Patrizia Sartori

Intravenous neural stem cells abolish nociceptive hypersensitivity and trigger nerve regeneration in experimental neuropathy

Summary In experimental neuropathic pain, intravenous neural stem cells induce a rapid remission of hyperalgesia and allodynia, followed by improvement in nerve morphology. Abstract A nonphysiological repair of the lesioned nerve leading to the formation of neurinomas, altered nerve conduction, and spontaneous firing is considered the main cause of the events underlying neuropathic pain. It was investigated whether neural stem cell (NSCs) administration could lead to a physiological nerve repair, thus to a reduction of neuropathic pain symptoms such as hyperalgesia and allodynia in a well‐established model of this pain (sciatic nerve chronic constriction injury [CCI]). Moreover, since we and others showed that the peripheral nerve lesion starts a cascade of neuroinflammation‐related events that may maintain and worsen the original lesion, the effect of NSCs on sciatic nerve pro‐ and antiinflammatory cytokines in CCI mice was investigated. NSCs injected intravenously, when the pathology was already established, induced a significant reduction in allodynia and hyperalgesia already 3 days after administration, demonstrating a therapeutic effect that lasted for at least 28 days. Responses changed with the number of administered NSCs, and the effect on hyperalgesia could be boosted by a new NSC administration. Treatment significantly decreased proinflammatory, activated antiinflammatory cytokines in the sciatic nerve, and reduced spinal cord Fos expression in laminae I‐VI. Moreover, in NSC‐treated animals, a reparative process and an improvement of nerve morphology is present at a later time. Since NSC effect on pain symptoms preceded nerve repair and was maintained after cells had disappeared from the lesion site, we suggest that regenerative, behavioral, and immune NSC effects are largely due to microenvironmental changes they might induce at the lesion site.

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.

Long term effects of lipopolysaccharide on satellite glial cells in mouse dorsal root ganglia

Abstract Lipopolysaccharide (LPS) has been used extensively to study neuroinflammation, but usually its effects were examined acutely (24 h<). We have shown previously that a single intraperitoneal LPS injection activated satellite glial cells (SGCs) in mouse dorsal root ganglia (DRG) and altered several functional parameters in these cells for at least one week. Here we asked whether the LPS effects would persist for 1 month. We injected mice with a single LPS dose and tested pain behavior, assessed SGCs activation in DRG using glial fibrillary acidic protein (GFAP) immunostaining, and injected a fluorescent dye intracellularly to study intercellular coupling. Electron microscopy was used to quantitate changes in gap junctions. We found that at 30 days post‐LPS the threshold to mechanical stimulation was lower than in controls. GFAP expression, as well as the magnitude of dye coupling among SGCs were greater than in controls. Electron microscopy analysis supported these results, showing a greater number of gap junctions and an abnormal growth of SGC processes. These changes were significant, but less prominent than at 7 days post‐LPS. We conclude that a single LPS injection exerts long‐term behavioral and cellular changes. The results are consistent with the idea that SGC activation contributes to hyperalgesia. HighlightsA single lipopolysaccharides injection activated glia in mouse dorsal root ganglia for 30 days.This was accompanied by increased communications by gap junctions among glia and by hyperalgesia.Glial activation and coupling may contribute to chronic pain.

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.

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.

3D-spheroids: What can they tell us about pancreatic ductal adenocarcinoma cell phenotype?

&NA; We aimed at analyzing the effect of the 3D‐arrangement on the expression of some genes and proteins which play a key role in pancreatic adenocarcinoma (PDAC) progression in HPAF‐II, HPAC and PL45 PDAC cells cultured in either 2D‐monolayers or 3D‐spheroids. Cytokeratins 7, 8, 18, 19 were differently expressed in 3D‐spheroids compared to 2D‐monolayers. Syndecan 1 was upregulated in HPAF‐II and PL45 3D‐spheroids, and downregulated in HPAC. Heparanase mRNA levels were almost unchanged in HPAF‐II, and increased in HPAC and PL45 3D‐spheroids. Hyaluronan synthase (HAS) 2 and 3 mRNA increased in all 3D‐spheroids compared to 2D‐monolayers. CD44 and CD44s were expressed to a lower extent in HPAF‐II and HPAC 3D‐spheroids. By contrast, the CD44s/v3 and the CD44s/v6 ratio increased in HPAC and PL45 3D‐spheroids, compared to 2D‐monolayers. The expression of MMP‐7 was strongly upregulated in 3D‐spheroids. STAT3 was similarly expressed 3D‐spheroids or 2D‐monolayers, while pSTAT3 was almost undetectable in 2D‐monolayers and strongly upregulated in 3D‐spheroids. These results suggest that 3D‐spheroids represent a cell culture model that allows the characterization of PDAC cell phenotype, adding new information that contributes to a better understanding of the biology and behavior of PDAC cells. Highlights3D‐spheroids allow the detection of key characteristics of PDAC cell phenotype.3D‐spheroids contribute to a better understanding of PDAC cell biology.the first study describing the relationship between MMP‐7 and STAT3 in PDAC 3D‐spheroids.

Extracellular matrix components affect cell migration and invasive potential of cultured human pancreatic ductal adenocarcinoma cells

The tumor microenvironment influences cancer cell behavior in relation to tumor progression, as well as cell proliferation and invasion. Pancreatic ductal adenocarcinoma (PDAC) is characterized by an intense desmoplastic reaction and extracellular matrix (ECM) components in the tumor microenvironment are involved in a cross-talk between tumor cells, stromal fibroblasts and ECM components, influencing tumor cell behavior. We aimed at analyzing in vitro the effect of the crosstalk between PDAC cells and the ECM of the microenvironment by culturing PDAC cells on different ECM proteins used as a substrate, in order to better understand the relationship between cancer cell phenotype and the proteins occurring in the desmoplastic tissue. For this purpose, we analyzed some epithelial-to-mesenchymal transition (EMT) markers and the migration and invasive potential in human HPAF-II, HPAC and PL45 PDAC cells cultured on collagen type I (COL), laminin (LAM) and fibronectin (FN). Interestingly, the expression of E-cadherin was not significantly affected, but some differences were revealed by the wound healing assay. In fact, migration of HPAF-II and PL45 cells was decreased on FN and LAM, and increased on COL, compared to control cells grown on plastic (NC). By contrast, HPAC was very rapid and unaffected by the substrate. SDS-zymography showed that COL induced a strong upregulation of MMP-2 activity in HPAF-II and HPAC cells, and of MMP-9 in HPAF-II and PL45 cells, compared to NC. These preliminary results suggest that ECM components could differently affect PDAC migration and invasion, possibly depending on the differentiation grade. The characterization of the mutual effects elicited by the tumor-stroma interplay on the cancer cell will contribute to better understand the influence of the stroma on PDAC cancer cell phenotype, in order to develop new therapeutic strategies.

Characterization of an in vitro model to study the role of human Polyomavirus BK in prostate cancer

Prostate cancer (PCa) is one of the most common male neoplasm in the western world, being the most commonly diagnosed non-skin cancer and the second leading cause of cancer death. Various potential risk factors exist for the initial triggering events, including exposure to infectious agents, such as the human Polyomavirus BK (BKV). BKV is a good candidate as risk factor of PCa because it naturally infects the human reno-urinary tract, it establishes latency, and encodes oncoproteins that interfere with tumor suppressors pathways, thus altering the normal progression of cell cycle. Previous studies suggested a potential association between BKV and PCa, revealing that the prevalence of BKV was significantly higher in cancer than in control tissues, with a significant association between viral expression and cancer. However, this hypothesis is controversial because BKV is not restricted to tumor tissues but also infects healthy individuals in a high percentage. Moreover, an in vitro model of BKV infection in prostate cells is not available to understand the role for BKV in pathogenesis of PCa. Our aims were to determine whether BKV a) could infect normal epithelial prostate cells, b) affects cell phenotype and c) affects the phenotype of human prostate tumor cell line PC3. For this purpose normal epithelial prostate cell line RWPE-1 and prostate cancer cells PC3 were infected with BKV for 21 days. Cell proliferation, epithelial-to-mesenchymal markers (EMT) and invasion potential were analyzed by, respectively, MTT, immunofluorescence and SDS-zymography. Our results show that cell proliferation was increased or decreased by BKV, respectively, in RWPE-1 and PC3 cells. BKV induced E-cadherin downregulation and vimentin expression in both control and BKV-infected cells RWPE-1, suggesting that uninfected cells underwent EMT. Matrix metalloproteinase-2 and 9 activity was increased in RWPE-1 cells after BKV infection. By contrast, BKV did not significantly modified the phenotype of PC3 cells. These preliminary results suggest that normal epithelial prostate cells RWPE-1 and PC3 are susceptible and permissive to BKV infection. However, RWPE-1 cells exhibit some phenotype modifications related to EMT, possibly induced by the papilloma virus used to obtain their immortalization, thus suggesting that further experiments will be necessary to define if they represent a good experimental model to study prostate cancer.