Solid Grafts Research Articles

Apoptosis in Primary Cultures of E14 Rat Ventral Mesencephala: Time Course of Dopaminergic Cell Death and Implications for Neural Transplantation

Transplantation using fetal nigral grafts has been performed by various groups worldwide in over 200 Parkinson's disease (PD) patients in an attempt to restore dopaminergic (DA) input to the striatum. However, the proportion of the implanted DA neurons that survives, whether using suspension, partially dissociated, or solid grafts, is small, often as low as 5 to 10%, which is insufficient to allow a full functional recovery. A significant proportion of the transplanted neurons in animal models of PD has been shown to die via apoptosis, but the reason for this is unclear. Since the methods used to prepare donor tissue for neural transplantation and in vitro culture are identical, we have looked at the time course of DA neuron loss following cell suspension preparation using an in vitro assay system and considered whether the procedures used may, in part, be responsible for the poor DA neuron survival. Primary dissociated cultures of E14 rat ventral mesencephala were incubated for different periods in serum-containing and serum-free media. After fixation, the TUNEL method, as well as ethidium bromide and acridine orange, were used to detect apoptosis, and DA neurons were localized immunocytochemically. Results showed that most apoptosis occurred during the first 24 h and that 50% of the DA neurons were lost in the first 8 h. Double-immunofluorescent labeling confirmed the presence of TUNEL+ve nuclei within DA neurons. There was no difference in either the extent or rate of loss between the serum-containing and serum-free medium during the first 32 h. We suggest, therefore, that existing methods used to prepare cell suspensions probably induce apoptosis and may need to be modified in order to increase the survival of DA neurons.

Cytokine gene expression following neural grafts.

A reverse-transcription polymerase chain reaction study of molecular events following solid neural tissue xenogeneic and syngeneic grafts into the mouse caudate nucleus was made between 1 day and 30 days post-grafting. Constitutive expression of interleukin (IL)-1 and transforming growth factor (TGF)-beta1 mRNAs was detected. The sham operation produced minimal cytokine upregulation, indicating that surgical trauma caused minimal damage. A similar picture was seen with syngeneic grafts, which showed good graft survival. Xenografts, however, were rejected within 30 days and cytokine mRNAs for IL-2, IL-2R, IL-4, IL-10 and interferon (IFN)-gamma were detected from 7 days post-graft, correlating with histological identification of a leukocytic infiltrate. This method provides a quick, comparative screen for detecting cytokine mRNA profiles in neural grafts and may assist the diagnosis of early rejection phenomena. As such, it is a potential analytical tool for strategies aimed at depleting/blocking the activity of different cell types and/or cytokines in future neural transplant therapy.

Long-term evaluation of bilateral fetal nigral transplantation in Parkinson disease.

Parkinson disease (PD) is associated with a progressive loss of nigrostriatal dopamine neurons. Medication therapy provides adequate control of symptoms for several years, but long-term treatment is complicated by progressive disability and the development of motor fluctuations and dyskinesias. In animal models of PD, fetal nigral transplants have been shown to survive grafting into the striatum, provide extensive striatal reinnervation, and improve motor function. In patients with PD, cell survival and clinical benefit have been observed following fetal nigral grafting, but results have been inconsistent. To evaluate the safety and efficacy of bilateral fetal nigral transplantation into the postcommissural putamen in patients with advanced PD complicated by motor fluctuations and dyskinesias. Six patients with advanced PD underwent bilateral fetal nigral transplantation. Each patient received solid grafts derived from donors aged 6 1/2 to 9 weeks after conception stereotactically implanted into the postcommissural putamen using 3 to 4 donors per side. Cyclosporine was administered for approximately 6 months to provide immune suppression. Clinical evaluations included the Unified Parkinson's Disease Rating Scale (UPDRS), Schwab-England Activities of Daily Living Scale, and timed tests of motor function conducted during both the "off' and "on" states at baseline and at 1, 3, 6, 9, 12, 18, and 24 months following transplantation. Percentage of time off and percentage of time on with and without dyskinesia were recorded at half-hour intervals using home diaries during the week prior to each evaluation. 18F-fluorodopa positron emission tomographic scans were performed at baseline, and at 6 months and 1 year following transplantation. Patients have been followed up for a mean+/-SD of 20.5+/-5.5 months. Complications related to surgery were mild and transient. Activities of daily living, motor, and total (activities of daily living plus motor) UPDRS scores during the off state improved significantly (P<.05, Wilcoxon signed rank test) at final visit in comparison with baseline. Mean total UPDRS off score improved 32%, and each patient experienced at least a 19% improvement. Mean percentage of time on without dyskinesia during the waking day improved from 22% to 60% (P<.05). Mean putamenal fluorodopa uptake on positron emission tomography increased significantly at 6 and 12 months in comparison with baseline (P<.001, 2-tailed t test). This increase correlated with clinical improvement. Two patients died 18 months after transplantation from causes unrelated to the surgical procedure. In both cases, histopathological examination showed robust survival of tyrosine hydroxylase immunoreactive cells and abundant reinnervation of the postcommissural putamen. Fetal nigral tissue can be transplanted into the postcommissural putamen bilaterally in patients with advanced PD safely and with little morbidity. In this open-label pilot study we observed consistent long-term clinical benefit and increased fluorodopa uptake on positron emission tomography. Clinical improvement appears to be related to the survival and function of transplanted fetal tissue.

Haematopoietic stem cells can induce specific skin graft acceptance across full MHC barriers.

Previously, we and others have demonstrated in several animal models that the establishment of stable haematopoietic chimerism through allogeneic bone marrow transfusion provides an effective means for the development of specific transplantation tolerance. However, a major limitation to the clinical application of allogeneic bone marrow transfusion in immunosuppressed recipients for induction of tolerance to solid grafts, is the risk of graft-versus-host disease (GVHD). Therefore, it is important to identify the cell population needed for the induction of mixed chimerism and tolerance. Haematopoietic stem cells have the capacity of self-renewal and multilineage differentiation, and have been shown to reduce the risk of GVHD. We studied transfusion of two rich sources of stem cells, namely allogeneic fetal liver cells and a subset of purified bone marrow-derived progenitor cells (c-kit+) into anti-T cell monoclonal antibody-treated, low-dose irradiated recipient mice. Our data revealed that stable multilineage mixed chimerism and permanent donor-specific tolerance for skin, even when transplanted directly following conditioning, can be successfully achieved in this way, with no signs of GVHD.

Bilateral Fetal Striatal Grafts in the 3-Nitropropionic Acid-Induced Hypoactive Model of Huntington’s Disease

We investigated the 3-nitropropionic acid (3-NP)–induced hypoactive model of Huntington’s disease (HD) to demonstrate whether fetal tissue transplantation can ameliorate behavioral deficits associated with a more advanced stage of HD. Twelve-week-old Sprague–Dawley rats were introduced to the 3-NP dosing regimen (10 mg/kg, i.p., once every 4 days for 28 consecutive days), and were then tested for general spontaneous locomotor activity in the Digiscan locomotor apparatus. All rats displayed significant hypoactivity compared to their pre-3-NP injection locomotor activity. Randomly selected rats then received bilateral intrastriatal solid grafts of fetal striatal (lateral ganglionic eminence, LGE) tissues from embryonic day 14 rat fetuses. Approximately 1/3 of each LGE in hibernation medium was infused into each lesioned host striatum. In control rats, medium alone was infused intrastriatally. A 3-mo posttransplant maturation period was allowed prior to locomotor activity testing. Animals receiving fetal LGE grafts exhibited a significant increase in locomotor activity compared to their post-3-NP injection activity or to the controls’ posttransplant activity. Surviving striatal grafts were noted in functionally recovered animals. This observation supports the use of fetal striatal transplants to correct the akinetic stage of HD. To the best of our knowledge, this is the first study that has investigated the effects of fetal striatal transplantation in a hypoactive model of HD.

Bilateral fetal striatal grafts in the 3-nitropropionic acid-induced hypoactive model of Huntington's disease.

We investigated the 3-nitropropionic acid (3-NP)-induced hypoactive model of Huntington's disease (HD) to demonstrate whether fetal tissue transplantation can ameliorate behavioral deficits associated with a more advanced stage of HD. Twelve-week-old Sprague-Dawley rats were introduced to the 3-NP dosing regimen (10 mg/kg, i.p., once every 4 days for 28 consecutive days), and were then tested for general spontaneous locomotor activity in the Digiscan locomotor apparatus. All rats displayed significant hypoactivity compared to their pre-3-NP injection locomotor activity. Randomly selected rats then received bilateral intrastriatal solid grafts of fetal striatal (lateral ganglionic eminence, LGE) tissues from embryonic day 14 rat fetuses. Approximately 1/3 of each LGE in hibernation medium was infused into each lesioned host striatum. In control rats, medium alone was infused intrastriatally. A 3-mo posttransplant maturation period was allowed prior to locomotor activity testing. Animals receiving fetal LGE grafts exhibited a significant increase in locomotor activity compared to their post-3-NP injection activity or to the controls' posttransplant activity. Surviving striatal grafts were noted in functionally recovered animals. This observation supports the use of fetal striatal transplants to correct the akinetic stage of HD. To the best of our knowledge, this is the first study that has investigated the effects of fetal striatal transplantation in a hypoactive model of HD.

Characteristics of human fetal spinal cord grafts in the adult rat spinal cord: influences of lesion and grafting conditions.

The present study evaluated the growth potential and differentiation of human fetal spinal cord (FSC) tissue in the injured adult rat spinal cord under different lesion and grafting conditions. Donor tissue at 6-9 weeks of gestational age was obtained through elective abortions and transplanted either immediately into acute resection (solid grafts) or into chronic contusion (suspension and solid grafts) lesions (i.e., 14-40 days after injury) in the thoracic spinal cord. The xenografts were then examined either histologically in plastic sections or immunocytochemically 1-3 months postgrafting. Intraspinal grafts in acute lesions demonstrated an 83% survival rate and developed as well-circumscribed nodules that were predominantly composed of immature astrocytes. Solid-piece grafts in chronic contusion lesions exhibited a 92% survival rate and also developed as nodular masses. These grafts, however, contained many immature neurons 2 months postgrafting. Suspension grafts in chronic contusion lesions had an 85% survival rate and expanded in a nonrestrictive, diffuse pattern. These transplants demonstrated large neuronally rich areas of neural parenchyma. Extensive neuritic outgrowth could also be seen extending from these grafts into the surrounding host spinal cord. These findings show that human FSC tissue reliably survives and differentiates in both acute and chronic lesions. However, both the lesion environment and the grafting techniques can greatly influence the pattern of differentiation and degree of host-graft integration achieved.

Permeability of the Blood–Brain Barrier to Protein and [3H]GABA in Intraparenchymal Fetal CNS Tissue Grafts

In the present study solid grafts of rat fetal neocortex or substantia nigra were transplanted into young adult rat cortex or striatum. To study the permeability to protein and potential changes in the blood–brain barrier (BBB), HRP was administered intravascularly or, to examine endogenous protein exudation, noninjected animals were evaluated after immunocytochemical stain for rat serum albumin. In addition, [3H]GABA, a neurotransmitter that does not cross the BBB, was systemically administered to cortical graft-bearing hosts in order to determine if a barrier to small potentially bioactive compounds was present. Postoperative periods ranged between 1 week and 15 months for the cortical grafts and between 1 week and 8 months for nigral grafts; the experiments were repeated several times at the same time point in order to determine the potential for variability in the model systems. The results show that a high percentage (80%) of both neocortical and nigral grafts lack a complete BBB to protein up to 2–3 weeks postoperative. After this time the majority of neocortical grafts were impermeable to protein yet 20–30% of longer-term specimens showed variable degrees of permeability to protein; permeability to [3H]GABA, which manifested in the transmitter being sequestered by graft neurons, was observed only up to 4 weeks postoperative. Nigral grafts after 4 weeks postoperative showed negligible permeability to serum protein. Although in all early CNS grafts protein permeation is extensive in both graft and adjacent host, the mature host brain resolved the exudation far more efficiently so that the reaction appeared exclusively in the graft or interface and not in the host by 2–3 weeks, suggesting that the graft tissue may lack appropriate mechanisms for clearance of extracellular materials. It is unclear why this phenomenon occurred consistently but in a minority percentage of cortical specimens. It is suggested that either frank pathology or immunopathology provides a subtle and incomplete rejection process that allows the interface vessel to be permeable over the life of the graft. Since nigral grafts appeared to have significantly less BBB dysfunction variation it is possible that the maturation levels or the ontogeny between different graft sources may play a role in differing responses to injury or vascular problems after transplant surgery.

Telencephalic transplants in mice: characterization of growth and differentiation patterns

Telencephalic grafting represents a powerful tool for developmental studies and for the investigation of biological features of transgenic brain tissue. The interpretation of grafting experiments, however, requires detailed knowledge of graft biology. Therefore, we have characterized growth rates, graft size, and differentiation of embryonic telencephalic tissue harvested at various developmental stages and grafted into the caudoputamen and lateral ventricles of histocompatible mice. A total of 164 grafts were analysed up to 500 days after transplantation. Of all transplants, 79.3% resulted in the formation of solid neural grafts. Grafted cells were identified by 3H-thymidine labelling and autoradiography. Proliferation was studied by bromodeoxyuridine incorporation and decreased from an initial 35% at 1-3 d after grafting to less than 1.6% after 40 days. The graft size was measured as a function of the embryonic age of the transplanted tissue. Our data indicate that telencephalic tissue harvested at embryonic day E 12.5 reproducibly yields large, fully differentiated neuroectodermal grafts. The parameters defined in this study will be useful for detailed analysis of neuroectodermal tissue from mice undergoing fatal neurodegeneration, such as knockout mice bearing lethal mutations.

Transplantation of human fetal tissue from spontaneous abortions to a rodent model of parkinson's disease

The use of human fetal tissue from elective abortions for cell transplantation therapies has been the subject of considerable controversy. Because of concerns regarding the use of tissue from elective abortions, tissue from spontaneous abortions has been suggested as an alternate donor source. In the present study we have evaluated human fetal tissue from spontaneous abortions to assess its viability, growth potential, and functional expression. Viable cells (Grade I) from a donor (7 wk postconception) were transplanted as a suspension into the striatum of rats with unilateral 6-OHDA lesions of the nigrostriatal pathway. A second group of animals received solid grafts of tissue from a Grade I donor 14 wk postconception. Tissue from Grade II and III specimens were not sufficiently viable for transplantation. Locomotor responses were monitored over a period of 15 wk and revealed an amelioration of rotational asymmetry by animals that received tissue from the 7 wk donor. Animals receiving tissue from the 14 wk donor showed no functional improvement. We found numerous graft-derived tyrosine hydroxylase (TH) immunopositive neurons contained within the transplantation site, and a rich plexus of TH-immunopositive fibers extending into the striatum of animals receiving tissue from the 7 wk donor. Animals receiving tissue from the 14 wk donor exhibited tissue necrosis at the transplant site and were devoid of TH-immunopositive neurons. These results suggest that human fetal ventral mesencephalic cells from spontaneous abortions can survive and develop after transplantation, and rectify locomotor deficits associated with experimental parkinsonism if the donor tissue is of the appropriate gestational age at the time of implantation. Our study further suggests, however, that the availability of tissue from spontaneous abortions of sufficient viability is quite limited and may thus restrict its potential use in cell transplantation therapies for Parkinson's disease.

Transplantation of human fetal tissue from spontaneous abortions to a rodent model of Parkinson's disease.

The use of human fetal tissue from elective abortions for cell transplantation therapies has been the subject of considerable controversy. Because of concerns regarding the use of tissue from elective abortions, tissue from spontaneous abortions has been suggested as an alternate donor source. In the present study we have evaluated human fetal tissue from spontaneous abortions to assess its viability, growth potential, and functional expression. Viable cells (Grade I) from a donor (7 wk postconception) were transplanted as a suspension into the striatum of rats with unilateral 6-OHDA lesions of the nigrostriatal pathway. A second group of animals received solid grafts of tissue from a Grade I donor 14 wk postconception. Tissue from Grade II and III specimens were not sufficiently viable for transplantation. Locomotor responses were monitored over a period of 15 wk and revealed an amelioration of rotational asymmetry by animals that received tissue from the 7 wk donor. Animals receiving tissue from the 14 wk donor showed no functional improvement. We found numerous graft-derived tyrosine hydroxylase (TH) immunopositive neurons contained within the transplantation site, and a rich plexus of TH-immunopositive fibers extending into the striatum of animals receiving tissue from the 7 wk donor. Animals receiving tissue from the 14 wk donor exhibited tissue necrosis at the transplant site and were devoid of TH-immunopositive neurons. These results suggest that human fetal ventral mesencephalic cells from spontaneous abortions can survive and develop after transplantation, and rectify locomotor deficits associated with experimental parkinsonism if the donor tissue is of the appropriate gestational age at the time of implantation. Our study further suggests, however, that the availability of tissue from spontaneous abortions of sufficient viability is quite limited and may thus restrict its potential use in cell transplantation therapies for Parkinson's disease.

Bilateral fetal nigral transplantation into the postcommissural putamen in Parkinson's disease.

We performed fetal nigral transplantations in 4 Parkinson's disease (PD) patients. Solid grafts were bilaterally implanted into the postcommissural putamen using 3 to 4 donors per side aged 6 1/2 to 9 weeks postconception. Transplant deposits were separated by no more than 5 mm in three dimensions. Cyclosporine was employed for a total of 6 months. Patients were evaluated at baseline and at 1, 3, and 6 months postoperatively. Striatal 18-fluorodopa uptake was assessed by positron emission tomography at baseline and at 6 months postoperatively. The procedure was well tolerated in all patients. One patient had a clinically asymptomatic superficial cortical hemorrhage along the needle tract and a second had transient postoperative confusion and hallucinations. All patients experienced clinically meaningful benefit. Significant improvement (p < 0.05) was detected in total UPDRS score during the "off" state, Schwab-England disability score during the "off" state, percent "off" time, and percent "on" time with dyskinesia. Increased striatal fluorodopa uptake was observed bilaterally in each patient, with mean increases of 53% on the right (p = 0.01) and 33% on the left (p = 0.08). Our study demonstrated clear and consistent improvement in clinical features and striatal fluorodopa uptake following fetal tissue transplantation in patients with advanced PD whose condition was not improved preoperatively by drug manipulation. These preliminary results are encouraging and support further studies to evaluate grafting strategies as a therapy for PD.

Neovascularization of rat fetal neocortical grafts transplanted into a previously prepared cavity in the cerebral cortex: a three-dimensional morphological study using the scanning electron microscope

Neovascularization within syngeneic rat fetal neocortical grafts transplanted into a previously prepared cavity in the cerebral cortex was studied 1 to 3 months after transplantation, utilizing scanning electron microscopy of vascular corrosion casts. The grafts were easily identified and the outer surface of the grafts, especially at the host-graft interface, was surrounded by large regenerated vessels of leptomeninges and connective tissue (e.g. dura). Large vessels originating from the choroid plexus also coated the grafts in animals whose lateral ventricles had been opened at the time of cavitation. These large regenerated vessels were mainly observed on the surface of the grafts, and they ramified markedly to form capillary networks in the vicinity of the host-graft interface. Occasionally several relatively large regenerated vessels were noted to extend into the grafts, and to ramify and connect with graft capillary networks having the same features as that of the host brain. Moreover, direct vascular connections between host capillaries and those within the grafts were observed. In some animals, arteries and arterioles which fed the grafts were identified in the perimeter of the grafts with their characteristic morphology. The interior microvasculature structure of the grafts was largely composed of the capillary network of graft origin, and of several relatively large penetrating vessels originating from the regenerated leptomeningeal vessels or the vessels of the choroid plexus. The present study demonstrated that the blood supply to the solid grafts transplanted into the previously prepared cavities originated primarily from the regenerated host vessels. These host vessels perfused the intrinsic graft vessels via new anastomoses which formed predominantly at the host-graft interface.

Neural transplantation and recovery of cognitive function

AbstractCognitive deficits were produced in rats by different methods of damaging the brain: chronic ingestion of alcohol, causing widespread damage to diffuse cholinergic and aminergic projection systems; lesions (by local injection of the excitotoxins, ibotenate, quisqualate, and AMPA) of the nuclei of origin of the forebrain cholinergic projection system (FCPS), which innervates the neocortex and hippocampal formation; transient cerebral ischaemia, producing focal damage especially in the CA1 pyramidal cells of the dorsal hippocampus; and lesions (by local injection of the neurotoxin, colchicine) of the granule cells of the dentate gyrus. Following chronic alcohol or lesions of the FCPS, transplants of cholinergically rich fetal brain tissue into the terminal areas (neocortex and/or hippocampus) restored performance almost to control levels, with a time course consistent with growth of the transplants and integration with host tissue; transplants of cholinergically poor fetal tissue (hippocampus) were without effect, as were transplants of cholinergically rich tissue into the region containing the nuclei of origin of the FCPS. Grafts of primary cells enriched in glia and cultured neuroblastoma cells into the terminal areas of the FCPS were equally effective, suggesting that there are multiple mechanisms by which neural transplants can restore cognitive function following diffuse cholinergic damage. In contrast, after ischaemia- or neurotoxin-induced damage to CA1 or dentate granule cells respectively, cholinergically rich fetal transplants into the damaged hippocampal formation were ineffective in restoring performance. After ischaemic damage, however, performance was restored by suspension grafts of CA1 cells but not by transplants containing CA3 pyramidal cells or granule cells; and after colchicine damage it was restored by solid grafts containing granule but not CA1 pyramidal cells. Furthermore, electrophysiological evidence has demonstrated functional, graft type-specific host-graft functional neuronal connectivity. Thus, restoration of cognitive function by neural transplants is possible after damage to either diffuse (cholinergic) or point-to-point (intrahippocampal) forebrain systems, but the transplant must be appropriate to the damage to be repaired. Because the different types of brain damage studied provide analogues of human alcoholic dementia, Alzheimer's disease, and heart attack, these results are encouraging with regard to the eventual application of neural transplant surgery to the treatment of cognitive deficits in humans.

The influence of donor age on the survival of solid and suspension intraparenchymal human embryonic nigral grafts.

In many species, graft survival and graft-derived behavioral recovery are affected by the embryonic donor age. We compared the ability of solid and suspension grafts of human embryonic mesencephalic dopaminergic (DA) neurons at different embryonic stages to survive intraparenchymal transplantation into 6-OHDA lesioned immunosuppressed rats. Suspension grafts survived best when donor age was between postconception (PC) days 34 and 56. Transplants displayed numerous healthy tyrosine hydroxylase immunoreactive (TH-IR) neurons which sent extensive neuritic processes into the host striatum. Suspension grafts survived poorly when donor age was greater than 65 days. Solid implants displayed comparable viability of TH-IR neurons when donor age was between 44 and 65 days. No solid grafts contained TH-IR cells when donor tissue was older than 72 days. The suspension and solid methods of transplantation resulted in comparable survival of robust grafts, but solid grafts resulted in more intergraft variability than suspension grafts, particularly among the more marginal implants. Our results demonstrate that the upper limit for survival of human embryonic DA suspension grafts correlates well with the period of development of the human nigrostriatal pathway. The "window" for donor age of solid human embryonic DA grafts appears to be extended by about 9 days in comparison to suspension grafts. These data suggest that the upper age limit for grafting human mesencephalic DA neurons should be PC day 56 for suspension grafts, and PC day 65 for solid implants. Older donors are likely to produce grafts with fewer surviving DA neurons.

The influence of donor age on the survival of solid and suspension intraparenchymal human embryonic nigral grafts

In many species, graft survival and graft-derived behavioral recovery are affected by the embryonic donor age. We compared the ability of solid and suspension grafts of human embryonic mesencephalic dopaminergic (DA) neurons at different embryonic stages to survive intraparenchymal transplantation into 6-OHDA lesioned immunosuppressed rats. Suspension grafts survived best when donor age was between postconception (PC) days 34 and 56. Transplants displayed numerous healthy tyrosine hydroxylase immunoreactive (TH-IR) neurons which sent extensive neuritic processes into the host striatum. Suspension grafts survived poorly when donor age was greater than 65 days. Solid implants displayed comparable viability of TH-IR neurons when donor age was between 44 and 65 days. No solid grafts contained TH-IR cells when donor tissue was older than 72 days. The suspension and solid methods of transplantation resulted in comparable survival of robust grafts, but solid grafts resulted in more intergraft variability than suspension grafts, particularly among the more marginal implants. Our results demonstrate that the upper limit for survival of human embryonic DA suspension grafts correlates well with the period of development of the human nigrostriatal pathway. The “window” for donor age of solid human embryonic DA grafts appears to be extended by about 9 days in comparison to suspension grafts. These data suggest that the upper age limit for grafting human mesencephalic DA neurons should be PC day 56 for suspension grafts, and PC day 65 for solid implants. Older donors are likely to produce grafts with fewer surviving DA neurons.

Angiogenesis and the blood-brain barrier in intracerebral solid and cell suspension grafts

Solid and suspension grafts of fetal central nervous system (CNS) tissue rapidly reform an intact blood-brain barrier (BBB), whereas solid grafts of peripheral nervous system (PNS) tissue fail to establish a BBB as detected by horseradish peroxide (HRP) leakage, administrated intravenously. We examined the acute changes in the BBB after grafting of fetal CNS tissue in solid and suspension form and superior cervical ganglion (SCG) and PNS tissue in the same manner. Adult rats (n = 20) received fetal (day 14–15) forebrain grafts (either solid or cell suspension) to their rostral corpus callosum bilaterally. A second group (n = 20) received SCG solid and cell suspension grafts at the same coordinates with the same technique. The animals were killed on first, third, seventh, and tenth days after grafting. Intravenous HRP (Sigma, type VI, 75 mg/5-g rat) was given 1 hour before perfusion with mixed aldehydes. Fifty-micron coronal sections were examined for the presence and location of the graft by cresyl violet and AChE staining and Mesulam's TMB method to detect HRP leakage. HRP leakage was detected in the parenchyma in all groups on the first and the third days post-transplantation indicating a disrupted BBB. No HRP reaction was seen at days 7 and 10 in groups receiving fetal forebrain tissue whether solid or cell suspension. Solid grafts of SCG consistently demonstrated HRP leakage from the first through the tenth day. However, cell suspension of SCG established a BBB by 7 days. These results suggest that within the solid grafts of CNS and PNS tissue, the permeability of the vessels is dictated by the transplanted tissue itself. When cell suspensions of the same tissue are introduced, host CNS tissue dominates as the local environment resulting in non-leaky vasculature within the graft.

Vascularization and microvascular permeability in solid versus cell-suspension embryonic neural grafts.

Vascularization and microvascular permeability were assessed in a comparative study of solid (organized) and cell-suspension (dissociated) fetal nigral grafts implanted in the dopamine-deprived striatum of adult rats. Both graft types were analyzed by chromogen detection of intravenously injected horseradish peroxidase (HRP), which outlined vessel walls, and, in cases in which the blood-brain barrier was compromised, permeated the graft and host parenchyma. Survival of graft-derived dopaminergic cells was assessed using tyrosine hydroxylase (TH) immunocytochemistry. Glial reactivity to cell-suspension grafts was similarly assessed with an antibody directed against glial fibrillary acidic protein. Morphometry revealed significantly higher microvessel density in the cell-suspension grafts (p < 0.001), which effectively equaled that found in the contralateral striatum despite rather prominent surrounding glial reactivity. Capillaries in the cell-suspension grafts were not permeable to blood-borne HRP at postimplantation study times of 7, 14, and 30 days whereas, in the solid grafts, permeability in some cases could be detected for up to 30 days. Large numbers of cells immunoreactive for TH were seen in cell-suspension grafts; in contrast, few if any were found in the majority of solid transplants. The multiple-fragment solid graft implant model used clinically compares poorly with the cell-suspension model because it lacks consistency in early revascularization and shows a greater (albeit temporary) tendency for blood-brain barrier dysfunction. Delayed and inadequate vascularization of the solid graft is likely to account for graft failure more often than in the cell-suspension graft. Similarly, a certain critical number of specific grafted cells are required to achieve sufficient expression to bring about a favorable response in the disabled host, and this expression appears to be achieved less consistently with the solid implant technique.

Expression of cholinergic phenotype by embryonic ventral horn neurons transplanted into the spinal cord in the rat

Solid grafts of E12 embryonic spinal ventral horn were transplanted into motoneuron-depleted adult lumbar spinal cord in the rat. A muscle was implanted parallel to the vertebral column with its nerve inserted into the lumbar cord at the site of transplantation so as to provide a target for innervation by the grafted neurons. Previous retrograde labelling studies have shown that modest numbers of grafted motoneuron-like cells participate in the muscle's reinnervation and these are often found outside the graft within the host spinal cord. However, Nissl stained sections show that larger numbers of neurons survive within tissue recognisable as being of graft origin. In this study we have examined the expression of acetylcholinesterase (AChE) and choline acetyltransferase (ChAT) by neurons within the graft. These enzymes are involved in cholinergic neurotransmission and are characteristic of motoneurons. Thirty-four to seventy days following transplantation the grafts contained numerous neurons with acetylcholinesterase (AChE) activity. Different patterns of AChE staining were observed which probably reflected the degree of differentiation and maturation within the graft. AChE positive neurons were found in isolation or in groups resembling developing motor pools. Most of the AChE-positive neurons appeared immature with scant cytoplasm. However, neurons could be found which appeared relatively mature with a regularly shaped nucleus, prominent nucleolus and Nissl bodies. The grafts contained few AChE-positive axons and no dense plexuses of varicose fibres around the neurons such as are found around motoneurons in the mature ventral horn. Comparisons between the size of AChE-positive neurons in the graft and the size of AChE-positive neurons in the developing ventral horn found that the size of grafted neurons to be intermediate between the sizes of spinal motoneurons at E19 and P0. Far fewer grafted neurons were found to be immunoreactive for choline acetyltransferase (ChAT) than histochemically reactive for AChE. This was consistent with our findings in the spinal cord during normal development where we found that fixation and staining procedures which labelled adult motoneurons failed to reliably demonstrate ChAT immunoreactivety in normal motoneurons prenatally, although AChE histochemical reactivity could be demonstrated as early as E16. We conclude that the grafts contain numbers of immature motoneurons which fail to proceed beyond a certain stage of development, perhaps because of a failure to form appropriate efferent and afferent connections.

Observations on the Survival of Grafted Embryonic Motoneurons in the Spinal Cord of Developing Rats

The sciatic nerve of newborn rats was injured unilaterally. Small solid grafts of ED-12 embryonic spinal cord prelabeled with 5-bromo-2′-deoxyuridine (BrDUr) were inserted into the host's hemicord on the side of the sciatic nerve injury on PD5-12. Each graft was connected to a neuromuscular implant, which in group 1 consisted of the soleus muscle and its nerve taken from the healthy leg of the same rat pup, whereas in group 2 the soleus neuromuscular implant was taken from an immunocompatible adult rat. Six to 12 weeks later the neurons extending axons into the nerve-muscle implants were retrogradely labeled with fast blue and diamidino yellow. The embryonic origin of these neurons was ascertained by visualizing BrDUr using an immunocytochemical method. The grafts survived well and contained many BrDUr-positive neurons. No retrogradely labeled neurons were found in group 1 animals. In group 2 (animals with adult implants) numerous retrogradely labeled neurons were present in the host's neuropil. Despite the access to the target there were no BrDUr-positive cells of embryonic origin that contained the retrograde labels. Indeed no motoneuron-like cells of embryonic origin were seen in the host neuropil in any of the animals which received a graft at the age of 5-12 days. This contrasts with previous studies where the grafts were introduced into the adult spinal cord and motoneuron-like cells of embryonic origin were present in the host neuropil. Thus, it appears that the environment of the developing spinal cord is unfavorable for survival of embryonic motoneurons.