联合培养胚鼠的黑质及纹状体LGE细胞脑内移植治疗帕金森病大鼠的实验研究

目的　观察并检测胚鼠纹状体外侧节突 (LGE)对多巴胺能 (DA)细胞存活性的促进和营养导向作用。方法　将帕金森病 (PD)模型随机分成四组 :Co -culture组 (n =12 ) ;Cograft组 (n =12 ) ;Solo -VM组 (n =12 ) ;Con trol组 (n =8)。将胚鼠LGE细胞和腹侧中脑组织 (VM )制成细胞悬液 ,植入Control组外的其他各组动物的尾壳核。 2周后进行PD鼠行为学检测 ,连续观察 2 4周 ,继之将各组大鼠处死 ,进行免疫组化染色。结果　Co -culture组和Co - graft组大鼠移植后旋转行为较Solo -VM组大鼠明显减少。CO -culture组和CO - graft组之间大鼠的旋转行为比较 ,无统计学差异。免疫组化观察证实LGE和VM离体培养移植和新鲜移植均能提高DA细胞的存活性 ,增加宿主纹状体内DA纤维重新支配的密度 ,并形成明显的DA细胞团。结论　LGE细胞对VM移植物有明显的营养导向作用 ,并可增强DA细胞的存活 ,促进移值后DA细胞功能持久维持 ,并增加DA细胞再支配的密度     

Co-grafts of embryonic dopamine neurons and adult sciatic nerve into the denervated striatum enhance behavioral and morphological recovery in rats

We have recently demonstrated that a diffusible factor(s) derived from explanted adult rat sciatic nerve can increase the number and neurite outgrowth of embryonic rat dopamine (DA) neurons in culture. The present study extends this finding to compare DA neuron-sciatic nerve co-grafts to grafts of DA-rich neural tissue alone for behavioral and morphological effects in rats with unilateral nigrostriatal lesions of the DA pathway. Our results indicate that the presence of a co-grafted segment of sciatic nerve increased the likelihood of rapid behavioral recovery and promoted complete recovery mediated by small grafts that yielded only modest behavioral changes in the absence of co-grafted nerve. These behavioral effects were accompanied by a modest increase in survival of grafted tyrosine hydroxylase-positive neurons in the striatum and a more pronounced increase in the area and density of striatal reinnervation provided by grafted DA neurons in co-grafted animals. This evidence supports the view that a diffusible product of explanted peripheral nerve acts as a growth-promoting factor for embryonic DA neurons and that the presence of this factor augments the behavioral efficacy of grafted DA neurons.     

Embryonic mesencephalic and striatal co-grafts: development of grafted dopamine neurons and functional recovery

Previous neural grafting studies have shown that embryonic dopamine neurons survive transplantation into the parenchyma of the brain; however, fiber outgrowth from those cells is often limited to the immediate vicinity of the graft. More extensive outgrowth is desirable for promoting and maintaining functional recovery of damaged neural systems in animal models as well as human neurodegenerative disorders. The present study examined the possibility of stimulating fiber outgrowth of grafted neurons by simultaneously grafting dopamine neurons with their embryonic target cells. Subsequent functional recovery was evaluated in concert with morphological characteristics of these grafts. Co-grafts of embryonic mesencephalic and striatal cells were implanted into the DA-denervated striatum of rats previously given unilateral 6-hydroxydopamine lesions of the nigrostriatal pathway. Two types of co-grafts were implanted into the DA-denervated striatum: mixed or separate cell suspensions. Tyrosine hydroxylase immunocytochemical analysis of brain sections containing co-grafts revealed extensive arborization of TH-positive neurons in both types of co-grafts. When mesencephalic and striatal nerve cells were implanted into separate sites, TH-positive neurons extended projections that appeared to preferentially reach regions occupied by embryonic striatal neurons. Moreover, the average size of TH-positive cell bodies found in mixed or separate co-grafts was significantly larger than the size of those found in single mesencephalic grafts. Amphetamine-induced rotational behavior was used to assess the degree of functional recovery. In the majority of co-grafted animals, rotational behavior was attenuated by 3 weeks and reversed (amphetamine-induced contralateral rotation) by 5 weeks.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pattern of synaptophysin immunoreactivity within mesencephalic grafts following transplantation in a parkinsonian primate model

The majority of investigations into the degree of restoration of neural circuitry following transplantation of the embryonic ventral mesencephalon to the striatum have focused upon the particular neurochemical subtypes of the fibers exchanged between graft and host. Visualization of neurites of specific neurotransmitter type while informative regarding the specificity of graft–host interactions, vastly underrepresents overall synaptogenesis as it may occur in the grafting situation. The present approach of using a molecular marker characteristic of all normal, functional synapses provides broader information about the synaptic remodeling that occurs after tissue grafting. Synaptophysin (SY), an integral membrane protein of the synaptic vesicle, is a reliable marker of nerve terminal differentiation. Immunohistochemical staining with antibodies directed against SY and the dopamine synthetic enzyme tyrosine hydroxylase (TH) was used to assess overall synaptic differentiation as well as the relationship between SY immunoreactivity and the distribution of grafted dopamine (DA) neurons and processes in mesencephalic grafts and mesencephalic-striatal co-grafts implanted in the striatum of MPTP-treated African green monkeys. Grafted embryonic cerebellar tissue was used as a comparison graft type that does not normally exchange prominent direct projections with striatum. Dense pericellular arrays of SY-positive terminals were associated with TH-positive neurons in mesencephalic grafts. In mixed mesencephalic-striatal co-grafts, TH-positive fiber patches within the striatal portion of the graft demonstrated a high degree of correspondence with SY immunoreactivity. In contrast, grafts of cerebellar tissue did not display the same pattern of prominent pericellular arrays of SY staining. These observations suggest that functional synapses are abundantly present within grafted mesencephalon, and that these contacts are enriched in areas of the graft occupied by DA neurons. Implantation of an inappropriate striatal target, the cerebellum, results in visibly diminished innervation. The pattern of SY labeling observed suggests that tissue grafts are extensively innervated, probably both from extrinsic and intrinsic sources, and that the pattern and density of this innervation corresponds to the appropriateness of the graft–host interaction.     

Co-grafts of muscle cells and mesencephalic tissue into hemiparkinsonian rats: behavioral and histochemical effects

Extracts from skeletal muscle cell cultures have been shown to increase levels of the enzyme tyrosine hydroxylase (TH) and promote survival of different types of developing neurons in vitro. To determine the effect of muscle cell co-grafts on the survival of dopamine neurons in a rat model of Parkinson's disease, we transplanted an embryonic day (ED)-15 rat mesencephalic cell suspension alone or with neonatal muscle cells into 6-hydroxydopamine (6-OHDA) denervated rat striatum. In parallel experiments conducted in vitro, we cultured ED-15 rat mesencephalon or rat striatum in conditioned medium from neonatal rat muscle cultures (MC-CM). Our results showed that: (A) in vitro, MC-CM increased the number of TH-immunoreactive (TH-IR) neurons in embryonic mesencephalic cultures but did not induce expression of TH in embryonic striatal cultures; (B) in vivo, animals with co-grafts of muscle cells and ED-15 mesencephalon had more TH-IR in the grafted striatum compared to animals that received mesencephalic cells grafts alone, although the graft-induced reversal of circling behavior in response to methamphetamine was the same in both transplanted groups; and (C) grafts of muscle cells alone did not induce TH-IR in the denervated striatum and did not reduce methamphetamine-induced circling. These findings suggest that in vivo, neonatal muscle cells secrete factors that promote survival and/or outgrowth of fetal midbrain dopamine cells and improve the levels of TH-IR in grafted striatum.     

Intrastriatal grafting of dopamine-containing neuronal cell suspensions: effects of mixing with target or non-target cells

The effects of target and non-target cells on the growth and function of intrastriatal grafts of mesencephalic dopamine neurons have been studied in rats with unilateral 6-hydroxydopamine-induced lesions of the nigrostriatal dopamine pathway. Cell suspensions of ventral mesencephalon from 14-15-day-old rat fetuses (rich in developing dopamine neurons) were either grafted alone or grafted after mixing with equivalent numbers of cells obtained from the striatum (a major dopamine target area) or spinal cord (a non-target area for the mesencephalic dopamine neurons). The combined mesencephalic and striatal grafts gave rise to a greater area of dense innervation in the host caudate-putamen than grafts of mesencephalic cells alone or grafts of mesencephalic cells mixed with spinal cord cells. The number of surviving catecholamine-containing neurons did not differ significantly in the different types of grafts. In addition, there was an altered outgrowth pattern in the combined mesencephalic-striatal grafts consisting of small round islands of intensely fluorescent catecholamine-containing fibres, often in close association with the grafted dopamine neurons. In a subsequent biochemical study it was found that combined mesencephalic-striatal grafts exhibited dopamine levels and turnover that did not differ from grafts containing mesencephalic cells only. The mesencephalic-striatal cografts showed a trend toward enhanced behavioural effect, in terms of greater reduction in amphetamine-induced rotation asymmetry, when compared to other graft groups. It is suggested that the addition of embryonic striatal target cells can exert stimulatory effects on morphological development, and possibly functional parameters, of fetal dopamine cells also in vivo after intrastriatal grafting.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional innervation of the striatum by ventral mesencephalic grafts in mice with inherited nigrostriatal dopamine deficiency

Weaver mutant mice are characterized by a decrease in striatal dopamine (DA), which is associated with a progressive loss of DA neurones in the substantia nigra. This mutant thus provides the opportunity to examine the functional effects of DA neurones grafted to the striatum in a genetic model of parkinsonism. Ventral mesencephalic tissue from normal foetuses was placed on the surface of the right dorsal striatum of adult weaver mutants. After grafting, animals were tested for methamphetamine-induced circling behaviour. Mutants with DA containing grafts displayed a significant circling bias toward the left, non-grafted side. Mutants without grafts did not display any rotational bias to either side. These results demonstrate that grafted DA containing neurones establish a functional innervation of the weaver striatum and suggest that grafting of neural tissue is a viable approach in restoring function in genetic degenerative disorders of the nigrostriatal system.     

Transplantation of Mesencephalic Cell Suspension in Dopamine-Denervated Striatum of the Rat: I. Effects on Spontaneous Activity of Striatal Neurons

These studies have examined the extent to which intrastriatal grafts of embryonic mesencephalic neurons induce recovery of normal discharge patterns in striatal neurons of rats after a unilateral 6-hydroxydopamine (6-OHDA) lesion of the nigrostriatal dopamine (DA) pathway. Lesioned rats were tested for rotational behavior induced by amphetamine and apomorphine. Animals which responded positively to these tests received two suspensions of mesencephalic embryonic neurons into the dorsal striatum (ST) ipsilateral to the denervated side. Sham-grafted rats received the suspension medium only. The vitality of the graft was assessed by the disappearance or reversion of rotational movements induced by amphetamine. Extracellular recordings of neurons located throughout the ST were carried out 3 months after grafting, when the animals reached the age of 6 months. The 6-OHDA-induced nigral lesion caused a net increase both in the number of striatal neurons spontaneously active and in their discharging rates. The signs of increased neuronal activity were also present in sham-grafted animals. The grafting of embryonal cells strongly reduced the number of active neurons and decreased significantly their discharging rate. The effects of the intrastriatal graft appeared to be present within a radius of 1.5–2 mm from the core of the grafted area. The presence of tyrosine-hydroxylase-immunopositive neurons innervating the host ST confirmed the viability of the grafts at the time of electrophysiological recording. The results show that besides compensating motor asymmetries caused by DA denervation, intrastriatally grafted dopaminergic neurons are able to only partially restore the electrophysiological action of DA in discrete striatal domains.     

An in vitro interval before transplantation of mesencephalic reaggregates does not compromise survival or functionality

Grafts of primary ventral mesencephalic tissue and cell suspensions to the denervated striatum are currently utilized as a treatment strategy for Parkinson's disease. Survival rates of grafted dopamine (DA) neurons are extremely poor (5-20%) and is even poorer in grafts to the aged striatum. Short pretreatment of grafted cells with various survival-promoting agents has elicited 2- to 3-fold improvements in these survival rates. However, the duration of pretreatment is limited by the necessity of implanting the embryonic cells within a critical period after tissue harvest, potentially limiting the beneficial effects of these interventions. This study details the use of a modified mesencephalic reaggregate culture system combined with striatal-derived trophic factor support to provide an extended ex vivo cell culture interval before grafting. Mesencephalic cell suspension grafts implanted immediately following dissociation were compared to grafts of an equivalent number of cells reaggregated in the presence of striatal oligodendrocyte-type-2 astrocyte (SO2A) conditioned medium for 3 or 7 days. All grafts were placed in the denervated striatum of young adult male Fischer 344 rats. Rotational assessment of amphetamine-induced rotations indicates that aggregates maintained for 3 days in culture present statistically similar functional recovery profiles as compared to cell suspension grafts. Grafts of mesencephalic reaggregates maintained in vitro for 7 days did not display significant improvements in functional recovery. Immunohistochemical analysis for tyrosine hydroxylase immunoreactive (THir) neurons conducted at 10 weeks post-grafting revealed equivalent survival rates of THir neurons in grafts of fresh cell suspensions and aggregates held in culture for 3 days. Grafts of reaggregates held in culture for 7 days possessed significantly fewer THir neurons, about 25% of the cell suspension or 3-day aggregate grafts. This ex vivo reaggregate system allows for extended pretreatment (3 days) of mesencephalic cells with survival-promoting agents and immunological screening of tissue before transplantation.     

Interference with anoikis-induced cell death of dopamine neurons: implications for augmenting embryonic graft survival in a rat model of Parkinson's disease

One promising therapy for the treatment of Parkinson's disease is transplantation of embryonic ventral mesencephalic tissue. Unfortunately, up to 95% of grafted cells die, many via apoptosis. In this study we attempted to prevent anoikis-induced cell death, which is triggered during the preparation of cells for grafting, and examine the impact on graft viability and function. We utilized the extracellular matrix molecule tenascin-C (tenascin) and an antibody (Ab) to the cell adhesion molecule L1 to specifically mimic survival signals induced by cell-matrix and cell-cell interactions. In vitro, both tenascin- and L1 Ab-treated cultures doubled the number of tyrosine hydroxylase immunoreactive (THir) neurons compared to control. Additionally, cell survival assays determined that tenascin and L1 Ab-treated cell suspensions yielded more metabolically active and fewer dead cells than control suspensions. In contrast to the culture results, tenascin- and L1 Ab-treated mesencephalic grafts did not yield an increase in the number of THir neurons using our standard grafting paradigm (3 microl of 100,000 cells/microl). However, under low-density conditions (3 microl of 3,000 cells/microl), tenascin augmented grafted THir neuron survival. These findings are consistent with the view that cell density can dramatically influence the degree of stress placed on THir neurons and consequently affect the success of survival strategies in vivo. In conclusion, pretreatment with tenascin may prove beneficial to prevent anoikis in dilute cell suspension grafts, while long-term in vivo delivery methods need to be explored to determine if L1 can prevent anoikis in grafts of mesencephalic dopamine neurons after transplantation.     

Increased cell suspension concentration augments the survival rate of grafted tyrosine hydroxylase immunoreactive neurons

The poor survival rate (5-20%) of grafted embryonic dopamine (DA) neurons is one of the primary factors preventing cell replacement from becoming a viable treatment for Parkinson's disease. Previous studies have demonstrated that graft volume impacts grafted DA neuron survival, indicating that transplant parameters influence survival rates. However, the effects of mesencephalic cell concentration on grafted DA neuron survival have not been investigated. The current study compares the survival rates of DA neurons in grafts of varying concentrations. Mesencephalic cell suspensions derived from E14 Fisher 344 rat pups were concentrated to 25,000, 50,000, 100,000 and 200,000 cells/microl and transplanted into two 0.5 microl sites in the 6-OHDA-denervated rat striatum. Animals were sacrificed 10 days and 6 weeks post-transplantation for histochemical analysis of striatal grafts. The absolute number of DA neurons per graft increased proportionally to the total number of cells transplanted. However, our results show that the 200,000 cells/microl group exhibited significantly higher survival rates (5.48+/-0.83%) compared to the 25,000 cells/microl (2.81+/-0.39%) and 50,000 cells/microl (3.36+/-0.51%) groups (p=0.02 and 0.03, respectively). Soma size of grafted DA neurons in the 200,000 cells/microl group was significantly larger than that of the 25,000 cells/microl (p<0.0001) and 50,000 cells/microl groups (p=0.004). In conclusion, increasing the concentration of mesencephalic cells prior to transplantation, augments the survival and functionality of grafted DA neurons. These data have the potential to identify optimal transplantation parameters that can be applied to procedures utilizing stem cells, neural progenitors, and primary mesencephalic cells.     

Dissociation between short-term increased graft survival and long-term functional improvements in Parkinsonian rats overexpressing glial cell line-derived neurotrophic factor

The present study was designed to analyse whether continuous overexpression of glial cell line-derived neurotrophic factor (GDNF) in the striatum by a recombinant lentiviral vector can provide improved cell survival and additional long-term functional benefits after transplantation of fetal ventral mesencephalic cells in Parkinsonian rats. A four-site intrastriatal 6-hydroxydopamine lesion resulted in an 80-90% depletion of nigral dopamine cells and striatal fiber innervation, leading to stable motor impairments. Histological analysis performed at 4 weeks after grafting into the GDNF-overexpressing striatum revealed a twofold increase in the number of surviving tyrosine hydroxylase (TH)-positive cells, as compared with grafts placed in control (green fluorescent protein-overexpressing) animals. However, in animals that were allowed to survive for 6 months, the numbers of surviving TH-positive cells in the grafts were equal in both groups, suggesting that the cells initially protected at 4 weeks failed to survive despite the continued presence of GDNF. Although cell survival was similar in both grafted groups, the TH-positive fiber innervation density was lower in the GDNF-treated grafted animals (30% of normal) compared with animals with control grafts (55% of normal). The vesicular monoamine transporter-2-positive fiber density in the striatum, by contrast, was equal in both groups, suggesting that long-term GDNF overexpression induced a selective down-regulation of TH in the grafted dopamine neurons. Behavioral analysis in the long-term grafted animals showed that the control grafted animals improved their performance in spontaneous motor behaviors to approximately 50% of normal, whereas the GDNF treatment did not provide any additional recovery.     

Optimal conditions for in vivo induction of dopaminergic neurons from embryonic stem cells through stromal cell-derived inducing activity

A method of inducing dopamine (DA) neurons from mouse embryonic stem (ES) cells by stromal cell-derived inducing activity (SDIA) was previously reported. When transplanted, SDIA-induced DA neurons integrate into the mouse striatum and remain positive for tyrosine hydroxylase (TH) expression. In the present study, to optimize the transplantation efficiency, we treated mouse ES cells with SDIA for various numbers of days (8-14 days). SDIA-treated ES cell colonies were isolated by papain treatment and then grafted into the 6-hydroxydopamine (6-OHDA)-lesioned mouse striatum. The ratio of the number of surviving TH-positive cells to the total number of grafted cells was highest when ES cells were treated with SDIA for 12 days before transplantation. This ratio revealed that grafting cell colonies was more efficient for obtaining TH-positive cells in vivo than grafting cell suspensions. When we grafted a cell suspension of 2 x 10(5), 2 x 10(4), or 2 x 10(3) cells into the 6-OHDA-lesioned mouse striatum, we observed only a few surviving TH-positive cells. In conclusion, inducing DA neurons from mouse ES cells by SDIA for 12 days and grafting cell colonies into mouse striatum was the most effective method for the survival of TH-positive neurons in vivo.     

Exogenous erythropoietin provides neuroprotection of grafted dopamine neurons in a rodent model of Parkinson's disease

Parkinson's disease (PD) is a neurodegenerative disease marked by severe loss of dopamine (DA) neurons in the nigrostriatal system, which results in depletion of striatal DA. Transplantation of embryonic ventral mesencephalic (VM) DA neurons into the striatum is a currently explored experimental treatment aimed at replacing lost DA in the nigrostriatal system, but is plagued with poor survival (5-20%) of implanted neurons. Here, we tested the ability of erythropoietin (Epo) to provide neuroprotection for embryonic day 14 (E14) VM DA neurons. Epo was tested in vitro for the ability to augment tyrosine hydroxylase-immunoreactive (TH-ir) neuron survival under normal cell culture conditions. In vitro, Epo did not increase the number of TH-ir neurons when administered at the time of plating the E14 VM cells in culture. We also tested the efficacy of Epo to enhance E14 VM transplants in vivo. Rats unilaterally lesioned with 6-hydroxydopamine received transplants that were incubated in Epo. Treatment with Epo produced significant increases in TH-ir neuron number, soma size, and staining intensity. Animals receiving Epo-treated grafts exhibited significantly accelerated functional improvements and significantly greater overall improvements from rotational asymmetry compared to control grafted rats. These data indicate that the survival of embryonic mesencephalic TH-ir neurons is increased when Epo is administered with grafted cells in a rodent model of PD. As direct neurotrophic effects of Epo were not observed in vitro, the mechanism of Epo neuroprotection remains to be elucidated.     

Diminished survival of mesencephalic dopamine neurons grafted into aged hosts occurs during the immediate postgrafting interval

The survival rate of dopamine (DA) neurons in mesencephalic grafts to young adult rats is poor, estimated at 5-20%, and even poorer in grafts to the aged striatum. Grafted cells die in young adult rats during the first 4 days after implantation. The present study was undertaken to determine whether the decreased survival of DA neurons in grafts to aged rats is (1) due to additional cell death during the immediate postgrafting interval or (2) due to protracted cell loss during longer postgrafting intervals. We compared survival rates of tyrosine hydroxylase-immunoreactive (THir) neurons in cell suspension grafts to young adult (3 months) and aged (24 months) male Fischer 344 rats at 4 days and 2 weeks after transplantation. At 4 days after grafting, mesencephalic grafts within the aged rat striatum contain approximately 25% of the number of THir neurons in the same mesencephalic cell suspension grafted to young adult rats. This corroborates the decreased survival of grafted DA neurons we have demonstrated previously at 10 weeks postgrafting. THir neurons in grafts to the intact striatum possessed a significantly shorter "long axis" than their counterparts on the lesioned side. No significant differences in the number of apoptotic nuclear profiles or total alkaline phosphatase staining between mesencephalic grafts to young and aged rats were detectable at 4 days postgrafting. In summary, the present study indicates that the exaggerated cell death of grafted DA neurons that occurs following implantation to the aged striatum occurs during the immediate postgrafting interval, timing identical to that documented for young adult hosts.     

Angiogenic and neurotrophic effects of vascular endothelial growth factor (VEGF165): studies of grafted and cultured embryonic ventral mesencephalic cells

The present series of experiments investigated the effects of vascular endothelial growth factor (VEGF165) on adult rat striatal cerebrovasculature and embryonic dopamine (DA) neuron allografts in a rat model of Parkinson's disease (PD). We examined VEGF165's ability to (1) alter the vascular network of the adult rat striatum, (2) influence the vascular growth of solid embryonic day 14 (E14) ventral mesencephalic (VM) grafts when placed into a VEGF-pretreated host striatum, (3) alter the function and survival of E14 VM grafts when transplanted into an adult DA-deleted striatum, and (4) influence cell survival and neurite growth in cultures of E14 VM cells. We demonstrate here that a single bolus injection of VEGF165 into the adult rat striatum significantly increases the amount of vasculature in the vicinity of the injection site in a delayed and transient manner when compared to saline controls. Transplanting solid E14 VM grafts into the VEGF165-pretreated striatum resulted in a homogeneous distribution of small blood vessels throughout the graft, a pattern that closely resembles mature adult vasculature. In contrast, grafts in the control condition contained a patchy distribution of heavily dilated vessels. Behavioral measurements indicate that VEGF pretreatment of the intrastriatal graft site accelerates recovery of amphetamine-induced rotational asymmetry in unilateral 6-OHDA lesioned rats. Unexpectedly, however, VEGF pretreatments failed to increase survival of tyrosine hydroxylase-immunoreactive (THir) neurons in the grafts. In contrast to this finding in vivo, adding VEGF165 to glial-reduced E14 rat VM cultures produced a fourfold increase in THir cell survival and a doubling in the length of THir neurites. We conclude that with the proper method of delivery, VEGF165 may prove to be one of several strategies necessary to significantly improve the survival and function of fetal VM tissue grafts.     

Evidence for target-specific nerve fiber outgrowth from subpopulations of grafted dopaminergic neurons: a retrograde tracing study using in oculo and intracranial grafting

Efforts have been made to counteract the symptoms of Parkinson's disease by substituting the loss of dopaminergic neurons with fetal ventral mesencephalic grafts. One of the postulated limiting factors in this treatment is the relatively poor cell survival and limited graft-derived fiber outgrowth. Recent results documenting enhanced survival of grafted dopaminergic neurons showed no positive correlation to enhanced innervation of the striatal target. Therefore this study was undertaken to investigate whether all surviving grafted dopaminergic neurons projected to the striatal target. Hence, fetal ventral mesencephalic tissue was implanted adjacent to mature versus immature striatal tissue using in oculo and intraventricular grafting techniques. In in oculo grafting, fetal ventral mesencephalon was implanted simultaneously with fetal lateral ganglionic eminence (immature striatal target) or to already matured striatal in oculo grafts (mature striatal target). Furthermore, fetal ventral mesencephalon was implanted into the lateral ventricle adjacent to mature dopamine-depleted striatum. The retrograde tracer fluorogold was injected into the striatal portion of the in oculo cografts and into reinnervated areas of the adult brain. Immunohistochemistry revealed that a significantly larger proportion of tyrosine hydroxylase-positive neurons in the ventral mesencephalic graft was innervating in oculo immature striatal tissue, and hence was fluorogold-positive, in comparison with the number of tyrosine hydroxylase-positive neurons innervating mature striatal tissue. Moreover, intracranial transplantations showed that tyrosine hydroxylase-positive neurons were distributed within the grafts in dense clusters of cells. In most clusters tyrosine hydroxylase-positive cells were fluorogold-negative but calbindin-positive. In a few tyrosine hydroxylase-positive cell clusters, neurons were coexpressing fluorogold but were calbindin-negative. In conclusion, significantly more dopamine neurons projected to immature than to mature striatal tissue and thus, a subpopulation of grafted dopaminergic neurons was not projecting into adult striatum. Thus, the results from this study show that further attempts to enhance survival of grafted dopamine neurons in purpose to enhance graft-derived fiber outgrowth and efficacy should also consider different subtypes of dopamine neurons.     

Time Course of Apoptotic Cell Death within Mesencephalic Cell Suspension Grafts: Implications for Improving Grafted Dopamine Neuron Survival

The vast majority (90%) of embryonic mesencephalic dopamine (DA) neurons die following transplantation to the striatum. Recent reports indicate that at least a subpopulation of grafted cells undergo apoptotic cell death at early times following implantation. This study examines the temporal pattern and magnitude of apoptotic cell death following the implantation of mesencephalic cell suspension grafts. Two techniques, a modified terminal deoxynucleotide-mediated nucleotide end labeling (TUNEL) technique and cresyl violet staining, are used to assess apoptotic cell death by detection of its biochemical and morphological identifiers, respectively. Male, Fischer 344 rats were examined at 1, 4, 7, and 28 days following implantation of embryonic day 14 (E14) ventral mesencephalic cells to the DA-denervated striatum. Results indicate that the overwhelming majority of apoptotic cell death occurs within the first 7 days after transplantation. However, the impact of the apoptosis that occurs over the first week following grafting only appears to limit grafted tyrosine hydroxylase-immunoreactive (THir) neuron survival during the first 4 days. No significant differences between the survival rates of THir neurons at 4 days after grafting and at 28 days after grafting were found. Therefore, it appears that the critical interval during which an estimated 90% of grafted DA neurons die is during the first 4 days postimplantation and that a major contributor to this cell death is apoptosis.     

Behavioral and electrophysiological correlates of human mesencephalic dopaminergic xenograft function in the rat striatum

While human fetal xenografts placed into immunocompromised animal hosts have been shown to survive and grow, their ability to function and influence the host tissue has not been fully examined. Therefore, we implanted grafts of human fetal mesencephalic tissue intracranially into rats with unilateral 6-hydroxydopamine lesions of their nigrostriatal dopaminergic innervation and tested the rats behaviorally for reductions in apomorphine-induced rotations. The purpose of this study was to test the ability of these grafts to provide a functional reinnervation by comparing the behavioral changes with the morphology and presence of electrophysiologically active dopaminergic neurons within the graft and with firing rates of host striatal neurons. Adult Sprague-Dawley rats that had been unilaterally lesioned and that showed a stable two peak pattern of apomorphine-induced rotations received grafts of human fetal mesencephalic tissue placed directly into the lesioned striatum. These rats were then further tested each month for five months for reductions in their turning behavior. At 5 to 6 months postgrafting, electrophysiological recordings were made of cells within the graft and within the host striatum. The rats were then examined immunohistochemically to evaluate graft survival and extent of reinnervation of the host tissue. The rats receiving mesencephalic dopaminergic grafts demonstrated a 79% reduction in their apomorphine-induced rotations. Electrophysiological recordings revealed spontaneously active dopaminergic neurons within the graft as well as host striatal cell firing rates consistent with those of dopamine-innervated cells. Furthermore, immunohistochemical studies confirmed graft survival and revealed marked fiber outgrowth from the graft into and throughout the striatum. Taken together these findings provide evidence that grafts of human fetal mesencephalic tissue are able to produce behavioral improvements in lesioned animals which are associated with the presence of dopaminergic neurons within the graft and are consistent with normal host striatal cell activity levels.     

Tirilazad Mesylate Improves Survival of Rat and Human Embryonic Mesencephalic Neuronsin 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 thein vitrocell viability of embryonic rat and human mesencephalic tissue, treated and dissociated in the same way as in clinical trials. After 7 daysin vitro,the number of tyrosine hydroxylase-immunopositive, presumed DA neurons was 140% higher in rat cultures treated with 0.3 μMtirilazad 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 neuronsin 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.