Intracerebral Grafting Research Articles

Blood Flow Changes Following 137 Cs Irradiation in a Rat Glioma Model

Blood flow changes in response to 20 Gy 137Cs whole brain irradiation were measured with quantitative autoradiography of [14C]iodoantipyrine (IAP) in intracerebral grafts of the 36B-10 rat glioma, the brain around tumor (BAT), the contralateral corpus callosum, and the contralateral cerebral cortex. Irradiations were delivered on Day 14 post-transplantation, and measurements of flow (F) were performed with IAP on Day 15 or Day 16. Mean values of F were determined in individual tumors and in treatment groups. In 15- and 16-day-old unirradiated control tumors, the group mean F was 0.31 ml.g-1.min-1. In both 15- and 16-day-old tumor groups irradiated on Day 14 (Day 1 and 2 postirradiation tumors) the mean F for each day's group was 0.52 ml.g-1.min-1, 68% higher than the control (P less than 0.01). Flow in the BAT and the contralateral corpus callosum similarly was increased at these times (P less than 0.01). Flow in the contralateral cerebral cortex was 1.1, 1.5, and 1.3 ml.g-1.min-1 in the control, 1 day postirradiated, and 2 day postirradiated groups, respectively, but these increases were not significantly different from the control. These data indicate that flow increases in the intracerebral gliomas as well as in normal brain regions during the 2 days following 20 Gy irradiation. Changes such as these following radiotherapy may have important effects on the bioavailability of chemotherapeutic drugs.

Development of intracerebral dopaminergic grafts: a combined immunohistochemical and autoradiographic study of its time course and environmental influences.

The aim of the study was to obtain a description of some aspects of the development of intracerebral dopaminergic grafts, namely, the time course of the glial reaction and its relation to cell division on one hand, and the development of graft-originated innervation and its dependence on adequate matching of the implanted neurons and target site on the other hand. Cell suspensions obtained from the mesencephalon or hypothalamus of embryonic day (ED) 14 rat embryos were implanted into the striatum or lateral hypothalamus of adult rats following the destruction of the nigrostriatal system of the hosts. Animals were sacrificed at different postimplantation times, and the development of the graft was followed by immunohistochemistry by using antisera directed against tyrosine hydroxylase (TH) or glial fibrillary acidic protein (GFA). Furthermore, the existence of cell division at various times following implantation was examined by performing autoradiography on immunostained sections after prior intraventricular administration of 3H-thymidine to the host. The first stage of the development of intracerebral grafts was characterized by the existence of intense cell division within the grafted tissue, lasting about 2 weeks, and also in the host tissue surrounding the graft, lasting only about 6 days. The cell division in the host tissue was paralleled by the existence of a strong glial reaction which, however, did not extend into the graft itself. Glial reaction in the host tissue gradually decreased at later times and disappeared by 4 weeks postimplantation without leaving behind a noticeable glial scar. The graft itself was, however, transiently filled with a population of reactive astroglial cells between 3 and 6 weeks postimplantation. Within grafts of mesencephalic tissue located in the striatum TH-positive neurons were distributed evenly at short times postimplantation (2-6 days). At later time a compartmentation could be observed, with TH-positive neurons being aligned along the graft-host interface or clustered within the graft itself. Innervation of the host tissue by TH-positive fibers increased between 1 and 6 weeks postimplantation. On the other hand, no compartmentation and reinnervation of surrounding host tissue was observed for intrahypothalamic grafts of mesencephalic tissue or intrastriatal grafts of hypothalamic tissue. This last observation indicates that adequate matching of implanted neurons and target tissue plays an important role in the development of intracerebral dopaminergic grafts.

Housing conditions modulate the effects of intracerebral grafts in rats with brain lesions

The aim of this study was to examine whether the behavioural effects of intracerebral grafts in rats with brain lesions can be modulated by postoperative housing conditions. Sixty-six young adult female rats were used. Twenty-two rats were sham-operated (S) and 44 rats sustained a complete aspirative lesion (L) of the fimbria-fornix and overlying tissue. The cavity was used in half the rats as the implantation site for solid fetal septal transplants (LT). Two days later, half the rats of each group were housed in an ‘enriched’ condition (S.EC, L.EC and LT.EC); the other half remained in standard condition (S.SC, L.SC and LT.SC). Two and 10 months later, the rats were tested in a Hebb-Williams maze. Two months after surgery, there was a highly significant lesion effect, no graft effect and no environmental effect except in S rats, S.EC rats making fewer initial errors than S.SC rats. Ten months after surgery, L rats still made more errors than S rats and S.EC rats made fewer initial errors than S.SC rats; however, the lesion-induced deficits were significantly attenuated in LT.EC rats, but not in LT.SC rats. These data show that both the enriched environment and the implant were unable alone to promote behavioural recovery but, applied together, they reduced the lesion-induced deficits. Dorsal hippocampus acetylcholinesterase (AChE)-staining intensity was reduced by the lesions, but this reduction was partially compensated for by the grafts; however, environmental conditions did not affect AChE-staining significantly. This latter observation suggests that the graft-derived partial cholinergic reinnervation of the hippocampus probably does not underly the behavioural improvement observed only in LT.EC rats.

Distorted development of intracerebral grafts: long-term maintenance of tyrosine hydroxylase-containing neurons in grafts of cortical tissue

Cortical cells obtained from rat embryos (ED14 to ED20) were implanted in various regions of rat brain and the presence of tyrosine hydroxylase (TH)-, neuropeptide Y (NPY)- and Met-Enkephalin (ENK)-immunoreactive neurons within the grafts were tested using an immunohistochemical approach. TH-like immunoreactive (TH-LI) neurons were present within the implants obtained from ED14, but not ED18 or ED20, embryos up to 10 months post-implantation and their presence was not dependent on the age of the host (adult or neonate) at the time of implantation. Furthermore, the density of such cells varied with the site of implantation, being the highest in the dorsomedial corner of the striatum. This distorted development seems to affect also other cell populations, such as NPY-LI neurons which could be observed within the implants in a density much higher than that found in the normal cortex and which presented generally a rather immature morphology. It has been described that the rat cortex contains TH-LI neurons only during a limited period of development. The survival of such neurons within intracerebral grafts of cortical tissue indicates that their disappearance during normal cortical development is dependent upon environmental cues. The survival of TH-LI cells in grafts implanted to neonatal hosts suggests that these cues are not some humoral factors appearing postnatally. On the other hand, the present observations are compatible with several other hypothesis concerning the nature of such cues: humoral factors present during the late embryonic period, signals dependent on neuronal connectivities (input and/or outputs) established during embryonic or postnatal life. Moreover, humoral factors present in the host brain could help the maintenance of TH-LI cells in the graft, as shown by the site dependency of their density.

Brain grafting may reverse loss of responsiveness to levodopa therapy in Parkinson's disease.

Loss of efficacy and response fluctuations develop in many patients with Parkinson's disease after long-term levodopa therapy. This may be due in part to near-total degeneration of the surviving nigrostriatal dopaminergic neurons during disease progression, with massive decreases in the capacity of the striatum to form and store dopamine from exogenous levodopa. It was recently suggested that intracerebral grafting of fetal nigral or adrenal chromaffin cells may be beneficial in advanced Parkinson's disease by reestablishing spontaneous dopaminergic neurotransmission or by secretion of trophic factors that promote sprouting of residual dopaminergic nerve-terminals. It is now hypothesized that intrastriatal transplantation of such cellular elements that contain the enzyme dopa decarboxylase and dopamine storage sites may significantly increase synthesis, storage, and release of dopamine from exogenous levodopa. It may therefore reverse loss of responsiveness and restore the initial smooth and stable beneficial effect of levodopa therapy.

Ultrastructural organization of normal and transplanted rat fascia dentata: II. A quantitative analysis of the synaptic organization of intracerebral and intraocular grafts.

As part of an ultrastructural analysis of the normal rat fascia dentata and intracerebral and intraocular dentate transplants the synapses in the dentate molecular layer were quantified. Hippocampal and dentate tissue from 21-day-old rat embryos were grafted into the brain of developing and adult rats and to the anterior eye chamber of adult rats. After 100 or 200 days of survival the recipient rat brains and the recipient eyes were processed for electron microscopy, and the graft dentate molecular layer with the adjacent granule cell layer selected for ultrastructural analysis. Tissue from the dentate molecular layer of normal adult rats served as controls. The dentate synapses were classified as asymmetric (Gray's type 1) or symmetric (Gray's type 2), and according to the postsynaptic element (cell body, dendritic shaft, dendritic spine). The spine synapses were further classified into simple and complex types according to the spine-terminal configuration. Also, the length of synaptic contacts of the individual synaptic types was measured in some grafts, just as the percentage of the cross sectional area of the neuropil covered by blood vessels. The results showed that the synaptic density, expressed as number per unit area of neuropil, to a large extent was the same within the different parts of the normal dentate molecular layer. Compared with this the synaptic density was reduced with 16.4% in dentate molecular layer of the intracerebral graft, primarily because of a 17.6% reduction of simple synapses on dendritic spines and almost halving of the symmetric synapses on dendritic shafts. The synaptic density was independent of the age of the recipient, the intracerebral location of the graft, and the survival time. Although the synaptic length of some of the individual synaptic types increased, this did not compensate for the loss of synapses. In the intraocular grafts the synaptic density was lower than in the intracerebral grafts. Despite the reduced synaptic density, which mainly involved two synaptic types, we conclude that grafted dentate granule cells can develop a remarkably normal, ultrastructural synaptic organization even in the absence of major afferent inputs. This outcome must accordingly be achieved by reorganization of the available intrinsic afferents.

Ultrastructural organization of normal and transplanted rat fascia dentata: I. A qualitative analysis of intracerebral and intraocular grafts.

Few studies have dealt with the general ultrastructure and synaptic organization of grafted brain tissue. This study was therefore performed to extend current light microscopic observations on intracerebral and intraocular grafts of hippocampal tissue to the ultrastructural level. Blocks of tissue containing the hippocampus and fascia dentata from day 21 embryonic rats were grafted into the brain of developing and adult rats and to the anterior eye chamber of adult rats. After 100 or 200 days of survival the recipient rat brains or eyes were processed for electron microscopy. Tissue containing the graft dentate molecular layer and adjacent granule cell layer was selected for ultrastructural analysis, together with a few samples of the hilus and CA3. Normal dentate tissue was analyzed as control. At the light microscopic level most intracerebral and intraocular grafts displayed an organotypic organization with clearly recognizable cell and neuropil layers. Under the electron microscope the grafted granule cells had normal-appearing dendrites bearing the normal types of spines and forming the normal types of synapses. This was the case even in the absence of the normal major extrinsic afferents like the perforant path. The graft dentate granule cells formed axons and terminals with characteristics of the normal mossy fiber system in the hilus and CA3, in addition to aberrant supragranular mossy fiber terminals known from light microscopic studies of dentate transplants. Abnormal structures included a few dendritic growth cones and an increased occurrence of polyribosomes in spines. Their occurrence indicates ongoing dendritic plasticity even 100 days after transplantation. There was also an increased density of glial elements, particularly in the intraocular grafts. In some of these grafts the granule cells displayed immature traits in terms of nuclear indentations. Dentate interneurons of the basket cell type were present in both the intracerebral and the intraocular grafts. We conclude that grafted dentate granule cells, in different surroundings and without the normal, major perforant path input, can develop a basically normal cellular morphology, which includes the normal ultrastructural characteristics of the dendrites with spines and synapses, and the mossy fibers and its terminals.

Chapter 86 Implantation of genetically engineered cells to the brain

Publisher Summary This chapter discusses the combination of gene transfer via efficient viral vectors, followed by intracerebral grafting of the genetically modified cells may constitute an effective approach to some disorders of central nervous system (CNS) function. Several prerequisites for a human disease to be suitable as a candidate recipient for transplantation of genetically engineered cells are also discussed, including the pathogenesis and pathophysiology of the disease must be sufficiently well understood for the identification of the relevant gene product to be introduced into defective cells, the relevant gene must be well characterized and available as a clone, or ideally, an animal model should be available. However, the major difficulty with most human diseases of the CNS is that these criteria cannot be satisfied, because the exact mechanisms of pathophysiology and pathogenesis in most human genetic diseases, such as phenylketoneuria and Lesch-Nyhan disease, are not adequately understood. Although, the present models are all very complex, and truly effective therapy is likely to be extremely difficult to achieve, but it seems probable that a number of conceptually new approaches to these previously inaccessible diseases will become feasible, and that such new approaches will include the implantation of genetically modified cells.

Regional Blood-to-Tissue Transport in an Irradiated Rat Glioma Model

To assess vascular permeability in intracerebral grafts of the 36B-10, F-344 rat glioma following 20 Gy 137Cs whole brain irradiation, the blood-to-tissue transport constant, K, of [14C]-alpha-aminoisobutyric acid (AIB) was measured with quantitative autoradiography. Mean, 90th percentile, and 95th percentile values of K were determined in individual tumors and in treatment groups. In 15-day-old unirradiated control tumors, mean, 90th percentile, and 95th percentile values of K were, respectively, 11.3, 18.4, and 20.8 ml kg-1 min-1. In 15-day-old tumors irradiated on Day 14 (Day 1 postirradiation tumors) the K values were 5.9, 9.4, and 10.4, all of which were significantly less than the respective control values (P less than 0.01). In 16-day-old tumors irradiated on Day 14 (Day 2 postirradiation tumors), the K values were 10.8, 15.0, and 16.0, respectively, none of which was significantly different from control tumors. Mean K values for Day 2 vs Day 1 postirradiation tumors (10.8 vs 5.9) yielded P less than 0.05, but the 90th percentile and 95th percentile values for Day 2 vs Day 1 yielded 0.05 less than P less than 0.10. Separate experiments measured AIB and 86RbCl uptake in 36B-10 cells in vitro 1 and 2 days following 20 Gy irradiation to assess whether this radiation dose reduced the capacity of tumor cells to trap AIB or Rb+. Irradiation did not reduce the accumulation of either tracer, but rather was associated with an increased accumulation of AIB. Therefore, the AIB transport data suggest that vascular permeability and/or surface area decreases significantly in the day following 20 Gy irradiation and that this decrease reverses by the second day following irradiation.

Angioarchitecture of the CNS, pituitary gland, and intracerebral grafts revealed with peroxidase cytochemistry.

Blood vessels of the fetal, neonatal, and adult subprimate and primate CNS, including circumventricular organs (e.g., median eminence, pituitary gland, etc.), and of solid CNS and nonneural (anterior pituitary gland) allografts placed within brains of adult mammalian hosts were visualized with peroxidase cytochemistry applied in three ways: to tissues from animals injected systemically with native horseradish peroxidase (HRP) or peroxidase conjugated to the lectin wheat germ agglutinin (WGA) prior to perfusion fixation; to tissues from animals infused with native HRP into the aorta subsequent to perfusion fixation; and to tissues from animals fixed by immersion and incubated for endogenous peroxidase activity in red cells retained within blood vessels. In neonatal and adult animals receiving native HRP intravascularly, non-fenestrated vessels contributing to a blood-brain barrier were outlined with HRP reaction product when tetramethylbenzidine (TMB) as opposed to diaminobenzidine (DAB) was used as the chromogen; fenestrated vessels of circumventricular organs were not discernible due to the density of extravascular reaction product. Fenestrated and non-fenestrated cerebral and extracerebral blood vessels exposed to bloodborne WGA-HRP were visible when incubated in TMB and DAB solutions. Native HRP infused into the aorta of fixed animals likewise labeled non- fenestrated vessels throughout the brain upon exposure to TMB or DAB but obscured fenestrated vessels of the circumventricular organs. Endogenous peroxidase activity of red cells, seen equally well with TMB and DAB, outlined blood vessels throughout the cerebral gray and white matter and all circumventricular organs in fetal, neonatal, and adult animals. Application of the three peroxidase cytochemical approaches to study the development or absence of a blood-brain barrier in intracerebral allografts demonstrated that the vascularization of day 16-19 fetal/1 day neonatal CNS allografts is not well defined prior to 7 days following intracerebral placement of the grafts. CNS allografts secured from donor sites expected to possess a blood-brain barrier exhibited blood vessels that were not leaky to HRP injected intravenously in the host. Fenestrated blood vessels associated with anterior pituitary allografts were evident prior to 3 days posttransplantation within the host brain and permitted blood-borne HRP in the host to enter the graft and surrounding host brain parenchyma.

Cryopreservation of fetal rat brain tissue later used for intracerebral transplantation

Intracerebral grafting of immature brain tissue is now widely used as a tool to study neuronal development and regeneration in the brain and spinal cord. This has stimulated the interest in methods for storage of such tissue before transplantation. In this study a method for cryopreservation of immature rat central nervous tissue is presented and discussed in relation to current cryobiological principles. The method was applied to brain tissue from 16- and 17-day-old fetal rats, including the neocortex, habenula, septum and basal forebrain, cerebellum, and retina. After storage in liquid nitrogen from 6 to 52 days the tissue was grafted into the brain of adult rats. The recipients survived for 23 to 673 days before their brains were processed by current neuroanatomical, histological methods. The presence of graft tissues was recorded and their cellular and connective organization was examined, including their exchange of nerve connections with the host brain. The results obtained were comparable with results from other studies where the same tissues were grafted immediately after removal from the donor, and a study of cryopreservation of developing hippocampal tissue. We conclude that cryopreservation is a reliable method for storage of immature neural tissue later to be used for intracerebral grafting.

Chapter 27 Grafts of fetal cholinergic neurons to the deafferented hippocampus

Publisher Summary The cholinergic component of the septohippocampal system has been employed with considerable success in the studies of intracerebral grafting. There are several reasons for this success. First, the chemical neuroanatomical organization is well established; specifically, the cholinergic cells of origin in the septal-diagonal band region and their routes of passage, as well as the characteristic pattern of terminal lamination within the hippocampal formation, are well described. Second, the principal and most characteristic electrophysiological property of the hippocampus is dependent on the anatomical and cholinergic integrity of the septohippocampal circuitry. Third, the hippocampal formation has long been associated with learning and memory and substantial evidence supports the importance of the cholinergic system in the mechanism by which the hippocampus may influence learning and memory. Together these components of the cholinergic septohippocampal system have provided an experimental model within which the anatomical, biochemical, electrophysiological, and behavioral effects of graft–host interactions are investigated. The results of the experiments directed at these issues are reviewed in the chapter.

Neonatal hippocampal neurons, retrogradely labeled with granular blue, survive intracerebral grafting and explantation to tissue culture

Previous studies have shown that developing neocortical neurons labeled in situ by retrograde axonal transport of the fluorescent dye Granular Blue can retain this dye for at least 2 months essentially without fading or leakage. In this study, retrograde labeling with Granular Blue was used to label neonatal rat hippocampal neurons prior to intracerebral grafting to uninjected littermates, and explantation as slice cultures. Hippocampal regio inferior and hilar neurons labeled through their developing commissural axons were found to survive axotomy and subsequent grafting and explantation for at least 3 and 8 weeks, respectively. The labeling helped define the developmental differentiation of the neurons at the time of manipulation and provides a new method for identification of a specific population of transplanted or explanted neurons.

Behavioural effects of human fetal dopamine neurons grafted in a rat model of Parkinson's disease.

The ventral mesencephalon, containing the developing dopaminergic neurons of the substantia nigra-ventral tegmental region, was obtained from aborted human fetuses of 9-19 weeks of gestation. The tissue was grafted into the striatum of rats previously subjected to a 6-hydroxydopamine lesion of the mesostriatal dopamine pathway. The graft recipients were immunosuppressed by daily injections of Cyclosporin A. Amphetamine-induced motor asymmetry was reduced, and finally totally reversed, only in rats receiving grafts from the 9-week old fetal donor. The fluorescence microscopic analysis revealed large numbers of surviving dopamine neurons, and extensive fiber outgrowth into the host striatum, in these rats. By contrast, rats receiving grafts from 11-19 week old donors had at most only few surviving dopamine neurons. These results indicate that human fetal mesencephalic tissue may be an efficient source of dopamine neurons for functional intracerebral grafting in patients with Parkinson's disease.

Non-cholinergic afferents determine the distribution of the cholinergic septohippocampal projection: a study of the AChE staining pattern in the rat fascia dentata and hippocampus after lesions, X-irradiation, and intracerebral grafting.

The acetylcholinesterase (AChE) activity of the rat hippocampus and fascia dentata depends on an intact septohippocampal connection, and histochemical staining for AChE is commonly used to monitor the distribution of the cholinergic septohippocampal projection. It is also characteristic that the laminae of low or moderate to dense AChE staining in the hippocampus and fascia dentata coincide with the terminal fields of the major non-cholinergic, afferent pathways. While studying lesion-induced collateral sprouting and aberrant axonal growth of these pathways we observed that the AChE staining pattern changed in accordance with the reorganized distribution of the non-cholinergic pathways, and this occurred even without direct interfering with the septohippocampal projection itself. Widening and narrowing of the medial perforant path and mossy fiber terminal zones thus resulted in corresponding changes in the bands of AChE staining normally associated with these zones. Expansion of the commissural-associational hippocampodentate projections and the lateral perforant path was in a similar way paralleled by a widening of the AChE-poor zones which normally overlap with the termination of these projections. Observations of the same kind were made in intracerebral transplants of fascia dentata innervated by various host afferents, and in rats subjected to neonatal X-irradiation, where the mossy fiber projection is reduced and aberrant perforant pathways project into CA3 due to a reduced formation of granule cells. The observed sets of changes with linkage between the different non-cholinergic projections and the activity of AChE in their respective terminal fields were accordingly reproduced under several different experimental conditions. It could not be explained alone by interaction between the septal afferents and their target cells. We therefore conclude that the density and laminar distribution of the AChE activities within the hippocampus and fascia dentata are determined at least in part by the major afferent, non-cholinergic nerve connections. We suggest that the effect occurs through direct axonal interaction or through changes in the receptiveness of the common dentate and hippocampal target cells.

Implantation of PC12 cells into the corpus striatum of rats with lesions of the dopaminergic nigrostriatal neurons

PC12 cells, which have the capacity to synthesize and release catecholamines, were implanted into the corpus striatum of adult rats with lesions of the dopaminergic nigrostriatal pathway. PC12 cells survived for 1–2 weeks in the host striatum and degenerated thereafter. The presence of these cells was associated with an attenuation of the apomorphine-induced circling behavior in rats with unilateral nigrostriatal lesions. These findings indicate that cells from a cell line are able, at least temporarily, to survive in the rat brain and to compensate functional deficits caused by lesions of specific neuronal systems. Cell lines might therefore be used for intracerebral grafting instead of neurons derived from fetal brains, if their survival can be prolonged and their proliferation in the host brain prevented.

Rat fetal brain tissue grafts survive and innervate host brain following five day pregraft tissue storage

Freshly dissected fetal basal forebrain tissue rich in cholinergic neurons was either dissociated into a cell suspension and injected into the cholinergically denervated hippocampus of adult rats, or stored in a preservative medium at 4°C. Five days later the stored tissue was dissociated into a cell suspension and injected in the denervated hippocampus in another group of animals. Six weeks later the grafts were evaluated with acetylcholinesterase histochemistry for the assessment of graft survival, graft tissue volume and extent of reinnervation of the denervated hippocampus. All grafts in both the freshly dissociated and the ‘stored’ group survived. The stored grafts were on the average 1 5 in volume but had an appropriate and extensive laminated reinnervation pattern within the denervated hippocampus. This method of storing cells for extended periods prior to grafting allows for experimental manipulation of fetal tissue as well as long distance transportation prior to intracerebral grafting.

Functional capacity of solid tissue transplants in the brain: evidence for immunological privilege.

The capacity of the mammalian brain to support the physiological function of allografts was assessed in parathyroidectomized Fischer strain rats bearing either isografts or immunogenic DA allografts of parathyroid glands implanted in their cerebral cortices. Established isografts and allografts survived indefinitely in the brain, maintaining normal serum calcium levels, with equal numbers of spontaneous failures (18-21%) in each group. Similarly, both MHC-compatible and incompatible skin allografts survived and were 'functional' at 40-50 days postgrafting as assessed by: continued formation of keratin; the presence of differentiated hair follicles and sebaceous glands; and frequent mitotic figures. No serum alloantibodies were induced by either MHC-incompatible parathyroid glands or skin in this site. However, both types of allografts were promptly rejected or failed to become established in the brains of specifically presensitized hosts. Furthermore, when Fischer hosts with long-established intracerebral DA parathyroid grafts received orthotopic DA skin grafts, their parathyroid grafts were rejected along with first-set rejection of the skin grafts. The tempo of this cellular immune response and the primary alloantibody response that accompanied it indicate that although the intracerebral grafts failed to induce detectable host sensitization or suppression, they remained susceptible to immune effectors. Thus, by using strongly immunogenic, adult tissues, we have established that the rat cerebral cortex is an immunologically privileged site, and the privilege is not dependent on lack of graft immunogenicity or alterations in host responsiveness. Furthermore, Ia+ (possible antigen-presenting) cells were rare in the cortical parenchyma sites used for transplantation though numerous in the choroid plexus of the ventricles and in certain areas of white matter. Therefore, privilege probably reflects deficient graft antigen presentation related to the paucity of Ia+ cells as well as to the brain's poor lymphatic drainage.

Irradiation and BCNU effects in the stroma in a rat transplanted glioma model: analysis of cerebral tumor bed effect.

F-344 rats were treated with whole-brain fractionated or unfractionated 137Cs at several time intervals up to 6 weeks before they received intracerebral grafts of an ethylnitrosourea-induced rat astrocytoma (36B-10). Rats treated with a single dose of 18 Gy at 3- and 2 week intervals prior to tumor implantation died earlier than unirradiated control tumor recipients (P less than 0.05, log rank sum test). Delivered 2 weeks prior to tumor grafting single doses of 10, 15, and 20, but not 5 Gy also reduced animal life span (P less than 0.05). Similarly, a total dose of 38 but not 28.5, 19, or 9.5 Gy, each delivered in five daily fractions 2 1/2 weeks before tumor grafting, shortened survival (P less than 0.05). Chemotherapy with 1,3-bis(2-chloroethyl)-1-nitrosourea (13.3 mg/kg) alone or in combination with a single-dose of irradiation of 18 Gy 3 weeks preceding transplantation showed no effect on animal survival. Unirradiated control tumors demonstrated sheets of densely-packed isomorphic astrocytes, perivascular brain parenchymal invasion, sparse, thin-walled vasculature, frequent mitoses, but little necrosis or hemorrhage. The tumors of rats irradiated before transplantation showed large areas of confluent necrosis associated with microvascular thrombosis, edema, and multifocal acute hemorrhages. These survival results and morphological changes indicate that the cerebral tumor bed effect involves early animal death and extensive morphological changes in the tumors; and they suggest that tumor cell death in irradiated brain tumors may, in part, result from radiation-induced vascular injury.

Monitoring of cell viability in suspensions of embryonic CNS tissue and its use as a criterion for intracerebral graft survival

Neuronal cell suspensions, prepared by trypsination and mechanical disruption from embryonic CNS tissue, are currently used for intracerebral neuronal grafting to deep brain sites. In the present study the viability of suspended neurons from different brain regions has been monitored with a fluorescent vital stain, and studied as a function of time after dissociation and age of the donor rat embryos. Subsequently, the validity of the in vitro viability rates as a criterion for the in vivo survivability of each individual suspension was tested for suspensions prepared from the developing mesencephalon, rich in dopamine-containing neurons. The results indicate that mechanically dissociated embryonic CNS neurons remain viable for several hours in a simple glucose-saline solution at room temperature. The in vitro viability scores declined faster in suspensions prepared from mesencephalon than in those prepared from telencephalon (striatum and basal forebrain), and they declined faster in suspensions prepared from older embryos. The fetal cells were sensitive to the mechanical trauma caused by excessive pipetting, and tissue from older embryos seemed generally more vulnerable in the trypsination-dissociation procedure. The grafting experiments showed a good correlation between the in vitro cell viability counts and in vivo neuronal survival after grafting, indicating that the vital stain, at least under certain conditions, can be used as a simple and practical routine test to check and standardize cell suspensions to be used in intracerebral grafting experiments.