Homotopic Grafts Research Articles

In situ analysis of neuronal dynamics and positional cues in the patterning of nerve connections

Recently developed imaging techniques permit individual cells to be uniquely labeled and followed over time as development proceeds in intact vertebrate embryos. Small groups of cells in the developing eye rudiment of the frog Xenopus have been labeled with the vital dyes DiI, lysinated fluorescein dextran (LFD) or lysinated rhodamine dextran (LRD). Individual optic axons and their growth cones were clearly visible in the intact living animal using confocal microscopy or epifluorescence microscopy with a low light level video camera and computer-based video image enhancement. To follow the dynamics of single optic nerve fiber terminal arborizations, small groups of cells, or even single retinal ganglion cells, were labeled with DiI, and the resulting labeled optic nerve fibers were imaged using a confocal microscope. The images show a profound alteration in morphology from day to day, demonstrating that optic nerve terminal arborizations are dynamic structures constantly extending and retracting branches. To follow the topography of the developing projection and analyze the cues that guide its formation, small groups of eyebud cells from LFD- and LRD-labeled donor embryos were grafted to an unlabeled host in either a location equivalent to that from which they had been removed (homotopic grafts) or a non-equivalent location (heterotopic grafts). Axons from homotopic grafts projected to the tectum as expected from the adult topography of the retinotectal projection. Dorsoventral topography was present from the time that the optic nerve fibers were observable in the tectum, in agreement with previous work. Nasotemporal topography was subtle or absent for the first few days, and then slowly refined. The importance of positional cues was tested by performing heterotopic eyebud grafts, in which the labeled eyebud cells are grafted to inappropriate places in the host eyebud. The heterotopic grafts appeared to integrate with the ectopic site in the eyebud in a functional manner. They should, therefore, project to the tectum together with their new neighbors if neighbor interactions or activity-based cues were of primary importance in the initial patterning of the map. However, the experiments showed that the axons from heterotopic grafts always behaved in a fashion appropriate to their position of origin in the donor, regardless of their final position in the host. These observations indicate that small groups of eyebud cells (as small as a single cell) possess positional information that plays a dominant role in guiding the optic nerve fibers to their target sites in the tectum.

Axotomized, adult basal forebrain neurons can innervate fetal frontal cortex grafts: A double fluorescent tracer study in the rat

The ability of axonal regeneration of identified adult basal forebrain (BFB) neurons was examined after homotopic grafting of fetal neocortical tissue to a lesion cavity in the frontal neocortex. Using a four step experimental procedure, adult rats first received an injection of the fluorescent dye Fluoro-Gold (FG) into the sensorimotor cortex in order to label those neurons with projections to the area by retrograde axonal transport. After one week the injection area was removed by aspiration, leaving a cavity in the neocortex. One week later a block of fetal (E14) frontal cortical tissue was placed in the cavity. The animals were then allowed to survive for 6 weeks before a second fluorescent tracer, Nuclear Yellow (NY), was injected into the transplant. The animals were sacrificed 24 h later and analyzed by fluorescence microscopy. Both single labeled, FG and NY containing neurons and double labeled neurons containing both tracers were found in the BFB. The results demonstrate that adult BFB neurons can reestablish cortical projections into fetal cortical grafts (double labeled neurons), and they suggest that other BFB neurons, not initially innervating the lesioned cortical area, have sprouted into the transplant (NY labeled neurons).

Chapter 21 Transplantation of fetal spinal cord tissue into acute and chronic hemisection and contusion lesions of the adultrat spinal cord

Publisher Summary This chapter discusses and examines: (1) the pattern of axonal connectivity established with fetal homotopic (that is, spinal cord) grafts into the acutely injured spinal cord, (2) the influence of an existing histopathology on survival of these transplants and their integration with the host spinal cord in chronic lesions, and (3) the feasibility of transplantation into chronic, contusion lesions. It is feasible to transplant embryonic central nervous system (CNS) tissue into various lesions of the adult spinal cord, including those, which may ultimately shed light on the potential clinical application of intraspinal transplantation. These studies also establish a useful neuroanatomical framework for physiological and behavioral tests of the functional impact of these grafts in acute and chronic hemisected and contused spinal cords. While, evidence exists for some axonal connectivity between host and graft, it is still uncertain that an optimal setting has been established under any of these lesion conditions, as indicated in part by the variable glial responses seen in individual transplant recipients. However, intraspinal transplantation has begun to stimulate new avenues of investigation, which will undoubtedly facilitate a more in-depth understanding of the complex biology of the injured spinal cord.

GABAergic neurons of the hippocampus: Development in homotopic grafts and in dissociated cell cultures

The hippocampus taken from E18-E19 rat embryos was dissociated into a cell suspension and was either grafted into the hippocampus of adult rats or cultured. The growth of GABAergic neurons was examined using a GABA directed antiserum. The implanted tissue was capable of survival and growth without exhibiting a laminar organization. Most of the various morphological neuronal types could be observed, establishing different types of synapses; however, granule neurons were rarely encountered. A substantial proportion of GABA-positive neurons was detected within the graft with profuse labelling of the neuropil. In cultures issued from the same cell suspension, GABA-immunoreactive neurons were numerous and had different morphologies. Altogether these data suggest that GABA neurons express a high potential for growth and sprouting in vitro and in vivo.

Fetal hypothalamic brain grafts reduce the obesity produced by ventromedial hypothalamic lesions

Bilateral destruction of the rat ventromedial hypothalamus (VMH) produces a syndrome characterized by hyperphagia and obesity. In the present study we examined whether grafts of fetal hypothalamus could reverse the effects of this lesion. Three groups of adult rats received bilateral electrolytic lesions of the VMH. The first group of animals was then implanted with embryonic day 14–16 hypothalamic tissue by stereotaxic injection into the lesion sites. The second series of animals received comparable-sized grafts from a variety of non-hypothalamic regions of the fetal CNS. The third group experienced similar VMH lesions but did not receive any tissue grafts. After surgery, body weight and food consumption were recorded daily for up to 8 weeks. These measures were compared with similar ones obtained from non-operated rats. Hyperphagia and obesity were consistently observed in all of the lesioned animals not bearing transplants. An initial period of weight gain was also observed in animals receiving hypothalamic grafts, but the duration of the ‘dynamic’ phase of this syndrome was reduced. Consequently, these graft recipients exhibited significantly less weight gain. This depressed weight gain, however, did not coincide with a statistically significant decrease in hyperphagia. Transplantation of non-hypothalamic tissue also caused an attenuation of the VMH-lesion effect but this was more modest than that induced by homotopic grafts. The results of this experiment show that homotopic transplants can alter the dynamics of weight gain induced by bilateral VMH lesions. However, lesion-induced hyperphagia was not completely reversed in these grafted animals. The fact that other regions can exert a similar effect, though of lesser magnitude, suggests that a more general property of fetal CNS tissue may be involved.

Vasopressin neuron survival in neonatal Brattleboro rats; critical factors in graft development and innervation of the host brain

Previously it was found that grafts of supraoptic plus paraventricular areas from 19-day-old foetal normal rats survived in the third ventricle of the brain of 4- to 6-day-old, vasopressin-deficient Brattleboro pups, but could not alleviate their polyuria. In the present series, factors important in graft development were analysed. Again using day-19 fetuses as donors, anterohypothalamus grafts as well as grafts placed near a crushed median eminence survived relatively poorly, but showed the presence of vasopressin neurons immunocytochemically one month post-grafting. Homotopic grafting in the supraoptic nucleus, however, even failed to show surviving vasopressin neurons. Graft survival was improved by the use of donor tissue of fetuses younger than day 19. Parvocellular vasopressin cells were frequently seen, organized into clusters resembling the normal suprachiasmatic nucleus. However, magnocellular neurons, as normally seen in supraoptic and paraventricular nuclei, only survived grafting when taken between days 11 and 15 of fetal age. It was concluded that only immature vasopressin neurons survived grafting under the condition employed. Magnocellular neurons had a limited fiber outgrowth into the host brain and median eminence. Most large neurons only stained with non-specific neurophysin antiserum, not with specific vasopressin-associated neurophysin antiserum. Thin fibers of the parvocellular vasopressin neurons provided only occasional and sparse innervation of the host median eminence and lateral septum (one case), but several examples of massive fiber bundles running dorsally from graft into host brain were observed. These fibers terminated in the thalamic periventricular area, a nucleus that is normally innervated by the vasopressin neurons of the suprachiasmatic nucleus. The failure of the grafts to provide adequate vasopressinergic innervation of the host median eminence probably explains why none of the nearly 200 Brattleboro neonates operated upon showed any sign of relief of their diabetes insipidus. It suggests, however, that the present procedures might be useful in restoring central vasopressinergic functions in the developing Brattleboro rat.

Experimental studies on a mutant gene ( e) preventing the differentiation of eye and normal hypothalamus primordia in the axolotl

The phenotype of axolotls (Ambystoma mexicanum) homozygous for the mutant gene e (“eyeless”) is different from normal in that (1) no optic vesicles develop in e e embryos, (2) e e larvae from posthatching onward are darker than normal white larvae, and (3) fully grown e e animals are sterile. Experiments reported here show that eyelessness in e e embryos results from a direct effect of the gene on presumptive forebrain ectoderm; not on the mesoderm that induces the ectoderm to form eyes. Homotopic grafts of normal presumptive ectoderm on e e blastula hosts differentiated complete eyes, but reciprocally grafted embryos were always eyeless. Similarly, grafts of either e e or normal presumptive prechordal mesoderm into normal hosts gave normal eyes, but in the mutant hosts no eyes developed. Thus the e gene affects only the ectodermal component of the inductive system for eye formation. Genetically eyeless (pigmented) cells, when interspersed prior to gastrulation among genetically eyed (albino) cells in the eye preprimordium, are induced to form clones of pigmented retinal epithelium in the albino host eye. The sterility of e e larvae appears also to be due to a direct effect of the e gene on the ectodermal (neural plate) primordium of the hypothalamus. Grafts of normal cells which included the hypothalamic, but not the optic or anterior pituitary primordia, always restored fertility to e e recipients. The mutant pigmentation phenotype was demonstrated to be a consequence of eyelessness and, therefore, an indirect effect of the gene. The pigment pattern of normal embryos from which both optic vesicles were removed resembles that of the mutants. In addition, implantation of a single full-sized, functional eye was able to restore the normal pigmentation, but not fertility, to e e recipients.

Systemic Influence on Activity of Hair Follicles in Skin Homografts

ABSTRACT Strips of skin from the flank were exchanged between rats aged 28 and 49 days respectively, so that their expected hair growth waves would be out of phase. Similarly sited autografts were used as controls. In the wave of growth immediately following grafting eruption of hairs on the homografts coincided with that on the donor’s autograft. In the older recipient this was significantly in advance and in the younger recipient significantly in retard of hair growth on their autografts. Thus, follicular activity has an inherent rhythm which continues for at least one cycle after homotopic grafting. In the succeeding waves of growth, when follicular activity in the recipient preceded that in the donor, the time of eruption of hair on the homograft was advanced, and by the third wave after grafting was completely in line with its host. When follicular activity in the recipient succeeded that in the donor the homografts tended to behave donor-wise. These results show clearly that both an inherent rhythm and a systemic stimulus are involved in follicular activity at a particular site.