H Widner

Bilateral fetal mesencephalic grafting in two patients with parkinsonism induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)

BACKGROUNDnIntracerebral transplantation of fetal dopaminergic neurons is a promising new approach for the treatment of Parkinsons disease. Patients with parkinsonism induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) have a relatively stable lesion limited to the nigrostriatal system, rendering them ideal candidates for transplantation. Improvement of motor function after neural grafting has previously been observed in nonhuman primates with MPTP-induced parkinsonism.nnnMETHODSnWe grafted human fetal tissue from the ventral mesencephalon (obtained six to eight weeks after conception) bilaterally to the caudate and putamen in two immunosuppressed patients with severe MPTP-induced parkinsonism, using a stereotaxic technique. The patients were assessed regularly with clinical rating scales, timed tests of motor performance, and [18F]fluorodopa positron-emission tomography during the 18 months before the operation and the 22 to 24 months after the operation.nnnRESULTSnBoth patients had substantial, sustained improvement in motor function and became much more independent. Postoperatively, the second patients maintenance dose of levodopa was decreased to 150 mg daily, which was 30 percent of the original dose. Striatal uptake of fluorodopa was unchanged 5 to 6 months postoperatively but was markedly and bilaterally increased at 12 to 13 and 22 to 24 months in both patients, closely paralleling the patients clinical improvement. There were no serious complications.nnnCONCLUSIONSnBilateral implantation of fetal mesencephalic tissue can induce substantial long-term functional improvement in patients with parkinsonism and severe dopamine depletion and is accompanied by increased uptake of fluorodopa by the striatum. The results in these patients resemble those obtained in MPTP-treated primates and suggest that this will be a useful model for the assessment of transplantation therapies in Parkinsons disease.

Immunological aspects of grafting in the mammalian central nervous system. A review and speculative synthesis

Transplantation immunology ...................................................................................................................... 3.1. Basic concepts in immunology ............................................................................................................... 3.2, Lymphocyte activation and antigen presentation ........................................................................................ 3.3. Transplanta&ion immunology ................................................................................................................ 3.4. Mechanisms of rejection ...................................................................................................................... 3.5. Mechanisms of action of immunosuppressive drugs ..................................................................................... 3.6. Lymphocyte circulation .......................................................................................................................

Human fetal dopamine neurons grafted in a rat model of Parkinson's disease : immunological aspects, spontaneous and drug-induced behaviour, and dopamine release

SummaryWe have used a rat model of Parkinsons disease (PD) to address issues of importance for a future clinical application of dopamine (DA) neuron grafting in patients with PD. Human mesencephalic DA neurons, obtained from 6.5–8 week old fetuses, were found to survive intracerebral cell suspension xenografting to the striatum of rats immunosup-pressed with Cyclosporin A. The grafts produced an extensive new DA-containing terminal network in the previously denervated caudate-putamen, and they normalized amphetamine-induced, apomorphine-induced and spontaneous motor asymmetry in rats with unilateral lesions of the mesostriatal DA pathway. Grafts from an 11.5-week old donor exhibited a lower survival rate and smaller functional effects. As assessed with the intracerebral dialysis technique the grafted DA neurons were found to restore spontaneous DA release in the reinnervated host striatum to normal levels. The neurons responded with large increases in extracellular striatal DA levels after the intrastriatal administration of the DA-releasing agent d-amphetamine and the DA-reuptake blocker nomifensine, although not to the same extent as seen in striata with an intact mesostriatal DA system. DA fiber outgrowth from the grafts was dependent on the localization of the graft tissue. Thus, grafts located within the striatum gave rise to an extensive axonal network throughout the whole host striatum, whereas grafted DA neurons localized in the neocortex had their outgrowing fibers confined within the grafts themselves. In contrast to the good graft survival and behavioural effects obtained in immunosuppressed rats, there was no survival, or behavioural effects, of human DA neurons implanted in rats that did not receive immunosuppression. In addition, we found that all the graft recipients were immunized, having formed antibodies against antigens present on human T-cells. This supports the notion that the human neurons grafted to the non-immunosuppressed rats underwent immunological rejection. Based on an estimation of the survival rate and extent of fiber outgrowth from the grafted human fetal DA neurons, we suggest that DA neurons that can be obtained from one fetus may be sufficient to restore significant DA neurotransmission unilaterally, in one putamen, in an immunosuppressed PD patient.

Can sensory stimulation improve the functional outcome in stroke patients

After obtaining informed consent, we randomized 78 patients with severe hemiparesis of the left or right side within 10 days of stroke onset: 40 to a control group receiving daily physiotherapy and occupational therapy, and 38 to a group that, in addition, we treated with sensory stimulation (acupuncture) twice a week for 10 weeks. The median age was 76 years for both groups. Motor function, balance, and ADL (Barthels Index) were assessed before the start of treatment and at 1 and 3 months after stroke onset; ADL was also assessed after 12 months. We assessed the quality of life (QL) using the Nottingham Health Profile 3, 6, and 12 months after stroke onset. Patients given sensory stimulation recovered faster and to a larger extent than the controls, with a significant difference for balance, mobility, ADL, QL, and days spent at hospitals/nursing homes. Whether acupuncture per se is responsible for the differences requires further study.

Intracerebral xenografts of dopamine neurons: the role of immunosuppression and the blood-brain barrier

SummaryFetal mesencephalic mouse tissue, rich in dopamine neurons, was xenografted as a dissociated cell suspension into the striatum of rats with unilateral 6-hydroxydopamine induced lesions of the mesostriatal pathway. The rats were either assigned to a 10-day, 21-day or 42-day Cyclosporin A (CyA) immunosuppression scheme, or given no immunosuppression. The functional effects of the grafts were followed over 6 months by monitoring changes in the recipient rats amphetamine-induced turning behaviour. Without immunosuppression no grafts were functional at the end of the experiment. In the 10-, 21-and 42-day CyA treatment groups there was a significant reduction of rotational asymmetry at some timepoint following grafting in 26 of the 33 rats. However, by 6 months only 8 grafts remained functional suggesting that in several rats an immunological rejection took place following the termination of immunosuppression. This was supported by catecholamine histofluorescence analysis which revealed evidence of surviving grafts only in the few rats which had shown sustained functional graft effects at 6 months after grafting. In animals in which the grafts had undergone rejection, there was scarlike tissue in the striatum which appeared more extensive in rats that had lost their grafts after several weeks compared to rats in which the grafts were rejected at an early time-point. In a subgroup of the grafted animals the humoral antibody response against major transplantation antigens present on the grafted cells was investigated. All the studied rats were found to be immunized against the grafted mouse tissue following the intrastriatal implantation. This occurred irrespective of prior immunosuppressive treatment. In a parallel group of rats, the leakage of the blood-brain barrier was studied following intrastriatal implantation of a syngeneic fetal neural cell suspension. Evans Blue was infused into rats 3–12 days following transplantation surgery. At the early time-points there was a marked barrier leakage at the implantation site. This subsided with time such that there was minor leakage after 7–8 days and no leakage after 12 days. In summary, the results indicate the CyA is effective in promoting survival of intracerebral xenografts of fetal neural tissue, but that cessation of immunosuppressive treatment in most cases results in rejection of the grafted tissue. Temporary CyA treatment, even exceeding the time it takes for the blood-brain barrier to reform after transplantation surgery, is thus not sufficient to reliably support long term survival of xenografted dopamine neurons.

Temporal pattern of host responses against intrastriatal grafts of syngeneic, allogeneic or xenogeneic embryonic neuronal tissue in rats

The host response to immunologically incompatible intrastriatal neural grafts was studied using immunohistochemical techniques. Dissociated ventral mesencephalic tissue from embryonic donors of either syngeneic, allogeneic or xenogeneic (mouse) origin was stereotaxically implanted into adult rats. The brains were analysed 4 days, 2 weeks or 6 weeks after grafting with antibodies against the following antigenic structures: major histocompatibility complex (MHC) class I antigens; MHC class II antigens; complement receptor (CR) 3 (marker for microglia and macrophages); helper T-lymphocyte antigen-cluster of differentiation (CD) 4; cytotoxic T-lymphocyte antigen-CD8; tyrosine hydroxylase (TH) (marker for transplanted dopaminergic neurons). The number of surviving TH-positive cells was not different at the various time points in either the syngeneic or allogeneic groups, whereas the xenogeneic cells were all rejected by 6 weeks.The host reactions were similar in character in the syngeneic and allogeneic groups. At 4 days after implantation, there were increased levels of expression of MHC class I and II antigens. In and around the grafts, there were cellular infiltrates consisting of activated microglia, macrophages, CD4- and CD8-positive lymphocytes. At 6 weeks, MHC expression was reduced and the cellular infiltrates had subsided with only low numbers of activated microglia cells and CD8-positive lymphocytes remaining. In the xenogeneic group, at 4 days, some grafts contained cavities, possibly reflecting acute rejection. At later stages, the xenografts were heavily infiltrated by macrophages, activated microglial cells and T-lymphocytes, and at 6 weeks all the xenografts were rejected.Taken together, the results suggest that there is an inflammation caused by the implantation process which leads to an accumulation of host defence cells. This, in turn, leads to increased MHC expression in and around the grafts. In syngeneic grafts, these reactions are short lasting and weak; for allografts slightly more pronounced and longer lasting than syngeneic grafts, but not sufficient to cause rejection. For xenografts, the reactions are more intense and lead to transplant rejection. Thus, a strong sustained inflammatory response may be an important determinator for the failure of histoincompatible neural grafts. It can be speculated that a short-term anti-inflammatory treatment of graft recipients may be a sufficient immunosuppressive regimen to allow long-term graft survival.

Sequential intrastriatal grafting of allogeneic embryonic dopamine-rich neuronal tissue in adult rats: Will the second graft be rejected?

An important issue in clinical neural grafting is whether a second instriatial allograft can survive well in a patient who has received an allograft before. In this study, the survival, immunogenicity and function of intrastriatal grafts of allogeneic or syngeneic embryonic dopamine-rich tissue in rats which had previously received either an intrastriatal allo- or syn-graft or sham injections were examined. The first graft tissue was taken from inbred Lewis or Sprague-Dawley rat embryos and grafted into an intact striatum of adult Sprague-Dawley rats subjected to a unilateral 6-hydroxydopamine lesion on the contralateral side. Eight weeks after the first transplantation, either allogeneic or syngeneic tissue was grafted as dissociated tissue into the dopamine depleted striatum. The function of the second grafts was assessed by rotational asymmetry at two different time points, i.e. eight and 14 weeks after the second transplantation. There were significant reductions of rotational asymmetry in all groups over time, but no significant difference between groups. Tyrosine hydroxylase immunocytochemistry was used to assess dopamine cell survival and graft size. Statistical analysis revealed no significant differnce in the mean number of tyrosine hydroxylase immunoreactive cells or the mean volume of the second grafts placed on the right side (lesioned side) between groups. Monoclonal antibodies were used to evaluate cellular immune reactions and the major histocompatibility complex class I and class II expression in and around grafts. No major histocompatibility complex class I expression was seen in any of the graft combinations. The expression of the major histocompatibility complex class II antigens was generally higher in patches in and around the second allograft of rats which had previously received an allograft than that in and around any other type of grafts. However, the expression of the major histocompatibility complex class II antigens was low throughout the grafts and did not appear as marked perivascular infiltrates. All the major histocompatibility complex class II positive cells displayed a microglia-like morphology, supported by the parallel microglia and macrophage-specific OX-42 immunostaining. The results show that there is no marked on-going immune reactions in or around the implantation site in any group fourteen weeks after a second transplantation. It may be concluded, therefore, that sequential allografting, using stereotaxic implantation of dissociated embryonic neural tissue into the striatal parenchyma, is possible to perform without a major risk of graft rejection, provided that an atraumatic technique is used.

Immune response in deep cervical lymph nodes and spleen in the mouse after antigen deposition in different intracerebral sites

Brain interstitial and cerebrospinal fluid drainage into the lymphatics was studied by injections of 5μl of packed sheep red blood cells (SRBC) injected into the caudate nucleus, the occipital lobe, and the lateral ventricle of the brain in mice. The number of plaque‐forming cells (PFC) was determined in the deep cervical lymph nodes, the axillar lymph nodes, and the spleen, and the number of PFC was compared with the response in the same tissues after Intravenous immunization with 0.1 ml 10% SRBC. The weight of the deep cervical lymph nodes increased 3.0 times on day 3 after injection in the brain parenchyma compared with the weight of these nodes after intravenous immunization. The antigen‐specific response peaked on day 5, 392±37 PFC/106 for IgG in the deep cervical lymph nodes after antigen deposition in the caudate nucleus, whereas only a minor peak in the antigen‐specific response was obtained after intraventricular antigen deposition, 127±79 PFC×106 for IgG on day 6. There were no increased PFC in any of the lymph nodes after intravenous immunization. The experiments show an antigen‐specific response in the deep cervical lymph nodes after intracerebral antigen deposition, whereas antigens deposited in the lateral ventricles drain preferentially to the blood, with a high response in the spleen.

Sequential intracerebral transplantation of allogeneic and syngeneic fetal dopamine-rich neuronal tissue in adult rats : will the first graft be rejected?

The immune response against intracerebral grafts of allogeneic fetal dopamine-rich tissue was assessed in adult rats. Sprague-Dawley rats, now outbred, but originating from an inbred stock, were given unilateral 6-hydroxy-dopamine lesions of the mesostriatal pathway, and grafted intrastriatally with mechanically dissociated ventral mesencephalic tissue (embryonic day 13-15) obtained from an inbred Lewis strain. Graft survival was assessed by functional recovery of amphetamine-induced rotational behavior on four different occasions postsurgery, and histologically using catecholamine histofluorescence and tyrosine hydroxylase immunohistochemistry. The following groups were analysed: long-term survival of a single allogeneic graft; survival of a first allogeneic graft with a syngeneic second graft; survival of a first allograft combined with a second allogeneic graft; the survival of bilateral allogeneic grafts following a subsequent orthotopic allogeneic skin graft. Evidence for recipient immunization was obtained using an indirect fluorescent antibody detection technique, Simonsens Spleen Index (S I) test. Viable grafts, giving rise to behavioral compensation, were present after 40 wk in rats from all groups. The “first” allograft always displayed good survival and function, even following a second intracerebral allograft. However, five of nine “second” allogeneic intracerebral grafts survived poorly. In contrast, all secondary syngeneic grafts survived well. Following the application of a subsequent orthotopic allogeneic skin graft in a subgroup of rats, there was a significantly lower survival of grafted dopamine neurons in the “first” graft.

Survival and immunogenicity of dissociated allogeneic fetal neural dopamine-rich grafts when implanted into the brains of adult mice

SummaryThe survival of grafts of dissociated allogeneic fetal neural dopamine (DA) rich tissue in the striatum has been studied after transplantation between inbred strains of mice differing at defined immunogenetical loci between donor and recipient. Six to 7 weeks and 15 weeks after grafting, surviving grafted DA neurons were found in the brains of all the recipients, albeit with a large variation in numbers, located either within the striatum or within the adjacent lateral ventricle. The mean number of surviving DA neurons did not differ between the syngeneic controls and the histoincompatible donor-host combinations, and there was no difference in survival between grafts that differed at single or multiple major histocompatibility complex (MHC) loci, and those that differed at multiple non-MHC loci. The amount of inflammatory cells in the graft area did not differ between the groups, and none of the animals showed massive infiltration of inflammatory cells. The in situ immunogenicity of the grafted neural tissue after intracerebral implantation was monitored by means of Simonsens alloimmunization test, at 6–7 weeks after transplantation, which provides a sensitive measure primarily of the cellular immunological response. Most, but not all, graft recipients showed immunization with a Spleen Index (S.I.) close to that seen in recipients of an orthotopical skin graft of the same histoincompatibility combination. In contrast to the prolonged survival of the intracerebral neural transplants, none of the skin grafts survived longer than 3 weeks, thus demonstrating the immunologically privileged status of the brain. We conclude that intracerebrally grafted allogeneic neural tissue is capable of provoking a cellular immune response. Despite host immunization, however, the dissociated fetal neural allografts survived for at least 15 weeks without any overt signs of rejection, regardless of the donor-host combination used.