Carla Martinelli

Quantitative changes in mitochondria of spinal ganglion neurons in aged rabbits

Within the context of our research on the age-related structural changes in spinal ganglia, we studied the mitochondria of the neuronal perikaryon in the spinal ganglia of 12-, 42-, and 79-month-old rabbits. Both the volume of the perikaryon and the total mitochondrial mass within the perikaryon increased significantly passing from young adult to old animals. Hence, there is no net loss of mitochondria in these neurons with age. Since, however, the volume of the perikaryon increased by more than 63% while the total mitochondrial mass within the perikaryon increased by only 18%, the mean percentage of perikaryal volume occupied by mitochondria decreased with age. This decrease is only in very minor part a consequence of lipofuscin accumulation, so that the ratio between the total mitochondrial mass and the functionally active volume of cytoplasm decreased with age. Possible causes of this decrease are discussed briefly. Moreover, while the mitochondrial structure did not change, mitochondrial size increased with age. Finally, in each of the three age groups both the mean percentage volume of mitochondria and the mean mitochondrial size were very similar in large light and in small dark neurons.

Age-related quantitative changes in mitochondria of satellite cell sheaths enveloping spinal ganglion neurons in the rabbit

We studied mitochondria in the satellite cell sheaths which envelope the spinal ganglion neurons of rabbits aged 12, 42, and 79 months. While the mean cytoplasmic volume of satellite cell sheaths did not change significantly with age, the mean percentage of cytoplasmic volume occupied by mitochondria decreased with age. This decrease is mainly due to a reduction in the total mitochondrial mass and only in minor part is a consequence of lipofuscin accumulation. Mitochondrial structure did not change, while mitochondrial size increased with age. Comparison between mitochondria in nerve cell bodies and those in satellite cell sheaths showed that: (1) the mean percentage of cytoplasmic volume occupied by mitochondria was greater in nerve cell bodies than satellite cell sheaths and the ratio between these two percentages remained constant with advancing age; (2) the total mitochondrial mass was much greater in nerve cell bodies than satellite cell sheaths and the ratio between these two values increased with age; (3) the extent of increase of mitochondrial size with age was similar in nerve cell bodies and satellite cell sheaths. The results of the present study suggest that: (1) the ability of satellite cell sheaths to produce energy decreases with age; (2) the decreased ability of sensory neurons in old animals to meet high energy demands may be partly due to the diminished contribution of their associated satellite cell sheaths.

Autophagy in Normal and Abnormal Early Human Pregnancies

Autophagy is an inducible catabolic process by which cells degrade and recycle materials to survive stress, starvation, and hypoxia. The aim of this study was to evaluate autophagy at the fetal–maternal interface, to assess autophagy involvement during the early phase of human gestation, and to explore autophagic modification in case of early abnormal pregnancy outcome. Specimens were collected from first-trimester normal gestations undergoing legal termination of pregnancy and first-trimester sporadic spontaneous miscarriages. Autophagy was studied in villous and decidual samples by transmission electron microscopy, immunohistochemistry, immunofluorescence, and Western blotting. Autophagy markers were found in cytotrophoblast, syncytiotrophoblast, extravillous trophoblast, and decidual stromal cells. Autophagy is physiologically involved in early normal gestation. Compared with normal pregnancy, spontaneous miscarriage presents an increase in autophagy expression in villous specimens due to an increment in concentration of autophagic vacuole in syncytiotrophoblast, suggesting a cytoprotective mechanism of the cells to respond to microenvironmental challenge.

Increase in number of the gap junctions between satellite neuroglial cells during lifetime: An ultrastructural study in rabbit spinal ganglia from youth to extremely advanced age

This study investigated quantitative aspects of the gap junctions between satellite neuroglial cells that envelope the spinal ganglion neurons in rabbits aged 1 year (young), 3.6 years (adult), 6.7 years (old), and 8.8 years (very old). Both the total number of gap junctions present in 30,000 microm2 of surface area occupied by perineuronal satellite cells, and the density of these junctions increased throughout life, including the extremely advanced age. By contrast, the mean length of individual gap junctions did not change with age. Thus, the junctional system which provides morphological support for the metabolic cooperation between satellite cells in rabbit spinal ganglia becomes more extensive as the age of the animal increases. These results support the hypothesis that the gap junctions between perineuronal satellite cells are involved in the spatial buffering of extracellular K+ and in neuroprotection.

Mitochondria in Perineuronal Satellite Cell Sheaths of Rabbit Spinal Ganglia: Quantitative Changes during Life

We studied quantitative changes in mitochondria of perineuronal satellite cell sheaths (SCSs) of rabbit spinal ganglia from young to extremely advanced age (1, 3.6, 6.7 and 8.8 years). The mitochondrial structure did not differ in the four age groups, while mitochondrial size increased progressively and significantly with age. The mean percentage of cytoplasmic volume occupied by mitochondria decreased progressively and significantly from young to old animals. This decrease was mainly due to a progressive and significant reduction in the total mitochondrial volume. Lipofuscin accumulation had a negligible influence on this reduction. These results suggest that the ability of SCSs to produce energy decreases with age and that the reduced ability of spinal ganglion neurons to respond to high energy demands in old age may be in part due to the diminished contribution of perineuronal satellite cells.

The perineuronal glial tissue of spinal ganglia. Quantitative changes in the rabbit from youth to extremely advanced age

The volumes of the nerve cell bodies and those of the enveloping satellite cell sheaths from spinal ganglia were determined by morphometric methods applied to electron micrographs in young, adult, old and very old rabbits. The mean volume of the nerve cell bodies increased progressively with age; this is probably related to the increase with age of the body size of the rabbits studied. The mean volume of the satellite cell sheaths did not differ significantly in young, adult and old animals, but was significantly smaller in very old animals. It is extremely unlikely that this marked reduction in the volume of the satellite cell sheath is the result of a pathological process. The mean value of the volume ratio between the satellite cell sheaths and the related nerve cell bodies did not differ significantly in young and adult animals, but was significantly smaller in old and very old animals. This ratio was particularly low in very old animals. Our analysis showed that in each age group the volume of the satellite cell sheath is linearly related to the volume of the related nerve cell body. This result suggests that in rabbit spinal ganglia the quantitative relations between glial and nervous tissue are tightly controlled throughout life. It is suggested that ganglionic neurons release signals to influence and control the volume of their associated glial tissue. Since satellite cells have important support roles for the neurons they surround, it is likely that the marked reduction in the volume of perineuronal sheaths in the extremely advanced age is accompanied by a reduction of those roles, with negative consequences for neuronal activity.

Tendon structure and extracellular matrix components are affected by spasticity in cerebral palsy patients

We studied the effect of spasticity-induced overload on tendons from the gracilis and semitendinosus muscles from cerebral palsy (CP) and healthy subjects (CT) stained with haematoxylineosin, Sirius red and Alcian blue. Vascularity was also characterized using an anti-CD34 antibody. Light microscopy analysis of haematoxylin-eosin stained sections revealed that the overall structure of tendons was maintained, characterized by parallel and slightly wavy collagen fibers in both CT and CP tendons. However, hypercellularity, cell rounding, increased vascularity and lipoid degeneration were observed in CP samples. Sirius red stained collagen fibers were more evident in CP tendons, suggesting an increased collagen content induced by spasticity. Alcian blue staining revealed an overall increase of glycosaminoglycans in CP tendons as observed in tendinopathy. Our results suggest that CP-induced spasticity may be considered as a chronic, persisting and repetitive loading of tendons, inducing ECM remodeling as adaptive response to increased functional demand. At the same time, the evidence of some tendinopathic-like markers in CP tendons suggests that the chronic nature of the CP condition could represent a pathologic condition, possibly leading to a transient weakness of the tissue making it more susceptible to damage from cumulative loading until an overt tendinopathy develops.

Malignant phenotype of renal cell carcinoma cells is switched by Ukrain administration in vitro

We investigated whether Ukrain modulates the malignant phenotype of clear cell renal cell carcinoma (ccRCC) cells Caki-1, Caki-2, and ACHN treated with four doses (5, 10, 20, and 40 &mgr;mol/l) for 24 and 48 h. The epithelial-to-mesenchymal transition markers E-cadherin, &bgr;-catenin, and vimentin were analyzed by immunofluorescence as well as actin and tubulin; matrix metalloproteinase-2 and matrix metalloproteinase-9 activity was analyzed by SDS-zymography, intracellular and secreted SPARC levels by western blot, and cell cycle by flow cytometry. Ukrain did not induce E-cadherin/&bgr;-catenin immunoreactivity at the cell–cell boundary, although it determined the actin cortical expression in Caki-2 and ACHN, and did not affect vimentin organization; however, in some Caki-1 and ACHN cells the perinuclear concentration of vimentin was consistent with its downregulation. Matrix metalloproteinase-2 and matrix metalloproteinase-9 activity was significantly downregulated 48 h after 20 &mgr;mol/l Ukrain administration. At this time point, Ukrain significantly decreased migration and invasion, and downregulated SPARC levels in cell supernatants at all doses in Caki-2, and at 20 &mgr;mol/l in Caki-1 and ACHN cells. Concomitantly, SPARC was upregulated in all ccRCC cells, suggesting that Ukrain could also affect cell proliferation by cell cycle inhibition, as supported by the cell cycle analysis, as SPARC also acts as a cell cycle inhibitor. Our results suggest that Ukrain may switch the epithelial-to-mesenchymal transition-related phenotype of ccRCC cells, and targets the two major aspects involved in RCC progression, such as tumor invasion/microenvironment remodeling and cell proliferation.

Pancreatic cancer cells retain the epithelial-related phenotype and modify mitotic spindle microtubules after the administration of ukrain in vitro

The aim of this study is to characterize the phenotype of pancreatic ductal adenocarcinoma (PDAC) cells in relation to the expression of epithelial-to-mesenchymal transition (EMT) markers and determine whether ukrain, an anticancer drug based on the alkaloids extracted from greater celandine, modulates in vitro the malignant behavior of PDAC cells in order to extend our understanding of its therapeutic potential. Three cell lines (HPAF-II, HPAC, and PL45) were treated with ukrain (5, 10, and 20 &mgr;mol/l) for 48 h or left untreated (control). Cell proliferation was assessed by growth curves. Apoptosis was determined by Hoechst nuclear staining and by cytochrome c and caspase-8 expressions. The EMT markers E-cadherin, &bgr;-catenin, and vimentin, as well as actin and tubulin cytoskeletons, were analyzed by immunofluorescence. Interphase and mitotic microtubules as well as abnormal mitotic figures were studied by fluorescence microscopy after tubulin immunolabeling. Ukrain strongly suppressed cell proliferation and induced apoptosis possibly through an extrinsic pathway as cytochrome c immunoreactivity suggested that the integrity of the mitochondria was not affected. Tubulin expression indicated an antiproliferative effect of ukrain on the basis of alterations in mitotic spindle microtubule dynamics, leading to abnormal mitosis. Membranous E-cadherin/&bgr;-catenin immunoreactivity was similarly expressed in control-treated and ukrain-treated cells, although the drug upregulated E-cadherin in cell lysates. Our results suggest that ukrain exerts its chemotherapeutic action on PDAC cells targeting mitotic spindle microtubules, leading to abnormal mitosis and apoptosis, and favoring cell cohesiveness. The differentiated epithelial phenotype of HPAF-II, HPAC, and PL45 cell lines concomitant with a highly invasive potential suggests that further experiments will be necessary to definitively clarify the role of EMT in PDAC progression.

Influenza del microambiente perineuronale sullo svüuppo délie propaggini del corpo delle cellule nervose dei gangli spinali

Per stabilire se lo svuuppo delle sottili propaggini che emergono dal corpo dei neuroni dei gangli spinali e controllato esclusivamente da fattori intrinseci al neurone o e influenzato anche dall’ambiente circostante, abbiamo approfittato di un particolare, seppur raro, tipo di organizzazione di tali neuroni. Mentre di solito, negli animali adulti, il corpo di ciascun neurone dei gangli spinali e awolto dal proprio involucro di cellule satelliti, a volte si trovano coppie di corpi neuronali che sono a diretto contatto reciproco per una porzione piu o meno estesa della loro superficie e che sono a contatto con 1e cellule satelliti per 1a parte rimanente della loro superficie. Nel medesimo corpo neuronale noi abbiamo potuto pertan-to confrontare lo svuuppo delle propaggini in porzioni della superficie che si trovano sotto il controllo dei medesimi fattori intrinseci, ma in rapporto con microambienti differenti. Con uno studio quantitativo con-dotto, in ratti adulti, su 104 corpi neuronali organizzati in coppie, abbiamo stabilito che lo sviluppo com-plessivo delle propaggini e significativamente maggiore nelle porzioni di superficie situate a contatto con le cellule satelliti che in quelle situate a contatto con un altro neurone. I risultati ottenuti fanno ritenere che lo svuuppo delle propaggini del corpo dei neuroni dei gangli spinali sia influenzato da fattori ambientali. Ulte-riori studi saranno necessari per l’identificazione di tau fattori.To investigate whether the outgrowth of the slender projections arising from the Perikaryon of spinal ganglion neurons is influenced by environmental factors or is solely controlled by factors intrinsic to the neuron, we have examined nerve cell bodies arranged in pairs. These nerve cell bodies, which can occasionally be found in spinal ganglia, are in immediate contact with each other along more or less extensive portions of their surfaces, while along the remaining portions of their surfaces they contact satellite cells. For each nerve cell body we have, therefore, quantitatively compared the extent of the projections on portions of the surface which are under the control of the same intrinsic factors, but are in contact with different microenvironments. By means of a quantitative study carried out on 104 paired neurons in the spinal ganglia of 3 adult rats, we have found that the overall development of the perikaryal projections is significantly greater in those portions of the surface in contact with satellite cells than in the portions in contact with another neuron. This result suggests that the outgrowth of perikaryal projections is influenced by environmental factors. Further investigations are required to characterize these factors.RiassuntoPer stabilire se lo svüuppo delle sottili propaggini che emergono dal corpo dei neuroni dei gangli spinali è controllato esclusivamente da fattori intrinseci al neurone o è influenzato anche dall’ambiente circostante, abbiamo approfittato di un particolare, seppur raro, tipo di organizzazione di tali neuroni. Mentre di solito, negli animali adulti, il corpo di ciascun neurone dei gangli spinali è awolto dal proprio involucro di cellule satelliti, a volte si trovano coppie di corpi neuronali che sono a diretto contatto reciproco per una porzione più o meno estesa della loro superficie e che sono a contatto con 1e cellule satelliti per 1a parte rimanente della loro superficie. Nel medesimo corpo neuronale noi abbiamo potuto pertan-to confrontare lo svüuppo delle propaggini in porzioni della superficie che si trovano sotto il controllo dei medesimi fattori intrinseci, ma in rapporto con microambienti differenti. Con uno studio quantitativo con-dotto, in ratti adulti, su 104 corpi neuronali organizzati in coppie, abbiamo stabilito che lo sviluppo com-plessivo delle propaggini è significativamente maggiore nelle porzioni di superficie situate a contatto con le cellule satelliti che in quelle situate a contatto con un altro neurone. I risultati ottenuti fanno ritenere che lo svüuppo delle propaggini del corpo dei neuroni dei gangli spinali sia influenzato da fattori ambientali. Ulte-riori studi saranno necessari per l’identificazione di tau fattori.