized in response to flash stimuli delivered to retinal transplants and to normal intact retinae. Stimulation of the intact eye induced significant cFos expression in various visual centers, including the stratum griseum superficiale of the superior colliculus and the pretectal area, but not in the dLGN, suprachiasmatic nucleus, or retina. Animals with functional transplants expressed cFos throughout the depth of the stratum griseum superficiale and stratum opticum of the superior colliculus, thus apparently activating an additional population of superior collicular cells. Like the eye-stimulated animals, animals with functional transplants failed to elicit significant cFos expression in the dLGN or transplanted retina. This study indicates that intracranial retinal transplants are capable of forming functional connections with the host superior colliculus which not only mediate transient changes in electrical activity but also may affect gene expression through the induction of the c-fos gene.
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Rapid enhancement of transplant-mediated pupilloconstriction after elimination of competing host optic input.
Retinae transplanted over the brainstem, when directly illuminated, cause pupilloconstriction in the host eye. This occurs even when the host eye is maintained in darkness. If the host optic nerve is then severed, the transplant-mediated response is substantially augmented within 1-2 days. It is suggested that this is due to enhancement of the signal-to-noise ratio resulting from elimination of dark discharge in the host optic nerve.
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Plasticity in innervation of the rat superior colliculus by transplanted retinae as a result of eye removal at maturity.
When the superior colliculus of a rat is innervated by inputs from both eyes as well as a retina transplanted intracranially over one tectum at birth, the tectal projection from the transplant is confined mainly to the superficial surface of the superior colliculus. The transplant-derived fibers possess a simple morphology, lacking terminal arborizations. If the contralateral eye is removed one month after transplantation, these fibers can be induced to arborize into the denervated portion of the superior colliculus over the next month. This demonstration of sprouting in a mature sensory relay system raises the possibility that an enhancement of behavioral responses mediated by transplanted retina might also occur. In turn, this may provide an ideal system to study the correlation between anatomical changes in transplant axons and changes in behaviors mediated by transplant activity.
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The impact of intracerebral retinal transplants on types of behavior exhibited by host rats.
Retinae transplanted over the midbrain of newborn rats establish functional connections with host brain centers, which provide a substrate for several distinct visual functions. These responses provide insight into the relationship between anatomy and behavior under normal conditions and after brain injury, as well as into the strategies used by an animal to extract significant information from its visual environment.
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Parameters of retinal graft-mediated responses are related to underlying target innervation.
Retinae taken from embryonic mice were transplanted to the brain of newborn rats. One eye was removed from the recipients at this time to promote innervation of host visual centers. After 4-6 weeks the optic nerve of the remaining eye was transected to eliminate all visual afferents of host origin and 4-6 days later the grafts were exposed surgically for testing. Nineteen animals with grafts located on the dorsal surface of the cerebellum form the basis of this study. Animals differing markedly in behavioral performance were selected for simultaneous immunohistochemical processing. Measurements of peak density and area of mouse-specific immunoreactivity associated with the olivary pretectal nuclei (OPN) were compared to pupillary response parameters using the Pearson correlation coefficient. This analysis was performed on the combined data from all groups and revealed a number of highly significant correlations between both area and density of labelling and pupillary response parameters. In all 7 co-processed groups, the best responders within each group showed the heaviest innervation of the olivary pretectal nucleus. These findings indicate therefore that the degree to which a transplanted retina innervates the appropriate host brain target is an important determinant of graft efficacy. The xenograft-mediated pupillary reflex provides a model which permits detailed analysis of the relationship between connectivity and behavior in a simple relay system.
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Fetal olfactory bulb transplants send projections to host olfactory cortex in the rat.
We are using the rat olfactory system to study developmental details of neurotransplantation. Tritiated [3H]thymidine-labeled fetal olfactory bulbs (OBs), were transplanted immediately into sites from which the neonatal host OB was removed. Subsequently, a small lesion was placed in the region of the transplanted OB and the tissue studied, using degeneration methods and autoradiography. Only OB's with extensive [3H]-label and precise lesions confined to the labeled areas were used. Degeneration was found mainly in the ipsilateral piriform cortex with lesser amounts at other nearby sites. The results demonstrate successfully transplanted donor OBs that send axons to specific and appropriate target areas of the host brain.
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Differential expression of class I and class II major histocompatibility complex antigen in early postnatal rats.
Cells expressing major histocompatibility complex (MHC) antigens are rarely found in normal mature brains, but cells resembling microglia can be induced to express these antigens following the onset of neural degeneration. In young rats, these cells show spontaneous expression of class I MHC antigens, which is further enhanced in the superior colliculus by the degeneration resulting from eye removal. By contrast, class II MHC antigen expression does not occur spontaneously and can only be induced by eye removal when the lesion is performed after the first postnatal week, when the optic tract begins to myelinate. We suggest that different signals are responsible for induction of class I and of class II MHC antigen expression.
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