Agneta Othberg

Platelet-derived growth factor promotes survival of rat and human mesencephalic dopaminergic neurons in culture

SummaryThe effect of two isoforms of platelet-derived growth factor (PDGF), PDGF-AA and PDGF-BB, was tested on dissociated cell cultures of ventral mesencephalon from rat and human embryos. PDGF-BB but not PDGF-AA reduced the progressive loss of tyrosine hydroxylase- (TH)-positive neurons in rat and human cell cultures. The mean number of TH-positive cells in the PDGF-BB-treated rat culture was 64% and 106% higher than in the control cultures after 7 and 10 days in vitro, respectively. Corresponding figures for human TH-positive neurons were 90% and 145%. The influence of PDGF-BB was specific for TH-positive neurons and not a general trophic effect, since no change of either total cell number or metabolic activity was found. In PDGF-BB-treated cultures of human but not rat tissue the TH-positive neurons had longer neurites than observed in control or PDGF-AA-treated cultures. These data indicate that PDGF-BB may act as a trophic factor for mesencephalic dopaminergic neurons and suggest that administration of PDGF-BB could ameliorate degeneration and possibly promote axonal sprouting of these neurons in vivo.

Viability and survival of hNT neurons determine degree of functional recovery in grafted ischemic rats

We recently reported behavioral improvements following intrastriatal transplantation of cryopreserved cultured human neuroteratocarcinoma-derived cells (hNT neurons) in rats with cerebral ischemia induced by occlusion of the middle cerebral artery. In the present study, the viability and survival of hNT neurons were evaluated immediately prior to the transplantation surgery and at 3 months post-transplantation in ischemic rats. Cryopreserved hNT neurons were routinely thawed, and trypan blue exclusion viability counts revealed 52–95% viable hNT neurons before transplantation. Monthly behavioral tests, starting at 1 month and extending to 3 months post-transplantation, revealed that ischemic animals that were intrastriatally transplanted with hNT neurons (4000) and treated with an immunosuppressive drug displayed normalization of asymmetrical motor behavior compared with ischemic animals that received medium alone. Within subject comparisons of cell viability and subsequent behavioral changes revealed that a high cell viability just prior to transplantation surgery correlated highly with a robust and sustained functional improvement in the transplant recipient. Furthermore, histological analysis of grafted brains revealed a positive correlation between number of surviving hNT neurons and degree of functional recovery. In concert with similar reports on fetal tissue transplantation, we conclude that high cell viability is an important criterion for successful transplantation of cryopreserved neurons derived from cell lines to enhance graft-induced functional effects.

BDNF makes cultured dentate granule cells more resistant to hypoglycaemic damage

The aim of this study was to explore whether brain-derived neurotrophic factor (BDNF) can improve neuronal survival in cell cultures of rat dentate gyrus subjected to a hypoglycaemic insult. Glucose deprivation for 15 h caused severe neuronal loss (about 70%). BDNF added either 24 h before or 4 h after onset of hypoglycaemia completely protected granule cells against this insult-induced damage. Nerve growth factor (NGF) had similar effects. These findings support the hypothesis that the rapid upregulation of BDNF mRNA in dentate granule cells after brief periods of hypoglycaemic coma and other insults is a local protective mechanism.

Specific effects of platelet derived growth factor (PDGF) on fetal rat and human dopaminergic neurons in vitro

The neurotrophic effects of the BB isoform of platelet-derived growth factor (PDGF) on rat and human fetal mesencephalic dopaminergic neurons have been characterized in vitro. A dose-response analysis demonstrated maximal responses at 30 ng/ml of PDGF-BB. This concentration resulted in a marked increase in the survival and neurite outgrowth from rat and human tyrosine hydroxylase-(TH) positive, presumed dopaminergic neurons after 7 days in vitro. The effects of PDGF-BB on survival of TH-positive neurons were comparable to those of brain-derived neurotrophic factor (BDNF), whereas neurite outgrowth was more pronounced after addition of BDNF. The combination of BDNF and PDGF-BB yielded no additive effects. Double immunohistochemical staining of rat cultures demonstrated PDGF β-receptors on about 90% of the TH-positive neurons. PDGF-BB treatment of rat mesencephalic cultures induced an upregulation of c-fos and TH mRNA with maximal levels after 0.5–2 h as assessed by quantitative PCR analysis. An increased number of Fos protein-positive cells was detected immunohistochemically after 4 h of PDGF-BB treatment. The present results provide further evidence for specific and direct effects of PDGF-BB on gene expression, survival and neurite outgrowth of mesencephalic dopaminergic neurons of rat and human origin.

Sertoli cells enhance the survival of co-transplanted dopamine neurons.

One of the major issues in neural transplantation is the low survival rate (<5%) of transplanted dopamine (DA) neurons [3]. Recently it has been shown that it is possible to enhance the survival of these neurons, which in turn may decrease the amount of tissue that is required for each transplantation patient. The present paper demonstrates a novel approach for enhancing neuronal survival by co-transplantation of neuronal tissue with Testis-derived Sertoli cells (SC). This strategy could improve neuronal survival through the provision of trophic support.

Sertoli cells decrease microglial response and increase engraftment of human hNT neurons in the hemiparkinsonian rat striatum.

Sertoli cells (SCs) provide immune protection and nutritive support to the developing germ cells in the testis. Sertoli cells have also been shown to provide immune protection to islets transplanted outside the testes. In this study, the ability of these cells to diminish the infiltration/activation of microglia into a neural graft implanted in the lesioned striatum of a hemiparkinsonian rat was investigated. Human neuron-like cells (hNT neurons) were implanted either alone or in combination with rat SCs. Three months later, the animals were sacrificed and immunohistochemistry was performed to determine the survival of the xenografted neurons as well as microglial infiltration/activation. Cotransplantation of the SCs with the hNT neurons increased graft survival and was associated with an increase in graft size. Furthermore, there were fewer microglia present in the grafted tissue of the cotransplantation groups. These results show that SCs retain their immunosuppressive ability even within the brain. As immune responses to grafted neural tissue within the central nervous system become better understood, this ability of the SCs to provide localized immunosuppression to the transplanted tissue may become more important. This is particularly true as the search for alternative sources of neural tissue to treat neurodegenerative diseases expands to encompass other species.

Trophic Effect of Porcine Sertoli Cells on Rat and Human Ventral Mesencephalic Cells and hNT Neurons In Vitro

The poor survival of embryonic dopaminergic (DA) neurons transplanted into patients with Parkinsons disease (PD) has encouraged researchers to search for new methods to affect the short- as well as long-term survival of these neurons after transplantation. In several previous rodent studies Sertoli cells increased survival of islet cells and chromaffin cells when cotransplanted in vivo. The aims of this study were to investigate whether porcine Sertoli cells had a positive effect on the survival and maturation of rat and human DA neurons, and whether the Sertoli cells had an effect on differentiation of neurons derived from a human teratocarcinoma cell line (hNT neurons). A significant increase of tyrosine hydroxylase (TH)-positive neurons of both rat and human ventral mesencephalic tissue was found when cocultured with Sertoli cells. Furthermore, there was a significantly increased soma size and neurite outgrowth of neurons in the coculture treated group. The Sertoli cell and hNT coculture also revealed an increased number of TH-positive cells. These results demonstrate that the wide variety of proteins and factors secreted by porcine Sertoli cells benefit the survival and maturation of embryonic DA neurons and suggest that cotransplantation of Sertoli cells and embryonic DA neurons may be useful for a cell transplantation therapy in PD.

Tirilazad Mesylate Improves Survival of Rat and Human Embryonic Mesencephalic Neurons in Vitro

The survival rate of embryonic dopamine (DA) neurons after transplantation to the striatum is only 5-20%. Therefore, mesencephalic tissue from several donors needs to be implanted in a parkinsonian patient to induce a therapeutic improvement. Lazaroids are a group of neuroprotective compounds which inhibit lipid peroxidation. Previously, two lazaroids (U-74389G and U-83836F) have been found to improve the survival of both cultured and grafted rat DA neurons. The only lazaroid approved for human use is tirilazad mesylate. The objective of the present study was to explore the effects of tirilazad mesylate on DA neuron survival in cultures of rat ventral mesencephalon and its capacity to promote the in vitro cell viability of embryonic rat and human mesencephalic tissue, treated and dissociated in the same way as in clinical trials. After 7 days in vitro, the number of tyrosine hydroxylase-immunopositive, presumed DA neurons was 140% higher in rat cultures treated with 0.3 microM tirilazad mesylate than that in control cultures. Rat and human cell suspensions supplemented with tirilazad mesylate maintained a high degree of viability for several hours longer than control suspensions. These results indicate that tirilazad mesylate promotes the survival of both rat and human embryonic mesencephalic neurons in vitro. Tirilazad mesylate can be administered clinically and may become a useful tool for increasing survival of grafted DA neurons in patients, thereby reducing the needed quantity of human donor tissue.

Effects of brain-derived neurotrophic factor on neuronal structure of dopaminergic neurons in dissociated cultures of human fetal mesencephalon

Brain-derived neurotrophic factor (BDNF) has been shown to promote the survival of cultured fetal mesencephalic dopaminergic neurons of rat and human origin. In the present study, BDNF was tested for its ability to influence neuronal structure of dopaminergic neurons in dissociated cultures of human fetal ventral mesencephalon after 7 days in vitro. Following immunocytochemical staining for tyrosine hydroxylase, all surviving dopaminergic neurons were counted. Computer-assisted three-dimensional reconstructions of uniform randomly selected neurons cultured with 50 ng/ml BDNF (n=120) or without BDNF (n=80) were made. BDNF increased the number of surviving human dopaminergic neurons by 76%. Mean soma profile area was significantly enlarged by 18% in BDNF-treated neurons as compared to controls. Analysis of parameters of neuritic size and complexity in these cultures revealed that combined neuritic length, combined neuritic volume, and neuritic field area were increased by 60%, 125% and 129%, respectively, and the mean number of segments per cell was increased by 41%. A change in neurite complexity in BDNF-treated cultures was further confirmed by the Sholls concentric sphere analysis. These results demonstrate that BDNF promotes development and differentiation of human fetal dopaminergic neurons in vitro.

Transplantation of testis-derived sertoli cells into the mammalian brain☆

NTRACEREBRAL transplantation therapy as a treatment for movement disorders, such as Parkinson’s disease, has accentuated research interest in the possibility of restoring CNS circuits to achieve some functional recovery following transplantation of fetal tissue.‘,’ The possible graft rejection due to CNS immune reactions have prompted investigations to find alternative methods for immunosuppression.3 Moreover, the logistical problems concerning tissue availability has focused research on the possibility of adding trophic factors to enhance the yield of tissue, as well as finding new sources for obtaining tissue for intracerebral transplantation. Recently, we demonstrated that cotransplantation of adrenal chromaffin cells with testis-derived Sertoli cells into the striatum of hemiparkinsonian rats produced localized immunosuppression and enhanced survival of the xenograft and, when transplanted alone, resulted in behavioral recovery.43”