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Increasing evidence suggests a pivotal role for neuroinflammation in the pathogenesis of Parkinson disease, but whether activated microglia participate in disease progression remains unclear. To clarify this issue, we determined the numbers of activated microglial cells in the substantia nigra pars compacta and ventral tegmental area of monkeys subacutely and chronically exposed to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Monkeys in the subacute MPTP treatment group were killed 1 week after the last MPTP injection; chronically treated monkeys were killed either 6 or 35 months after the last MPTP injection. Subacute MPTP administration induced loss of dopaminergic neurons in the substantia nigra pars compacta and ventral tegmental area and microglial activation in the same areas. Chronic MPTP treatment resulted in greater dopaminergic neuron depletion in both treatment groups. Both groups of chronic MPTP-treated monkeys showed increased numbers of activated microglial cells in the substantia nigra pars compacta that were similar to those of the subacute MPTP treatment group. These results indicate that microglial activation seems to be induced mainly by the toxic effects of MPTP and that it does not further progress once the toxin administration has been terminated. This suggests that the progressive degeneration of nigral cells in Parkinson disease may not necessarily be associated with progressively increased microglial activation.

Recent studies indicate that carotid body (CB) could be a suitable cell source for cell therapy in Parkinson's disease. We have isolated and successfully expanded in culture as monolayer adult CB-derived cells using a modification of the culture medium employed for bone marrow multipotent adult progenitor cells (MAPCs). These cells express variable amounts of tyrosine hydroxylase (TH), beta-III tubulin and Sox2. In addition, CB-derived cells showed high expression of Sox2 related to a high rate of proliferation and consistent with an undifferentiated state. Under culture conditions that reduced cell proliferation, Sox2 expression decreased while TH and beta-III tubulin expression was increased. This could indicate that the differentiation of some cells occurs in the culture, thus accounting for a certain neural differentiation potential of CB-derived cells.

To elucidate the role of the prostaglandin synthase cyclooxygenase-2 (Cox-2) and the mechanisms of dopaminergic (DA) neurodegeneration, monkeys were injected subacutely or chronically (n = 5/group) with 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine. Chronically treated animals developed parkinsonian signs and were killed 6 months after the last treatment; tyrosine hydroxylase-expressing neurons decreased in all substantia nigra (SN) cell groups in both treatment groups. In untreated controls (n = 3), there was low Cox-2 expression in ventral SN DA neurons and high expression in ventral tegmental area neurons. In subacutely treated monkeys, Cox-2 expression increased in surviving DA cells, particularly in the ventrolateral SN. In chronically treated monkeys, enhanced Cox-2 expression appeared only in surviving ventral tegmental area and ventral SN neurons. Thus increased Cox-2 did not persist in other SN neurons after discontinuing 1-methyl-4-phenyl-1,2,36-tetrahydropyridine. Some DA neurons in treated but not control monkeys expressed the active nuclear form of phospho-c-Jun, but not the active form of nuclear factor-κB. We conclude that Cox-2 expression does not confer vulnerability to neurodegeneration in DA neurons and that it is unlikely that a subacute insult to DA neurons can perpetuate degeneration through Cox-2 activation. Other mechanisms, probably through the Jun N-terminal kinase cascade, lead to DA cell death in this model. © 2008 by the American Association of Neuropathologists, Inc.

Progenitor cells generated in the subventricular zone (SVZ) migrate toward the olfactory bulb (OB), where they differentiate into neurons. Growth factors have been shown to promote neurogenesis in the SVZ/OB-system while dopaminergic lesion exerts an opposite effect. As carotid body (CB) cells express growth factors here we study the impact of intrastriatal CB graft on migration and differentiation of neural progenitor cells in the hemiparkinsonian rat SVZ/OB-system. Bromodeoxyuridine (BrdU) was given to intact, 6-hydroxydopamine (6-OHDA)-lesioned and 6-OHDA-lesioned animals transplanted with vehicle or rat CB cells. The migration of progenitor cells was assessed by the quantification of BrdU-labeled cells in the SVZ/OB-system and the neuronal differentiation by the proportion of newborn neurons in the OB. The graft survival was confirmed by CB cell morphology and their tyrosine hydroxylase expression. Some of these CB cells were stained with BrdU, thus indicating their ability for self-renewal. Grafted glomus cells also expressed brain derived neurotrophic factor (BDNF), glial derived neurotrophic factor (GDNF), epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF). The migration of neural progenitor cells was significantly decreased in 6-OHDA-lesioned respect to intact animals. We found a similar number of BrdU-labeled cells in sham-operated than in CB-grafted animals, suggesting that CB graft has no effect on progenitor cell migration. CB-grafted animals exhibited a significantly larger percentage of newborn cells (BrdU/Neuronal Nuclei-labeled cells) respect to 6-OHDA-lesioned and sham-operated animals. This study suggests that striatal CB graft might promote differentiation of SVZ progenitor cells into neurons, probably by the growth factors contained in CB cells.

We studied the histochemical phenotype of carotid body (CB) cells in the adult rat. In addition to tyrosine hydroxylase (TH), type I cells expressed numerous growth factors such as glial cell line-derived neurotrophic factor (GDNF), basic fibroblast growth factor (bFGF), brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), insulin-like growth factor-I (IGF-I), epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha), as well as the receptors p75, Ret, epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor-alpha (PDGFR-alpha). Type II cells expressed the glial fibrillary acid protein (GFAP), vimentin, the trophic factor bFGF and receptors p75, EGFR and PDGFR-alpha. Both types I and II cells exhibited a positive immunoreaction to markers of neural progenitor cells such as the polysialylated form of the neural cell adhesion molecule (PSA-NCAM) and nestin, respectively, suggesting that CB contain some immature cells even at the adult stage. The possibility that these cells can be expanded and differentiated into mature neurons should be explored.

Excitotoxicity has been suggested to play a pivotal role in the pathogenesis of Parkinson disease (PD). As subthalamic nucleus (STN) neurons express glutamate and are overactivated in parkinsonism, it seems that in PD dopaminergic (DA) neurons are under the influence of abnormally high levels of glutamate and consequently might be more vulnerable to neurodegeneration. To determine the contribution of the overactivated STN-SN pathway to the progression of PD, we studied the effect of prior unilateral STN lesion on the toxicity induced by subsequent administration of 1-methyl-4-phenyl-1,2,3,6, tetrahydropyridine (MPTP) to non-human primates. In animals from group 1, kainic-induced lesion of the STN was performed prior to the administration of MPTP whereas in animals from group 2, STN lesion was caused after animals had been chronically treated with MPTP. The lesion of the STN elicited a contralateral hemiballism in animals from group 1, and they developed an asymmetrical parkinsonism after being exposed to MPTP. The STN lesion produced an improvement in the contralateral parkinsonism and mild choreic movements in animals from group 2. Cell counting of tyrosine hydroxylase immunoreactive (TH-ir) cells was performed by stereology and showed a similar loss of TH-ir cells (approximately 85%) in the ipsilateral and contralateral SN to the lesioned STN. These data indicate that the surgical removal of the excitatory drive from the STN to SN neurons does not protect dopaminergic neurons against a chronic and extended toxic effect of MPTP and do not support the assumption that STN blockade might delay the progression of PD.

Recent publications have shown that the lateral wall of the lateral ventricles in the Macaca fascicularis brain, in particular the subventricular zone (SVZ), contains neural stem cells throughout adulthood that migrate through a migratory pathway (RMS) to the olfactory bulb (OB). To date, a detailed and systematic cytoarchitectural and ultrastructural study of the monkey SVZ and RMS has not been done. We found that the organization of the SVZ was similar to that of humans, with the ependymal layer surrounding the lateral ventricles, a hypocellular GAP layer formed by astrocytic and ependymal expansions, and the astrocyte ribbon, composed of astrocytic bodies. We found no cells corresponding to the type C proliferating precursor of the rodent brain. Instead, proliferating cells, expressed as Ki-67 immunoreactivity, were predominantly young neurons concentrated in the anterior regions, and occasional astrocytes of the ribbon. We observed displaced ependymal cells of still unknown significance. New neurons tended to organize in chain-like structures, which were surrounded by astrocytes. This pattern was highly reminiscent of that observed in rodent RMS, but not in humans. These chains spread from the frontal SVZ along a GAP-like layer, uniquely composed of astrocytic expansions, to the olfactory bulb (OB). The number of neuronal chains and the number of chain-forming cells decreased gradually upon reaching the OB. The purpose of this work is to provide a reference for future studies in the field of adult neurogenesis that may lead to an understanding of the fate and functionality of newborn neurons in primates, and ultimately in humans. J. Comp. Neurol. 514:533–554, 2009. © 2009 Wiley-Liss, Inc.

We assessed the presence of degenerating neurons in the substantia nigra pars compacta (SNpc) and ventral tegmental area (VTA) of parkinsonian monkeys. For this purpose, we used two histological markers of cellular death, Fluoro Jade B (FJB) staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL). Eight monkeys were subacutelly treated with four to six 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) injections (1–1.5 mg/kg, cumulative dose) and sacrificed 1 week and 11 months after last MPTP injection. Eight additional monkeys were chronically exposed to MPTP (4.5–15.3 mg/kg, cumulative dose) and sacrificed 6–35 months after last MPTP dose. Three intact monkeys served as controls. The number of tyrosine hydroxylase (TH)- and TUNEL-positive cells was quantified in SNpc and VTA and colocalization of FJB-positive and TUNEL-positive cells with neuronal (TH, NeuN, MAP2) and glial markers (human ferritin, GFAP) assessed on doubly labelled tissue sections. Only MPTP monkeys with 1-week survival displayed few doubly FJB-TH-labelled cells. Both groups of subacute MPTP monkeys, but not chronic MPTP monkeys, showed a significant increased number of TUNEL-positive cells in SNpc. TUNEL-positive cells exhibited morphological features and histological markers indicative of glial cells, whereas TUNEL/NeuN or TUNEL/MAP-2 colocalization was not observed. Our results indicate that MPTP treatment produced a nonapoptotic cell death of dopaminergic cells and the activation of the apoptotic cascade in glial cells. More importantly, we failed to demonstrate the existence of a delayed neurodegenerative process in the dopaminergic neurons after concluding MPTP injection thus, casting doubt on the validity of the “progressive model” created by repeated MPTP administration to monkeys. © 2008 Wiley-Liss, Inc.

We investigated the impact of the nigrostriatal lesion on the olfactory tyrosine hydroxylase-immunoreactive (TH-ir) cells in monkeys. The majority of these TH-ir cells appeared in the glomerular layer of the olfactory bulb and many were immature but functional dopaminergic neurons. In parkinsonian monkeys the number of olfactory dopaminergic neurons increased up to 100% as compared to controls, but their phenotype did not change. This increased TH-ir cell population might be a direct consequence of the nigral cell loss and contribute to the hyposmia reported by Parkinson's disease patients.

In non-human primates, striatal tyrosine hydroxylase-immunoreactive (TH-ir) cells are increased in number after dopamine depletion and in response to trophic factor delivery. As carotid body cells contain the dopaminotrophic glial cell line-derived neurotrophic factor (GDNF), we evaluated the number, morphology and neurochemistry of these TH-ir cells, in the anterior and posterior striatum of five monkeys treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) which received a graft of carotid body cell aggregates (CBCA) (n = 3) or sham surgery (n = 2), and six MPTP-monkeys that were sacrificed 6 months and 3 years after the last MPTP dose [MPTP I (n = 3) and MPTP II (n = 3), respectively]. Three intact monkeys served as controls. A disability rating scale was used for the assessment of parkinsonism in all lesioned animals, both before and after surgery. For the neurochemical examination, tissue sections were double-labelled with antibodies to TH, dopamine transporter, dopa decarboxylase-67, vesicular monoamine transporter 2, glutamic acid decarboxylase -67, calbindin, parvalbumin, calretinin, neuronal nitric oxide synthase and GDNF. Only animals receiving CBCA graft showed a moderate but significant recovery of parkinsonism that persisted 12 months after the graft. The grafted striatum contained the greatest TH-ir cell density (120.4 +/- 10.3 cells/100 mm2), while the control striatum displayed the lowest (15.4 +/- 6.8 cells/100 mm2), and MPTP I, MPTP II and sham-operated monkeys showed a similar intermediate value (66.1 +/- 6.2, 58.3 +/- 17.2 and 57.7 +/- 7.0 cells/100 mm2, respectively). In addition, in the post-commissural striatum, only CBCA graft induced a significant increase in the TH-ir cell density compared to control animals (47.9 +/- 15.9 and 7.9 +/- 3.2, respectively). Phenotypically, TH-ir cells were striatal dopaminergic interneurons. However, in the grafted animals, the phenotype was different from that in control, MPTP and sham-operated monkeys, with the appearance of TH/GDNF-ir cells and the emergence of two TH-ir subpopulations of different size as the two main differentiating features. Our data confirm and extend previous studies demonstrating that striatal CBCA grafts produce a long-lasting motor recovery of MPTP-monkeys along with an increase in the number and phenotype changes of the striatal TH-ir interneurons, probably by the action of the trophic factors contained in carotid body cells. The increased number of striatal TH-ir cells observed in the grafted striatum may contribute to the improvement of parkinsonism observed after the graft.

Adrenomedullin (AM) immunoreactivity has been found in granules of the glomus (type I) cells of the carotid bodies in rats. The identity of these cells was ascertained by colocalization of immunoreactivities for AM and tyrosine hydroxylase in their cytoplasm. Exposure of freshly isolated carotid bodies to synthetic AM resulted in a concentration- and time-dependent degranulation of glomus cells as measured by dopamine (DA) release. DA release reached a zenith 30 min after exposure to AM (94.2% over untreated controls). At this time-point, the response to AM was similar to the one elicited by 5 min of exposure to 100 mM K+. Nevertheless, injection of 1 micro l 60 nM AM/g body weight into the tail vein of the rats did not induce statistical differences in DA release from the carotid bodies. Exposure of the oxygen-sensitive cell line PC-12 to hypoxia elicited an increase in AM mRNA expression and peptide secretion into serum-free conditioned medium. Previous data have shown that elevation of AM expression under hypoxia is mediated through hypoxia-inducible factor-1, and that exposure of chromaffin cells to AM results in degranulation. All these data suggest that AM is an important autocrine regulator of carotid body function.