Targeted cell reprogramming produces analgesic chromaffin-like cells from human mesenchymal stem cells.

Transplantation of allogeneic adrenal chromaffin cells demonstrated the promise of favorable outcomes for pain relief in patients. However, there is a very limited availability of suitable human adrenal gland tissues, genetically well-matched donors in particular, to serve as grafts. Xenogeneic materials, such as porcine and bovine adrenal chromaffin cells, present problems; for instance, immune rejection and possible pathogenic contamination are potential issues. To overcome these challenges, we have tested the novel approach of cell reprogramming to reprogram human bone marrow (BM)-derived mesenchymal stem cells (hMSCs) using cellular extracts of porcine chromaffin cells. We produced a new type of cell, chromaffin-like cells, generated from the reprogrammed hMSCs, which displayed a significant increase in expression of human preproenkephalin (hPPE), a precursor for enkephalin opioid peptides, compared to the inherent expression of hPPE in naive hMSCs. The resultant chromaffin-like cells not only expressed the key molecular markers of adrenal chromaffin cells, such as tyrosine hydroxylase (TH) and methionine enkephalin (Met-enkephalin), but also secreted opioid peptide Met-enkephalin in culture. In addition, intrathecal injection of chromaffin-like cells in rats produced significant analgesic effects without using immunosuppressants. These results suggest that analgesic chromaffin-like cells can be produced from an individual's own tissue-derived stem cells by targeted cell reprogramming and also that these chromaffin-like cells may serve as potential autografts for chronic pain management.

Knockdown of RhoGDIα induces apoptosis and increases lung cancer cell chemosensitivity to paclitaxel.

This study aimed to investigate the effects of RhoGDIα knockdown on apoptosis and the chemosensitivity of lung cancer cells to paclitaxel. The signaling proteins involved were also assessed. RhoGDIα expression was assessed by RT-PCR, Western blotting and immunohistochemistry. Apoptosis was determined by flow cytometric assessment, and cell viability was measured with the MTT assay. Phosphorylation levels of signaling proteins, ERK, JNK, Akt, Bad and IκBα were tested by Western blotting and immunohistochemistry. Positivity for RhoGDIα in lung cancer tissues was significantly higher than in paracancerous tissues. Downregulation of RhoGDIα was associated with significantly increased apoptosis and repressed cell viability. This effect could be due to the consequent upregulation of p-JNK, as well as decreased levels of p-ERK, p-Bad and p-IκBα. Knockdown of RhoGDIα strengthened the effect on apoptosis and inhibition of cell viability induced by paclitaxel treatment. This chemosensitization effect could be a result of the intensification of pro-apoptotic JNK activation, and repression of anti-apoptotic p-ERK, p-Bad and p-IκBα expression stimulated by paclitaxel. In summary, our study indicated that RhoGDIα could be a promising therapeutic target, and the combination of RhoGDIα siRNA and paclitaxel might be a valuable potential therapy for lung cancer treatment.

AAV9-mediated erythropoietin gene delivery into the brain protects nigral dopaminergic neurons in a rat model of Parkinson's disease.

We have recently shown that intrastriatal injection of recombinant human erythropoietin (EPO) protects dopaminergic (DA) neurons in the substantia nigra (SN) from 6-hydroxydopamine (6-OHDA) toxicity in a rat model of Parkinson's disease. However, systemic administration of EPO did not protect nigral DA neurons, suggesting that the blood-brain barrier limits the passage of EPO protein into the brain. In the present study, we used an adeno-associated viral (AAV) serotype 9 (AAV9) vector to deliver the human EPO gene into the brain of 6-OHDA-lesioned rats. We observed that expression of the human EPO gene was robust and stable in the striatum and the SN for up to 10 weeks. EPO-immunoreactive (IR) cells were widespread throughout the injected striatum, and EPO-IR neurons and fibers were also found in the ipsilateral SN. Enzyme-linked immunosorbent assay and western blot analyses exhibited dramatic levels of EPO protein in the injected striatum. As a result, nigral DA neurons were protected against 6-OHDA-induced toxicity. Amphetamine-induced rotational asymmetry and spontaneous forelimb use asymmetry were both attenuated. Interestingly, we also observed that intrastriatal injection of AAV9-EPO vectors led to increased numbers of red blood cells in peripheral blood. This highlights the importance of using an inducible gene delivery system for EPO gene delivery.

Intrastriatal administration of erythropoietin protects dopaminergic neurons and improves neurobehavioral outcome in a rat model of Parkinson's disease.

Erythropoietin (EPO), a hematopoietic cytokine, has recently been demonstrated to protect nigral dopaminergic neurons in a mouse model of Parkinson's disease (PD). In the present study, we tested the hypothesis that recombinant human erythropoietin (rhEPO) could protect dopaminergic neurons and improve neurobehavioral outcome in a rat model of PD. rhEPO (20 units in 2 microl of vehicle) was stereotaxically injected into one side of the striatum. 6-hydroxydopamine (6-OHDA) was injected into the same side 1 day later. Another group of rats received rhEPO (5000 u/kg, i.p.) daily for 8 days, and unilateral injection of 6-OHDA in the striatum 3 days after systemic administration of rhEPO. We observed that intrastriatal administration, but not systemic administration of rhEPO significantly reduced the degree of rotational asymmetry. The rhEPO-treated rats also showed an improvement in skilled forelimb use when compared with control rats. The number of tyrosine hydroxylase (TH)-immunoreactive (IR) neurons in the ipsilateral substantia nigra (SN) was significantly larger in intrastriatal rhEPO-treated rats than that in control rats. TH-IR fibers in the 6-OHDA-lesioned striatum were also increased in the intrastriatal rhEPO-treated rats when compared with control rats. In addition, there were lower levels of expression of major histocompatibility complex (MHC) class II antigens and a smaller number of activated microglia in the ipsilateral SN in intrastriatal rhEPO-treated rats than that in control rats at 2 weeks, suggesting that intrastriatal injection of rhEPO attenuated 6-OHDA-induced inflammation in the ipsilateral SN. Our results suggest that intrastriatal administration of rhEPO can protect nigral dopaminergic neurons from cell death induced by 6-OHDA and improve neurobehavioral outcome in a rat model of PD. Anti-inflammation may be one of mechanisms responsible for rhEPO neuroprotection.

Rat nigral xenografts survive in the brain of MHC class II-, but not class I-deficient mice.

We have examined the role of the indirect pathway of antigen recognition and T cells in neural xenografts rejection by using major histocompatibility complex (MHC) class II-deficient mice as xenograft recipients. Dissociated embryonic ventral mesencephalic tissue from Sprague-Dawley rats was stereotaxically injected as a cell suspension into the striatum of MHC class II-deficient adult mice as well as MHC class I-deficient and wild-type mice as controls. All of the MHC class II-deficient mice had surviving grafts in the striatum 4 weeks post-grafting. In contrast, only a few of the MHC class I-deficient mice exhibited very small grafts and none of the wild-type mice had any surviving grafts. The mean number of surviving transplanted dopamine neurons in the MHC class II-deficient group was significantly larger than that observed in the other two groups. Moderate levels of MHC class I antigen expression were seen in the transplantation sites of some animals in the MHC class II-deficient group. No helper or cytotoxic T cells were observed infiltrating into the graft sites of this group. However, there were markedly increased levels of expression of MHC class I and class II antigens, and a number of T cells infiltrating in the graft sites in both the MHC class I-deficient and wild-type groups. These results show that rat embryonic nigral tissue can survive transplantation in the brain of the MHC class II-deficient mice for at least 4 weeks without any overt signs of rejection, suggesting that the indirect pathway of foreign antigen recognition mediated by host MHC class II molecules and helper T cells plays an important role in the rejection responses to intracerebral xenografts.

Survival of intrastriatal xenografts of ventral mesencephalic dopamine neurons from MHC-deficient mice to adult rats.

Previous studies of neural xenografts have used immunosuppressive agents to prevent graft rejection. In the present study we have examined the survival of mouse dopamine neurons lacking either MHC class I or MHC class II molecules transplanted into rat brains and the host immune and inflammatory responses against the xenografts. Survival of neural grafts was immunocytochemically determined at 4 days, 2 weeks, and 6 weeks after transplantation by counting tyrosine hydroxylase (TH)-positive cells in the graft areas. In addition, the host immune and inflammatory responses against neural xenografts were evaluated by semiquantitatively rating MHC class I and class II antigen expression, accumulation of macrophages and activated microglia, and infiltration of CD4- and CD8-positive T-lymphocytes. For the negative controls, the mean number of TH-positive cells in rats that received wild-type mouse tissue progressively decreased at various time periods following transplantation. In contrast, intrastriatal grafting of either MHC class I or MHC class II antigen-depleted neural xenografts resulted in a prolonged survival and were comparable to cyclosporin A-treated rats that had received wild-type mouse tissue. These results indicate that genetically modified donor tissue lacking MHC molecules can be used to prevent neural xenograft rejection.

Enhancement of nigral graft survival in rat brain with the systemic administration of synthetic fibronectin peptide V.

A major obstacle in neural transplantation is a severe loss of neurons in grafts soon after implantation. In the present study, we have investigated whether the systemic administration of synthetic fibronectin peptide V can increase the survival of neural grafts. Synthetic fibronectin peptide V is derived from the 33,000 mol. wt carboxyl-terminal heparin-binding domain of fibronectin. Previous studies have shown that these polypeptides possess anti-inflammatory properties. However, it is currently unknown whether this peptide has anti-apoptotic properties. Dissociated neural grafts were prepared from the ventral mesencephalon of pregnant Sprague-Dawley rats and were stereotaxically injected as a cell suspension into the striatum of adult Sprague-Dawley rats. A group of recipient rats received i.v. injections of peptide V (5mg/kg, dissolved in saline) at 24 and 4h prior to transplantation, at the time of transplantation, and 24, 48 and 72h post-transplantation. Saline-treated rats served as controls. The rats were killed at two, four and 42 days post-grafting and the brain tissue was immunologically processed for tyrosine-hydroxylase, major histocompatibility complex class I and class II antigens, complement receptor type 3 and leukocyte common antigen immunocytochemistry, and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay. We found a significant increase (approximately twofold) in the number of dopamine neurons in the grafts for the peptide-treated group at four and 42 days compared with the controls. In contrast, there was no significant difference in the patterns of inflammation using different immunocytochemical markers in the two different groups. The levels of expression for these markers, however, were reduced over time. Interestingly, the number of apoptotic cells in the graft areas was significantly smaller in the peptide-treated group than in the control group two days after grafting. The results demonstrate that the systemic administration of synthetic fibronectin peptide V can dramatically increase the survival of nigral grafts in the brain and substantially reduce the number of apoptotic cells in the graft site, suggesting that this peptide may exert a beneficial effect on survival of nigral grafts through an anti-apoptotic mechanism.

Discordant xenografts: different outcome after mouse and rat neural tissue transplantation to guinea-pigs.

Embryonic neural tissue obtained from other species has been considered as a donor tissue source in repair strategies for human neurodegenerative disorders. The neuro- and immunobiology of distantly related species combinations, discordant xenografts, need to be characterised. For this purpose, a small animal model would be an important research tool. Adult guinea-pigs, and adult rats as controls, received intrastriatal grafts of either mouse or rat embryonic ventral mesencephalic tissue. The survival rates and types of host immune response were assessed at 2 weeks after grafting using stereological techniques and semi-quantitative evaluations. In the mouse-to-guinea-pig group, all transplants were rejected and no tyrosine hydroxylase-immuno reactive (TH-IR) cells remained. In the rat-to-guinea-pig group, there was good survival of TH-IR cells (5050 SEM+/-1550), similar to that in the rat-to-rat group (4900 SEM+/-1540). In the mouse-to-rat group, half of the animals had no surviving TH-IR cells (520 SEM+/-230 for the whole group). These species combinations offer inexpensive, efficient, and suitable conditions to study important survival factors for discordant xenogeneic neural tissue transplants. The factors responsible for the divergent graft outcomes between the two combinations might provide clues on how to manipulate xenogeneic tissue to increase survival rates in the future.

Quinolinic acid-induced inflammation in the striatum does not impair the survival of neural allografts in the rat.

It has been suggested that inflammation related to intracerebral transplantation surgery can affect the survival of intrastriatal neural allografts. To test this hypothesis, we transplanted dissociated embryonic mesencephalic tissue from one of two rat strains, Lewis (allogeneic grafts) or Sprague-Dawley (syngeneic grafts), to the striatum of Sprague-Dawley rats. The target striatum was either intact or had received a local injection of quinolinic acid 9 days earlier, in order to induce a marked inflammation. At 6 or 12 weeks after transplantation, there was no significant difference between the different groups regarding the number of surviving grafted tyrosine hydroxylase immunoreactive neurons. However, the graft volume of both the syngeneic and allogeneic implants was significantly larger in the quinolinate-lesioned than in the intact striatum. There were dramatically increased levels of expression of major histocompatibility complex class I and II antigens, marked infiltrates of macrophages, activated microglia and astrocytes, and accumulation of large numbers of CD4 and CD8 positive T-lymphocytes in the quinolinate-lesioned striatum. In contrast, these immunological markers were much less abundant around both syngeneic and allogeneic grafts placed in intact striatum. We conclude that severe inflammation caused by quinolinic acid does not lead to rejection of intrastriatal neural allografts.

Rat intrastriatal neural allografts challenged with skin allografts at different time points.

The present study was designed to address two questions. First, can an intrastriatal neural allograft exhibit long-term survival (18 weeks) if the host is immunized by an orthotopic skin graft 6 weeks after neural transplantation (the 6w-Long group)? Second, can an intrastriatal neural allograft survive when the host is challenged by an orthotopic skin allograft either simultaneously (Sim) with the intracerebral graft surgery or 2 (2w) weeks later? Dissociated embryonic ventral mesencephalic tissue from Lewis rats was stereotaxically injected into the striatum of Sprague-Dawley rats with unilateral 6-hydroxydopamine lesions. Six weeks after neural grafting, no reduction in amphetamine-induced motor asymmetry was observed in the Sim and 2w groups. At 6 weeks after skin grafting, the mean motor asymmetry scores had returned to the initial pretransplantation levels in the 6w-Long group. All the neural allografts in the Sim group were completely rejected, and the mean number of tyrosine hydroxylase immunoreactivity neurons in the grafts was significantly reduced in the 2w and the 6w-Long group, when compared to the no-skin control group. There were very high levels of expression of MHC class I and II antigens, marked cellular infiltrates containing macrophages and T-lymphocytes, and several activated microglia and astrocytes in and around the surviving intracerebral transplants in the 2w and the 6w-Long groups. The results suggest that intrastriatal neural allografts are more likely to be rejected rapidly if the host is efficiently immunized with the same alloantigens simultaneously or soon after the neural transplantation than at a later time point. When established neural allografts are subjected to a strong immunological challenge, they undergo protracted rejection.

Addition of allogeneic spleen cells causes rejection of intrastriatal embryonic mesencephalic allografts in the rat.

To address the importance of antigen-presenting cells for the survival of intracerebral neural allografts, allogeneic spleen cells were added to the graft tissue before transplantation. Dissociated embryonic, dopamine-rich mesencephalic and adult spleen tissues were prepared from either inbred Lewis or Sprague-Dawley rats. A mixture of neural and spleen cells was sterotaxically transplanted into the right striatum of adult Sprague-Dawley rats. Controls were neural allografts without addition of allogeneic spleen cells and syngeneic neural grafts with or without the addition of syngeneic spleen cells. Six weeks after transplantation, brain sections were processed immunocytochemically for tyrosine hydroxylase, specific for grafted dopamine neurons, and a bank of markers for various components in the immune and inflammatory responses. The neural allografts which were mixed with allogeneic spleen cells were rejected. In these rats, there were high levels of expression of major histocompatibility complex class I and II antigens, intense cellular infiltration including macrophages and activated microglial cells, and a presence of cluster of differentiation 4- and 8-immunoreactive cells in the graft sites. Moreover, there were increased levels of intercellular adhesion molecule-1, tumour necrosis factor-alpha and interleukin-6 in and around the grafts which were undergoing rejection. In contrast, syngeneic neural grafts survived well regardless of whether they were mixed with syngeneic spleen cells or not, and control neural allografts also exhibited unimpaired survival. No significant difference was observed in the number of grafted dopamine neurons among these three latter groups. The levels of expression of the different markers for inflammation and rejection were generally lower in these grafts than in implants of combined allogeneic neural and spleen cells. In summary, intrastriatal neural allografts, which normally survive well in our animal model, were rejected if allogeneic spleen cells from the same donor were added to the graft tissue. The added spleen cells caused strong host immune and inflammatory responses. The study gave support to the notion that immunological privilege of the brain does not provide absolute protection to immunogenetically histoincompatible neural grafts.

Methylprednisolone prevents rejection of intrastriatal grafts of xenogeneic embryonic neural tissue in adult rats.

We studied the effects of high-dose methylprednisolone on the survival of intrastriatal neural xenografts and the host responses against them. Dissociated mesencephalic tissue from inbred mouse (CBA-strain) embryos was transplanted to the intact striatum of adult Sprague-Dawley rats. The rats received either daily injections of methylprednisolone (30 mg/kg), or cyclosporin A (10 mg/kg), or no immunosuppressive treatment. Two or six weeks after transplantation, there was good survival of xenografts in both the methylprednisolone- and cyclosporin A-treated rats. In contrast, the xenografts in untreated control rats were all rejected by six weeks. There was no marked difference in the degree of expression of MHC class I and II antigens and the accumulation of activated astrocytes and microglial cells/macrophages between the three groups. However, both methylprednisolone and cyclosporin A reduced infiltration of T lymphocytes to the transplantation sites. The expression of pro-inflammatory cytokines (interferon-gamma, tumour necrosis factor-alpha, interleukin-6) in and around the grafts was lower in the methylprednisolone- and cyclosporin A-treated groups than in untreated control rats. Although high-dose methylprednisolone caused significant body weight loss, we conclude that this treatment can prevent rejection of intrastriatal grafts of xenogeneic embryonic neural tissue in the adult.

Immune reactions following systemic immunization prior or subsequent to intrastriatal transplantation of allogeneic mesencephalic tissue in adult rats.

We have previously found that dissociated mesencephalic tissue, which differs from the host at both major histocompatibility complex and non-major histocompatibility complex gene loci, can survive stereotaxic transplantation to the striatum of adult rats. We have now studied the outcome of intrastriatal neural allografts in rats that were systemically immunized by an orthotopic skin allograft either prior or subsequent to intracerebral implantation surgery. Dissociated mesencephalic tissue from Lewis rat embryos was stereotaxically injected into the dopamine-depleted striatum of hemi-parkinsonian Sprague-Dawley rats. One group was immunized by an orthotopic allogeneic skin graft of the same genetic origin as the neural graft, six weeks before the neural transplantation (the pre-immunized group). Another group was post-immunized by an orthotopic skin allograft, six weeks after the neural transplantation (the post-immunized group). A control group of rats was not challenged by a skin allograft. Marked behavioural recovery was observed in six of seven rats in the control group, in six of eight rats in the post-immunized group, and in none of the pre-immunized rats. Tyrosine hydroxylase-immunopositive cells were found in rats from the two behaviourally compensated groups, but not in the pre-immunized group. The immune responses were evaluated by OX-18 (monoclonal antibody against major histocompatibility complex class I antigen), OX-6 (major histocompatibility complex class II antigen), OX-42 (microglia and macrophages), glial fibrillary acidic protein (astrocytes), OX-8 (cytotoxic T-lymphocytes) and W3/25 (helper T-lymphocytes) immunocytochemistry. All the neural allografts in the pre-immunized group were rejected, leaving scars only. There were more intense immune responses to the allografts in the post-immunized group than the control group, in terms of immunocytochemically higher expression of major histocompatibility complex class I and II antigens and more intense cellular reactions consisting of macrophages, activated microglia and astrocytes, in addition to CD8- and CD4-positive lymphocytes. In summary, the results show the following: (i) systemic pre-immunization leads to complete rejection of intrastriatal neural allografts, implying that the status of the host immune system before transplantation determines the outcome for intrastriatal neural allografts; (ii) established intrastriatal neural allografts can survive for at least six weeks after systemic immunization, in spite of increased host immune responses in and around the allografts; (iii) there are no marked immune reactions against intrastriatal neural allografts 13 weeks after implantation in rats which have not been systemically immunized by a skin allograft; (iv) pre-immunized rats may provide a very useful animal model to investigate the role of inflammatory lymphokines in immune rejection and to test alternative immunosuppressive drugs.

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.

Lazaroids improve the survival of grafted rat embryonic dopamine neurons.

In rodent models of Parkinson disease in which transplants of dissociated rodent and human embryonic mesencephalic tissue, rich in dopamine neurons, have been studied, only 5-20% of the dopamine neurons survive the implantation procedure. We have investigated the effects of inhibiting free radical generation with two lazaroids, U-74389G and U-83836E, on the survival of embryonic rat dopamine neurons. U-74389G is a 21-aminosteroid, and U-83836E combines the piperazinyl pyrimidine portion of 21-aminosteroids with the antioxidant ring of alpha-tocopherol. In an initial study, we found that the lazaroids markedly prolonged the period after tissue dissociation that an embryonic mesencephalic cell suspension exhibits high cell viability in vitro, as assessed by using a dye exclusion method. In a second series of experiments, addition of lazaroids to dissociated mesencephalic graft tissue increased the yield of surviving rat dopamine neurons 2.6-fold after implantation in the dopamine-denervated rat striatum. The improved survival correlated with an earlier onset of graft-induced functional effects in the amphetamine-induced rotation test. Thus, inhibition of free radical generation can significantly increase the yield of grafted embryonic dopamine neurons. Addition of lazaroids to the graft preparation is a relatively simple modification of the transplantation protocol and could readily be applied in a clinical setting. Moreover, since iron-dependent lipid peroxidation has been suggested to play a role in the death of nigral dopamine neurons in Parkinson disease and lazaroids are particularly potent inhibitors of such processes, the findings may have implications for the pathogenesis of this disease.

Platelet-derived growth factor receptor expression after neural grafting in a rat model of Parkinson's disease.

Platelet-derived growth factor (PDGF) has trophic effect on dopaminergic neurons in vitro. We have previously shown dynamic changes in the expression of PDGF in embryonic mesencephalic grafts and surrounding host striatal tissue following intracerebral transplantation in a rat model of Parkinson's disease. In this study the expression of the PDGF receptors was examined in the same model using immunohistochemistry. Most ventral mesencephalic (VM) cells from E13-E15 rat embryos possessed both PDGF alpha- and beta-receptors before implantation. Double immunofluorescence staining revealed that about 10% of the cells also expressed tyrosine hydroxylase (TH). The PDGF alpha-receptor was detectable in the graft up to 1 wk after transplantation but had disappeared at 3 wk. In the host tissue, scattered glial cells were positive for the alpha-receptor but the expression was unchanged following transplantation. The beta-receptor expression almost completely disappeared from the grafted tissue by 4 h following transplantation, and only a few cells of the host striatum showed immunoreactivity. However, after 3 wk beta-receptor positive cells were again detectable in the graft. These cells appeared to be endothelial cells as identified by an antibody against von Willebrand's factor. Our data suggest that PDGF might act locally on embryonic dopaminergic cells in an autocrine or juxtacrine manner before and shortly after transplantation, and on surrounding glial cells in a paracrine manner after transplantation. Furthermore, PDGF-BB might influence neovascularization in the graft.

Human embryonic dopamine neurons xenografted to the rat: effects of cryopreservation and varying regional source of donor cells on transplant survival, morphology and function.

When grafting human mesencephalic tissue to patients suffering from Parkinson's disease, the number of surviving dopamine (DA) neurons in the graft is probably crucial. It may be possible to increase the number of DA neurons available for grafting to a patient by pooling tissue from many human embryos collected over several days or by obtaining more DA neurons from each embryo. We have addressed these issues by cryopreserving human mesencephalic DA neurons prior to transplantation and also by grafting human embryonic diencephalic DA neurons. The effects of cryopreservation were assessed 4-15 weeks after xenografting ventral mesencephalic tissue into the DA-depleted striatum of immunosuppressed rats with unilateral 6-hydroxydopamine lesions of the mesostriatal pathway. Control rats grafted with fresh mesencephalic tissue displayed robust reductions in amphetamine-induced turning following transplantation. Functional effects of the cryopreserved mesencephalic grafts were only observed in the one rat out of nine which contained the largest graft in this group. The number of tyrosine hydroxylase immunoreactive neurons in animals transplanted with cryopreserved tissue was significantly reduced to 9% of fresh tissue control grafts. Morphological analysis showed that cryopreserved DA neurons were approximately 22% and 28% smaller regarding the length of the long and short axis, respectively, when compared to the neurons found in fresh grafts. In the second part of the study, the survival and function of human embryonic diencephalic DA neurons were examined following xenografting into the DA-depleted rat striatum. A reduction of motor asymmetry was observed in two out of seven diencephalon-grafted rats. This finding was consistent with a good graft survival in these particular rats, which both contained large grafts rich in tyrosine hydroxylase immunoreactive neurons. Moreover, there was immunopositive staining for graft-derived fibers in the rat striatum containing tyrosine hydroxylase and human neurofilament, both in rats grafted with mesencephalic and diencephalic DA neurons. These findings suggest that cryopreservation, using the current technique, is not a suitable storage method for use in clinical trials of DA neuron grafting in Parkinson's disease. On the other hand, the application of alternative sources of DA neurons may in the future develop into a strategy which can increase the number of neurons obtainable from each human embryo.

Transmitter release from transplants of fetal ventral mesencephalon or locus coeruleus in the rat frontal cortex and nucleus accumbens: effects of pharmacological and behaviorally activating stimuli.

The present study was performed in order to establish whether dopamine (DA) release from behaviorally functional intracerebral DA transplants is dependent on changes in neuronal impulse flow, and is under control of the host brain. Rats were subjected to combined intraventricular and ventral tegmental injections of 6-hydroxydopamine (6-OHDA) in order to obtain a severe bilateral lesion of the ascending mesocorticolimbic DA projections. Cell suspension grafts of fetal ventral mesencephalic neurons were thereafter implanted into the medial frontal cortex (MFC) and the nucleus accumbens (NAc). Since the neurotoxin injections removed also the ascending noradrenergic systems, fetal locus coeruleus neurons were added to the graft suspension in one group of animals. Age-matched lesion-only and normal animals served as controls. The lesion-induced alterations in spontaneous, amphetamine- and apomorphine-induced locomotor activity and in a skilled paw reaching task were evaluated before transplantation, and at 3 and 6 months post-grafting. Microdialysis probes were finally implanted in the MFC and NAc in order to monitor extracellular DA and noradrenaline (NA) levels (i) during administration of pharmacological agents which augment or depress catecholamine release in the intact brain; (ii) during exposure of the rats to stressful manipulations (handling and immobilization) or appetitive stimuli (eating) known to enhance cortical and limbic DA or NA release in intact animals. The lesion-induced reduction in amphetamine-induced locomotor activity was reversed in all grafted animals, which also showed a higher than normal spontaneous overnight activity. Daytime spontaneous locomotor activity (which was reduced in the lesion-only rats) as well as apomorphine-induced hyperactivity was reversed by the grafts of DA neurons only. By contrast, the lesion-induced impairment in skilled forelimb use was not alleviated by the grafts. The grafted DA neurons restored normal steady-state DA overflow in the NAc, whereas they enhanced cortical DA overflow to significantly higher than normal levels. Restoration of both cortical and striatal NA overflow was observed in the group that received mixed DA and NA grafts, whereas animals that received DA grafts only did not differ from the lesioned controls. The changes in extracellular DA and NA levels measured in the grafted MFC and NAc under potassium depolarization (100 mM KCl), inhibition of terminal catecholamine reuptake (10 microM nomifensine), and sodium channel blockade (1 microM TTX) indicated that graft-derived DA or NA release had normal neuronal properties, and was dependent on an intact axonal impulse flow.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

Restoration of complex sensorimotor behavior and skilled forelimb use by a modified nigral cell suspension transplantation approach in the rat Parkinson model.

While intrastriatal transplants of dopamine-rich ventral mesencephalic tissue are effective in reversing a variety of drug-induced behaviors in the rat Parkinson model, previous studies have failed to obtain significant graft-induced effects on deficits in certain aspects of complex sensorimotor behaviors. In the present study we have applied a modified cell suspension transplantation procedure, which allows more reproducible and consistent ventral mesencephalic transplants of large size, as well as more wide-spread distribution of the ventral mesencephalic tissue over multiple graft sites within the denervated caudate-putamen. Using this approach it has for the first time been possible to obtain significant amelioration of the lesion-induced deficits in skilled forelimb use and in the rats ability to switch from one behavior (eating) to another (orientation towards tactile stimuli), so-called disengage behavior. Rats with unilateral 6-hydroxydopamine lesions of the mesostriatal dopamine pathway received a total of 450,000 fetal ventral mesencephalic cells, implanted either as two large deposits along a single injection tract ("Macro" grafts), or as 18 small deposits along six injection tracts in the head of the denervated caudate-putamen ("Micro" grafts) and the behavioral changes were studied up to three months after transplantation. On the drug-induced tests, both types of transplants reversed amphetamine- and D1-receptor agonist-induced turning, and produced a partial (50-75%) reduction in apomorphine-induced and D2-receptor agonist-induced turning. On the spontaneous sensorimotor tests, both types of grafts reversed the deficit in simple sensorimotor orientation. In addition, the Micro-grafted animals (which produced the most extensive reinnervation of the denervated striatum) showed a significant improvement in skilled forelimb use and in response latency in the disengage behavior test. Although the large sized Macro-grafted animals showed a similar trend, it did not reach significance. Moreover, the Micro grafts had a more pronounced effect on spontaneous turning behavior in a conditioned response test. The improvement in response latency in the disengage test was significantly correlated with the dopamine level in the nucleus accumbens, whereas the magnitude of the conditioned turning response was significantly correlated with the dopamine levels in the head of the caudate-putamen. The results show that intrastriatal nigral transplants, despite their ectopic placement, can ameliorate lesion-induced deficits also in more complex sensorimotor behaviors. This improved graft effect is likely to depend on both extensive dopaminergic reinnervation throughout the head of the caudate-putamen, as well as on closer integration of the grafted nigral tissue with the host striatal circuitry.