Both apoptosis and necrosis occur early after intracerebral grafting of ventral mesencephalic tissue: a role for protease activation.

Neural transplantation is an experimental treatment for Parkinson's disease. Widespread clinical application of the grafting technique is hampered by a relatively poor survival (around 10%) of implanted embryonic dopamine neurones. Earlier animal studies have indicated that a large proportion of the grafted cells die during graft tissue preparation and within the first few days after intracerebral implantation. The present study was designed to reveal the prevalence of cell death in rat intrastriatal grafts at 90 min, 1, 3, 6 and 42 days after implantation. We examined apoptotic cell death using semi-thin and paraffin sections stained with methylene blue and an antibody against activated caspase 3, respectively. We identified abundant apoptotic cell death up to 3 days after transplantation. In addition, we studied calpain activation using an antibody specific for calpain-cleaved fodrin. We report a peak in calpain activity 90 min after grafting. Surprisingly, we did not observe any significant difference in the number of dopaminergic neurones over time. The present results imply that grafted cells may be victims of either an early necrotic or a later apoptotic cell death and that there is substantial cell death as early as 90 min after implantation

Mechanisms and time course of cell death in embryonic nigral tissue and intrastriatal transplants.

Parkinson’s disease (PD) is the second most common neurodegenerative disorder and severely affects about one in a hundred persons over the age of fifty years. In PD, there is a reduced striatal concentration of the neurotransmittor dopamine, mainly caused by a degeneration of the dopaminergic neurons in the substantia nigra. Transplantation of embryonic dopamine neurons has been developed as an alternative treatment for PD. Donor tissue dissection, mechanical dissociation, and intracerebral injection, can be divided into four phases that range from removal of embryos from the uterus to graft maturation. The transplantation procedure can be devided into four phases; i.e. embryo retrieval (phase 1); tissue dissection and dissociation (phase 2); injection of donor tissue into a recipient and a few days after injection (phase 3); and finally graft maturation (phase 4). Only a small fraction (5-20%) of the implanted dopaminergic neurons, grafted as a cell suspension into adult rats, will survive the transplantation procedure. The aim of the present thesis was to investigate the cause of cell death of the transplanted neurons during the four phases and to develop strategies to increase the neuronal survival. Several different methods were used to detect cell damage and death at various time points after tissue dissection and dissociation. We used transmission electron microscopy, vital staining, and electron paramagnetic resonance. The graft tissue was also evaluated at different time points after intrastriatal implantation, with markers for calpain activity and fragmanted DNA. The main results of the present thesis can be summarised as follows: 1) cell death occurs before implantation of the embryonic ventral mesencephalic tissue; 2) the majority of the transplanted dopaminergic neurons die within a few days after grafting; 4) addition of the antioxidant resveratrol to cell suspensions or transplantation into hypothermic hosts can result in an increased survival of embryonic mesencephalic neurons

Grafts of Embryonic Dopamine Neurons in Rodent Models of Parkinson's Disease

This chapter exemplifies rodent models of Parkinson's disease that have been used in transplantation studies and recounts basic transplantation methods. It describes morphological features of grafts containing dopamine neurons and focuses on the role of nigral grafts that are implanted homotopically in the mesencephalon, for example, near their location in the normal brain. The neurotoxin 6-hydroxydopamine (OHDA) is selectively taken up by catecholaminergic neurons and kills them through a mechanism that is still not fully elucidated, but may well include oxidative stress. When injected in adequate amounts into the brain, it can cause extensive and permanent damage to catecholaminergic neurons, such as those giving rise to the nigrostriatal pathway. The survival of dopamine neurons placed in a 6-OHDA denervated striatum is reflected by restoration of striatal dopamine levels to around 10 to 30% of normal. Examination of the ratio between levels of the dopamine metabolite DOPAC and dopamine reveals ratios that are 50 to 200% higher in grafted striata compared to normal, suggesting that the grafted neurons have an increased transmitter turnove

Characterisation of cell damage and death in embryonic mesencephalic tissue: a study on ultrastructure, vital stains and protease activity.

Dissociated embryonic ventral mesencephalic tissue is a source of dopaminergic neurones in both cell culture and neural transplantation studies. Around 90% of grafted dopaminergic neurones die within 1 week after transplantation. Little is known about when the cell death is triggered and what forms of cell death predominate. Using electron microscopy, we characterised ultrastructural changes in dissected embryonic day 14 rat mesencephalic tissue before and after tissue dissociation. In addition, cell viability was evaluated using Trypan Blue and Hoechst/Ethidium Homodimer. Several cells exhibited leaky outer membranes (permitting entry of vital stains) and ultrastructural degeneration already immediately after the mesencephalon was dissected, and before it was mechanically disrupted. After 2 h at room temperature, 90% of the remaining cells had intact outer membranes. However, when estimating cells lost acutely in the tissue dissociation, in addition to cells exhibiting condensed chromatin and organellar changes, we suggest that only around 14% of the cells initially dissected in the mesencephalic tissue pieces remained healthy after 2 h. There was a peak in calpain activity (specific cleavage of fodrin) immediately following tissue dissociation, and it subsided during the next few hours. Caspase-3 activity was initially low, but increased almost 20-fold 4 h after tissue disruption. Interestingly, extensive degradation of caspase-3 occurred already directly after dissection and was at least partly calpain-dependent. Our data suggest that, in addition to cells undergoing primary necrosis, some cells undergo apoptotic or related changes soon after tissue harvesting, and eventually undergo a secondary necrosis. In summary, embryonic mesencephalic cells exhibit multiple degenerative changes very early on in the neural transplant/tissue culture preparation protocol

Ultrastructural characterization of dissociated embryonic ventral mesencephalic tissue treated with neuroprotectants.

Poor survival and differentiation of grafted dopamine neurons limits the application of clinical transplantation in Parkinson’s disease. The survival of grafted dopamine neurons is only improved by a factor of 2–3 by adding neuroprotectants during tissue preparation. We used dye exclusion cell viability and electron microscopy to investigate the effects of the caspase inhibitor ac-YVAD-cmk and the lazaroid tirilazad mesylate on ultrastructural changes in dissociated embryonic mesencephalic cells. In addition, we examined whether the neuroprotectants selectively counteracted specific signs of neurodegeneration. Cell viability decreased significantly over time in both control and treated cell suspensions, but the number of viable cells remaining was significantly higher in tirilazad mesylate-treated cell suspensions. In control samples, the proportion of cells with an ultrastructure consistent with healthy cells decreased from 70%, immediately after dissociation, to 30% after 8 h of incubation. Similar changes were also observed in cell suspensions treated with neuroprotectants. Thus, the neuroprotectants examined did not block the development of specific morphological signs of neurodegeneration. However, when also taking into account that dead cells lysed and disappeared from each cell suspension with time, we found that the total number of remaining viable cells with healthy nuclear chromatin or intact membrane integrity was significantly higher in the tirilazad mesylate-treated group. The results indicate that tirilazad mesylate protects only a small subpopulation of embryonic mesencephalic cells from degeneration induced by mechanical trauma during tissue dissection and dissociation

Comparison between survival of lazaroid-treated embryonic nigral neurons in cell suspensions, cultures and transplants.

Death of transplanted dopaminergic neurons is induced both during preparation of donor tissue and after intrastriatal grafting. Oxidative stress is thought to be partly responsible for this cell death. In the present study we compared the effects of three lipid peroxidation inhibitors, the lazaroids Tirilazad mesylate, U-83836E and U-101033, on survival of embryonic mesencephalic neurons in different paradigms. The lazaroids were equally potent in preventing serum deprivation-induced death of cultured dopaminergic neurons. In a second set of experiments, mesencephalic suspensions were pretreated with lazaroids and cell survival was analyzed immediately after dissociation, after 2 or 24 h in culture or after intrastriatal transplantation. Lazaroid pretreatment failed to protect mesencephalic neurons in the in vitro paradigms and U-101033E did not protect grafted dopaminergic neurons in contrast to the neuroprotective effects previously reported for U-83836E and Tirilazad. Pretreatment with the iron chelator deferoxamine mesylate did not protect cultured or grafted dopaminergic neurons, nor did it improve neuronal survival in the serum deprivation model. U-83836E and U-101033E, but not Tirilazad, prevented cell death induced by the pro-oxidant tert-butyl hydroperoxide in suspensions. In a final experiment, we found that systemic treatment of the graft recipient rat with Tirilazad mesylate (before and during the first 3 days after grafting) improved survival of transplanted dopaminergic neurons to 180% of control values. Our results show that systemic treatment with a lipid peroxidation inhibitor for 3 days can promote graft survival, but also highlights the poor correlation between neuroprotective effect of pharmacological compounds in vitro and in grafts

Patterns of cell death and dopaminergic neuron survival in intrastriatal nigral grafts

Previous studies indicate that 80-95% of grafted dopamine neurons die following implantation of embryonic ventral mesencephalic tissue into the striatum. It is believed that the majority die within the first 1-3 weeks after surgery. The aim of this study was to study when and where the implanted neurons die, using the novel fluorescent stain Fluoro-Jade. Fluoro-Jade has recently been shown to stain cell bodies, dendrites, axons, and terminals of degenerating neurons. We transplanted dissociated ventral mesencephalic tissue from embryonic day 14 rat embryos into intact adult rat striatum. After perfusion and sectioning of the implanted rat brains, the number and distribution of Fluoro-Jade and tyrosine hydroxylase-positive neurons were evaluated at 6, 10, 14, and 42 days posttransplantation. Intensely Fluoro-Jade stained neurons were numerous in the grafts at 6 and 10 days after graft surgery; appeared in reduced numbers at 14 days; and had disappeared by the 42-day time point. The number of surviving tyrosine hydroxylase-positive, dopaminergic neurons in the grafts did not change between 6 and 42 days and the low survival rate confirmed that over 90% of these neurons had died during the first week. Assessment of the distribution of neurons positive for Fluoro-Jade or tyrosine hydroxylase revealed higher numbers of neurons stained for these markers located at the periphery than the center of the grafts, and this pattern did not change over time. This study indicates that transplanted neurons continue to die up to 14 days after grafting. Since the majority of transplanted tyrosine hydroxylase-positive neurons most probably die before 6 days after transplantation, neuroprotective strategies should primarily focus on the transplantation procedure and the first week after implantation

Grafting of nigral tissue hibernated with tirilazad mesylate and glial cell line-derived neurotrophic factor

Transplantation of embryonic ventral mesencephalon is a potential therapy for patients with Parkinson's disease. As only around 5-10% of embryonic dopaminergic neurons survive grafting into the adult striatum, it is considered necessary to use multiple donor embryos. To increase the survival of the grafted dopaminergic neurons, the clinical transplantation program in Lund currently employs the lipid peroxidation inhibitor, tirilazad mesylate, in all solutions used during tissue storage, preparation, and transplantation. However, the difficulty in obtaining a sufficient number of donor embryos still remains an important limiting factor for the clinical application of neural transplantation. In many clinical transplantation programs, it would be a great advantage if human nigral donor tissue could be stored for at least 1 week. This study was performed in order to investigate whether storage of embryonic tissue at 4 degrees C for 8 days can be applied clinically without creating a need to increase the number of donors. We compared the survival of freshly grafted rat nigral tissue, prepared according to the clinical protocol, with tissue transplanted after hibernation. Thus, in all groups tirilazad mesylate was omnipresent. One group of rats was implanted with fresh tissue and three groups with hibernated tissue with or without addition of glial cell line-derived neurotrophic factor (GDNF) in the hibernation medium and/or the final cell suspension. Earlier studies have suggested that GDNF improves the survival of hibernated nigral transplants. We found no statistically significant difference between the groups regarding graft survival after 3 weeks. However, there was a nonsignificant trend for fewer surviving dopaminergic neurons in grafts from hibernated tissue compared to fresh controls. Furthermore, we show that the addition of GDNF to the hibernation medium and/or to the final cell suspension does not significantly increase the survival of the dopaminergic neurons

Increased survival of embryonic nigral neurons when grafted to hypothermic rats

Hypothermia can reduce neuronal death caused by ischemia and traumatic brain injury. We therefore investigated whether mild hypothermia in rats receiving a transplant of embryonic mesencephalic rat tissue increases survival of the implanted dopaminergic neurons. Mild hypothermia (32-33°C) during graft implantation and for the following 90 min significantly increased the survival of transplanted dopaminergic neurons to 171% of control values in normothermic (37°C) rats. This demonstrates that treatment of the graft recipient for a relatively short period during and after surgery has a favorable effect on the survival of grafted dopaminergic neurons. These findings may be of importance for clinical neural transplantation trials which are in need of procedures that improve transplant survival. (C) 2000 Lippincott Williams and Wilkins

Ultrastructure of alpha-synuclein-positive aggregations in U373 astrocytoma and rat primary glial cells

Abnormal alpha-synuclein-positive glial cytoplasmic inclusions are found in Parkinson's disease, multiple system atrophy and dementia with Lewy bodies. We have recently developed an in vitro model of alpha-synuclein-immunoreactive aggregations in U373 astrocytoma cells. We have additionally overexpressed wild-type and a C-terminally truncated form of alpha-synuclein in primary rat glial cells. Astrocytes and oligodendrocytes were found to form alpha-synuclein-positive aggregations in vitro perinuclearly or in the processes of the cells. The morphological studies presented here demonstrate that the aggregations we have observed in vitro are not limited by a membrane but have unclear borders. They have an amorphous dense core that is intensely alpha-synuclein-immunopositive and a predominantly filamentous halo around. Mainly filamentous structures at the border area between the halo and the core are alpha-synuclein-immunoreactive. We conclude that this in vitro model of alpha-synuclein-positive glial aggregations mimics the morphology of the abnormal glial inclusions described in neuroclegenerative disorders and could be a suitable model for studying their role in the pathogenesis of these diseases. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved