Glial influence on nerve fiber formation from rat ventral mesencephalic organotypic tissue cultures

Rat fetal ventral mesencephalic organotypic cultures have demonstrated two morphologically different dopamine nerve fiber growth patterns, in which the initial nerve fibers are formed in the absence of astrocytes and the second wave is guided by astrocytes. In this study, the presence of subpopulations of dopamine neurons, other neuronal populations, and glial cells was determined. We used "roller-drum" organotypic cultures, and the results revealed that beta-tubulin-positive/tyrosine hydroxylase (TH)-negative nerve fibers were present as early as 1 day in vitro (DIV). A similar growth pattern produced by TH-positive neurons was present from 2 DIV. These neurites grew to reach distances over 4 mm and over time appeared to be degenerating. Thin, vimentin-positive processes were found among these nerve fibers. As the first growth was retracted, a second outgrowth was initiated and formed on migrating astrocytes. TH- and aldehyde dehydrogenase-1 (ALDH1)-positive nerve fibers formed both the nonglia-associated and the glia-associated outgrowth. In cultures with membrane inserts, only the glia-associated outgrowth was found. Vimentin-positive cells preceded migration of NG2-positive oligodendrocytes and Iba-1-positive microglia. Oligodendrocytes appeared not to be involved in guiding neuritic growth, but microglia was absent over areas dense with TH-positive neurons. In conclusion, in "roller-drum" cultures, nerve fibers are generally formed in two sequences. The early-formed nerve fibers grow in the presence of thin, vimentin-positive processes. The second nerve fiber outgrowth is formed on astroglia, with no correlation to the presence of oligodendrocytes or microglia. ALDH1-positive nerve fibers, presumably derived from A9 dopamine neurons, participate in formation of both sequences of outgrowth.     

MPP+ selectively affects calcium homeostasis in mesencephalic cell cultures from embryonal C57/B16 mice

Summary 1-Methyl-4-phenylpyridinium (MPP⁺), the active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) serves as a valuable tool in animal models of Parkinson's disease. Primary cell cultures of mesencephalon from C57/B16 mice were used to investigate the effects of various dopaminergic neurotoxins on the intracellular calcium metabolism. MPP⁺ was compared to its precursor MPTP and a structural analogue paraquat (methylviologen). Direct addition of these neurotoxins (10 M) to fura-2-labeled cells did not change intracellular calcium concentrations in the presence of 1 mM extracellular calcium. When mesencephalic neurons were exposed to the compounds for 24 hours, only MPP⁺ led to an increase in calcium concentration in the absence and presence of extracellular calcium (36%, p<0.05 and 47%, p<0.01 versus control group). Intracellular calcium concentrations in cortical cultures devoid of dopaminergic cells were not changed by the above neurotoxins. Thus MPP⁺ is shown to selectively increase intracellular calcium concentrations in mesencephalic cultures.     

小胎龄鼠的腹侧中脑神经前体细胞体外培养研究

目的体外培养小胎龄的胎鼠腹侧中脑神经前体细胞,根据多巴胺能神经元产量,确定较好的研究对象,为细胞移植治疗帕金森病奠定基础。方法取不同胎龄胎鼠(E10、E11、E12)的腹侧中脑神经前体细胞,体外加碱性成纤维细胞生长因子扩增7d后用含AA-2P的分化增养液培养7 d,行免疫荧光染色比较多巴胺能神经元形成情况。结果E10、E11、E12细胞经培养后细胞总数分别增加76.39倍、59.67倍、51.85倍。多巴胺能神经元占所有细胞的比例在E11、E10、E12依次显著降低。对于多巴胺能神经元占所有神经元的比例,E11显著高于E10和E12。结论来自胎龄11 d的胎鼠腹侧中脑神经前体细胞是较好的体外培养对象,可为细胞移植研究提供较为充足的细胞资源。     

Neuronal protective and rescue effects of deprenyl against MPP+ dopaminergic toxicity

Summary Intranigral infusion of 1-Methyl-4-phenylpyridinium ion (MPP⁺, 2.1–16.8 nmol) dose-dependently injured nigral neurons as reflected by reduced dopamine levels in the ipsilateral striatum four days after the infusion of this toxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Coadministration of deprenyl (4.2 nmol) with MPP⁺ into the substantia nigra protected against MPP⁺-induced moderate (20–50%) but not severe (over 70%) nigral injury as reflected in striatal dopamine reductions. However, supplementary treatment with deprenyl (0.25 mg/kg, s.c., twice daily for 4 days) after intranigral infusion of MPP⁺ significantly rescued nigral neurons from more severe damage caused by a higher MPP⁺ does (8.4 nmol) manifested by a lesser striatal dopamine decrease (–31%) compared to the non-deprenyl treated group (–70%). Thus, in addition to the blockade of bioactivation of MPTP, deprenyl can protect and/or rescue nigral neurons from MPP⁺-induced dopaminergic neurotoxicity. These in vivo data add further evidence to suggest that deprenyl, a putative and clinically unproven neuroprotective agent, may be of value in slowing the progressive nigral degeneration in early Parkinson's disease, but may prove to be less so in its terminal stages.     

Attenuation of levodopa-induced toxicity in mesencephalic cultures by pramipexole

Summary The direct-acting dopamine (DA) agonist pramipexole (2 amino-4,5,6,7-tetrahydro-6-propyl-amino-benzthiazole-dihydrocfiloride) was evaluated for its ability to attenuate levodopa-induced loss of tyrosine hydroxylase immunoreactive (THir, a marker for dopamine neurons) cells in mesencephalic cultures. Pramipexole reduced levodopa-induced THir cell loss in a dosedependent and saturable fashion (ED₅₀=500 pM), its inactive stereoisomer was significantly less potent in this regard and pergolide and bromocriptine had negligible cytoprotective effects. Culture media from mesencephalic cultures incubated with pramipexole for 6 days increased THir cell counts in freshly harvested recipient cultures. The magnitude of this effect was directly proportional to the amount of pramipexole in the donor cultures and heatinactivation of the media abolished the growth promoting effect. The results from this exploratory set of experiments suggest that pramipexole may be cytoprotective to dopamine neurons in tissue culture. Pramipexole's affinity for DA receptors, its antioxidant action or its ability to enhance mesencephalic trophic activity could be responsible for this effect.     

Comparison of GDF5 and GDNF as neuroprotective factors for postnatal dopamine neurons in ventral mesencephalic cultures

Loss of dopamine neurons is associated with the motor deficits that occur in Parkinson's disease. Although many drugs have proven to be useful in the treatment of the symptoms of this disease, none has been shown to have a significant impact on the development of the disease. However, we believe that several neurotrophic factors have the potential to reduce its progression. Glial cell line‐derived neurotrophic factor (GDNF), a member of the transforming growth factor‐β superfamily of neurotrophic factors, has been extensively studied in this regard. Less attention has been paid to growth/differentiation factor 5 (GDF5), another member of the same superfamily. This study compares GDNF and GDF5 in dissociated cultures prepared from ventral mesencephalon and in organotypic co‐cultures containing substantia nigra, striatum, and neocortex. We report that both GDNF (10–500 ng/ml) and GDF5 (100–500 ng/ml) promoted the survival of dopamine neurons from the substantia nigra of postnatal rats, although GDNF was considerably more potent than GDF5. In contrast, neither factor had any significant effect on the survival of dopamine neurons from the rat ventral tegmental area. Using organotypic co‐cultures, we also compared GDF5 with GDNF as chemoattractants for the innervation of the striatum and the neocortex by dopamine neurons from the substantia nigra. The addition of either GDF5 or GDNF (100–500 ng/ml) caused innervation by dopamine neurons into the cortex as well as the striatum, which did not occur in untreated cultures. Our results are consistent with similar findings suggesting that GDF5, like GDNF, deserves attention as a possible therapeutic intervention for Parkinson's disease. © 2014 Wiley Periodicals, Inc.     

Erythropoietin and GDNF enhance ventral mesencephalic fiber outgrowth and capillary proliferation following neural transplantation in a rodent model of Parkinson's disease

Low dopaminergic cell survival and suboptimal fiber reinnervation are likely major contributing factors for the limited benefits of neural transplantation in Parkinson's disease (PD) patients. Glial cell lined-derived neurotrophic factor (GDNF) has been shown to enhance dopaminergic cell survival and fiber outgrowth of the graft site as well as promote behavioral recovery in rodent models of PD, while erythropoietin (EPO) can produce dopaminergic neuroprotective effects against 6-hydroxydopamine (6-OHDA) exposure on cultured neurons and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mice. The aim of this study was to determine if fetal ventral mesencephalic (FVM) tissue exposed to hibernation media containing a combination of GDNF and EPO could enhance dopaminergic graft survival, striatal reinnervation and functional recovery in a 6-OHDA rodent model of PD. FVM tissue was dissected from 14-day-old rat fetuses and placed for 6 days in hibernation media alone, and in hibernation media that received either a daily administration of GDNF, EPO or a combination of GDNF and EPO. Following hibernation, FVM cells were transplanted as a single cell suspension into the striatum of unilateral 6-OHDA-lesioned rats. Rotational behavioral assessment revealed animals that received FVM tissue exposed to GDNF, EPO or the combination of both drugs had accelerated functional recovery. Immunohistochemical and stereological assessment revealed a significant increase in graft fiber density and angiogenesis into the graft when compared with control. These findings suggest that the hibernation of FVM tissue in a combination of GDNF and EPO can enhance graft efficacy and may have important implications for tissue preparation protocols for clinical neural transplantation in PD.     

Dopamine release in organotypic cultures of foetal mouse mesencephalon: effects of depolarizing agents, pargyline, nomifensine, tetrodotoxin and calcium

Organotypic mesencephalic cultures provide an attractive in vitro alternative to study development of the nigrostriatal system and pathophysiological mechanisms related to Parkinson's disease. However, dopamine (DA) release mechanisms have been poorly characterized in such cultures. We report here endogenous DA release (assessed by high-performance liquid chromatography) in organotypic cultures of foetal mouse (E12) midbrain following single or multiple challenges (1-h incubations) with high K⁺ or veratridine in the presence or absence of pargyline, nomifensine, calcium and/or tetrodotoxin (TTX). Basal (i.e. spontaneous) DA release was only detected in the presence of pargyline and nomifensine (PN), and was highly dependent on calcium and sensitive to TTX. Basal DA release increased 2.4-fold between week 3 (1st DA release experiment) and week 4 in vitro (3rd DA release experiment), DA tissue levels increased 1.6-fold and DA release expressed as a percentage of total DA (medium + tissue contents) increased from 20% to 34% during this growth period in vitro . Co-treatments with high K⁺ or veratridine did not cause major changes in percentages of DA release. Tyrosine hydroxylase activity was increased by high K⁺, but not by the other drug treatments. The acute (single or multiple) treatments with depolarizing agents did not affect the survival of dopaminergic neurons, but chronic low-level veratridine treatments were toxic.     

Striatal expression of GDNF and differential vulnerability of midbrain dopaminergic cells

Glial cell line-derived neurotrophic factor (GDNF) is a member of the transforming growth factor-beta superfamily that when exogenously administrated exerts a potent trophic action on dopaminergic (DA) cells. Although we know a lot about its signalling mechanisms and pharmacological effects, physiological actions of GDNF on the adult brain remain unclear. Here, we have used morphological and molecular techniques, and an experimental model of Parkinson's disease in rats, to investigate whether GDNF constitutively expressed in the adult mesostriatal system plays a neuroprotective role on midbrain DA cells. We found that although all midbrain DA cells express both receptor components of GDNF (GFRalpha1 and Ret), those in the ventral tegmental area (VTA) and rostromedial substantia nigra (SNrm) also contain GDNF but not GDNFmRNA. The levels of GDNFmRNA are significantly higher in the ventral striatum (vSt), the target region of VTA and SNrm cells, than in the dorsal striatum (dSt), the target region of DA cells in the caudoventral substantia nigra (SNcv). After fluoro-gold injection in striatum, VTA and SNrm DA cells show triple labelling for tyrosine hydroxylase, GDNF and fluoro-gold, and after colchicine injection in the lateral ventricle, they become GDNF-immunonegative, suggesting that GDNF in DA somata comes from their striatal target. As DA cells in VTA and SNrm are more resistant than those in SNcv to intracerebroventricular injection of 6-OHDA, as occurs in Parkinson's disease, we can suggest that the fact that they project to vSt, where GDNF expression is significantly higher than in the dSt, is a neuroprotective factor involved in the differential vulnerability of midbrain DA neurons.     

Angiotensin II increases differentiation of dopaminergic neurons from mesencephalic precursors via angiotensin type 2 receptors

In addition to the well-known actions of the humoral renin-angiotensin system, all components of this system are present in many tissues, including the brain, and may play a major role in brain development and differentiation. We investigated the possible effects of angiotensin II on the generation of dopaminergic phenotype neurons from proliferating neurospheres of mesencephalic precursors. We observed immunoreactivity for both angiotensin type 1 and type 2 (AT(1) and AT(2)) receptors in the cell aggregates. Double immunolabeling studies revealed that both receptor types are located in neurons and astrocytes. Interestingly, neurons with a dopaminergic phenotype (i.e. tyrosine hydroxylase activity) showed double labeling for AT(1) and AT(2) receptors although the labeling for AT(2) was more intense. Treatment of the neurospheres with angiotensin II (100 nm) during the differentiation period induced a marked increase (about 400%) in the generation of dopaminergic neurons. This was not affected by treatment with the AT(1) antagonist ZD 7155 but was blocked by treatment with the AT(2) antagonist PD 123319. This suggests that AT(2) receptors mediate the stimulatory effect of angiotensin II on the generation of dopaminergic neurons. Apoptotic cell death studies and bromodeoxyuridine immunohistochemistry indicated that the increase in generation of dopaminergic neurons is not due to increased survival or proliferation of dopaminergic cells during treatment with angiotensin and suggested that angiotensin induces increased differentiation of mesencephalic precursors towards the dopaminergic phenotype. Manipulation of the renin-angiotensin system may be useful for increasing production of dopaminergic neurons for transplantation in Parkinson's disease.     

如何提高帕金森病中脑细胞移植中的细胞存活率？

虽然胚胎中脑细胞移植对帕金森病的疗效已得到广泛证实,但在这项技术广泛应用于临床之前仍有一些问题亟待解决。其中主要是移植物存活率低和宿主纹状体神经支配恢复有限。迄今为止,人们尝试了很多方法来解决这些问题,包括神经营养因子的广泛应用,以及神经和(或)非神经来源组织的联合移植。本文将对目前的胚胎中脑细胞移植术中所用的神经保护手段及其局限性进行简要的介绍。     

Glia-dependent neurotoxicity and neuroprotection in mesencephalic cultures

Dopaminergic neurotoxicities of 6-hydroxydopamine (6-OHDA) and the lipopolysaccharide (LPS) were compared in rat mesencephalic cultures plated on poly-l-lysine or on glial monolayers. In the neuron-enriched cultures plated on polylysine, 6-OHDA killed 89% of the tyrosine hydroxylase (TH)-immunopositive neurons, but LPS was not neurotoxic. Conversely, in mixed neuron/glial cultures, 6-OHDA killed only 27% of the TH-immunopositive neurons while LPS killed 70%. The mixed neuronal/glial mesencephalic culture offers a better in vitro model for studying possible mechanisms involved in Parkinson's disease.     

Shock stress protects mice against amphetamine-induced dopaminergic toxicity

The effect of tail shock (ten, 2.0 mA/0.15 s shocks) on amphetamine-induced dopaminergic toxicity in adult, male BALB/c mice was assessed. Fifteen minutes following a single shock session, mice received amphetamine (50-mg/kg) or saline as follows: Shock/Saline; NoShock/Saline; Shock/Amphetamine; No Shock/Amphetamine. Amphetamine caused a 60% dopamine depletion in the No Shock/Amphetamine group. Tail shock provided neuroprotection against amphetamine-induced dopamine depletion, an effect likely related to the stress response.     

Inhibition of proteoglycan synthesis affects neuronal outgrowth and astrocytic migration in organotypic cultures of fetal ventral mesencephalon

Grafting fetal ventral mesencephalon has been utilized to alleviate the symptoms of Parkinson's disease. One obstacle in using this approach is the limited outgrowth from the transplanted dopamine neurons. Thus, it is important to evaluate factors that promote outgrowth from fetal dopamine neurons. Proteoglycans (PGs) are extracellular matrix molecules that modulate neuritic growth. This study was performed to evaluate the role of PGs in dopamine nerve fiber formation in organotypic slice cultures of fetal ventral mesencephalon. Cultures were treated with the PG synthesis inhibitor methyl-umbelliferyl-beta-D-xyloside (beta-xyloside) and analyzed using antibodies against tyrosine hydroxylase (TH) to visualize dopamine neurons, S100beta to visualize astrocytes, and neurocan to detect PGs. Two growth patterns of TH-positive outgrowth were observed: nerve fibers formed in the presence of astrocytes and nerve fibers formed in the absence of astrocytes. Treatment with beta-xyloside significantly reduced the distance of glial-associated TH-positive nerve fiber outgrowth but did not affect the length of the non-glial-associated nerve fibers. The addition of beta-xyloside shifted the nerve fiber growth pattern from being mostly glial-guided to being non-glial-associated, whereas the total amount of TH protein was not affected. Further, astrocytic migration and proliferation were impaired after beta-xyloside treatment, and levels of non-intact PG increased. beta-Xyloside treatment changed the distribution of neurocan in astrocytes, from being localized in vesicles to being diffusely immunoreactive in the processes. To conclude, inhibition of PG synthesis affects glial-associated TH-positive nerve fiber formation in ventral mesencephalic cultures, which might be an indirect effect of impaired astrocytic migration.     

Diffusible factor(s) from adult rat sciatic nerve increases cell number and neurite outgrowth of cultured embryonic ventral mesencephalic tyrosine hydroxylase-positive neurons

Dissociated embryonic rat ventral mesencephalon containing the developing A8-A10 dopamine (DA) neurons was cultured alone or in the presence of a 10 mm segment of adult rat sciatic nerve that had been explanted and maintained in separate culture for 72 hours prior to introduction to mesencephalic cultures. Nerve segments were contained in a co-culture basket, so that midbrain cells and nerve shared medium but were not in physical contact. The number and morphology of cultured DA neurons was assessed via immunocytochemistry for tyrosine hydroxylase (TH). Co-cultures of ventral midbrain tissue and nerve exhibited an increased number of TH-positive neurons, with larger neuronal perikarya, and increased length and complexity of neurites, than cultures of midbrain tissue alone. Increased number and growth of TH-positive neurons was obtained with as little as 2 days of exposure to nerve. This evidence suggests that a diffusible, soluble factor(s) from sciatic nerve can enhance the number and development of TH-positive neurons detected in cultures of embryonic ventral mesencephalon.     

The lazaroid U-83836E improves the survival of rat embryonic mesencephalic tissue stored at 4°C and subsequently used for cultures or intracerebral transplantation

We assessed the effects of addition of the lazaroid U-83836E to a preservation medium on the survival of rat dopamine neurons stored before culturing or intracerebral transplantation. Embryonic ventral mesencephalic tissue was preserved at 4°C for 8 days with or without the addition of 0.3 μM of U-83836E to a chemically defined “hibernation” medium. Freshly dissected mesencephalic tissue was used in control groups. For culture experiments, the mesencephalic tissue was dissociated and grown in serum-containing medium. Following 24–48 h in vitro, the number of dopamine neurons in cultures derived from tissue hibernated without the lazaroid was 40% of fresh control, compared with 67% of control in cultures prepared from tissue stored in the presence of U-83836E. When mesencephalic tissue was transplanted to the dopamine-depleted striatum of hemiparkinsonian rats following 8 days storage at 4°C in a medium without U-83836E, the mean number of surviving dopamine neurons in the grafts was significantly reduced to 40% of control. In contrast, grafts of tissue which had been hibernated in U-83836E-containing medium contained as many dopamine neurons as transplants of freshly dissected tissue. High yields of surviving grafted dopamine neurons were correlated to a significantly faster onset of functional recovery of amphetamine-induced motor asymmetry. We conclude that the storage period for rat mesencephalic tissue can be prolonged up to 8 days when using lazaroid-supplemented hibernation medium. As lazaroids have undergone clinical safety testing, the application of lazaroids for tissue storage in clinical transplantation trials can be envisaged.     

Dodecylglycerol provides partial protection against glutamate toxicity in neuronal cultures derived from different regions of embryonic rat brain

Primary cultures enriched in neurons dissociated from embryonic rat cerebral cortex, cerebellum, or hippocampus were treated in a chemically defined serum-free media with either vehicle, dodecylglycerol (DDG, 3 μM), or glutamate (75 μM), or preincubated with DDG for 4 or 24 h, and further incubated with glutamate. Their morphological and biochemical assessments (lactate dehydrogenase [LDH] release in the culture media, neuronal viability and intracellular Ca²⁺ mobilization) were made. Neurotoxic effects of glutamate and glutamate-mediated increases in intracellular Ca²⁺ were maximal in neurons from cerebellum and minimal in neurons from cortex. Cotreatment of cells with DDG and glutamate failed to provide significant neuronal protection against glutamate in the three brain regions. Pretreatment of cells with DDG for 4 or 24 h prior to glutamate treatment provided significant neuroprotection as judged by morphological changes and a decrease in LDH activity. Neuroprotection of approximately 15–35% was observed following 4 h of DDG pretreatment, increasing to 60–85% protection after 24 h of DDG pretreatment. Although the mechanism of DDG’s neuroprotective action remains to be elucidated, these results demonstrate that both glutamate and DDG have differential specificity for anatomical regions of the brain.