Magdalena Gewartowska

Perinatal exposure to lead (Pb) induces ultrastructural and molecular alterations in synapses of rat offspring

Lead (Pb), environmentally abundant heavy-metal pollutant, is a strong toxicant for the developing central nervous system. Pb intoxication in children, even at low doses, is found to affect learning and memorizing, with devastating effects on cognitive function and intellectual development. However, the precise mechanism by which Pb impairs synaptic plasticity is not fully elucidated. The purpose of this study was to investigate the effect of pre- and neonatal exposure to low dose of Pb (with Pb concentrations in whole blood below 10μg/dL) on the synaptic structure and the pre- and postsynaptic proteins expression in the developing rat brain. Furthermore, the level of brain-derived neurotrophic factor (BDNF) was analyzed. Pregnant female Wistar rats received 0.1% lead acetate (PbAc) in drinking water from the first day of gestation until weaning of the offspring, while the control animals received drinking water. During the feeding of pups, mothers from the Pb-group were continuously receiving PbAc. Pups of both groups were weaned at postnatal day 21 and then until postnatal day 28 received only drinking water. 28-Day old pups were sacrificed and the ultrastructural changes as well as expression of presynaptic (VAMP1/2, synaptophysin, synaptotagmin-1, SNAP25, syntaxin-1) and postsynaptic (PSD-95) proteins were analyzed in: forebrain cortex, cerebellum and hippocampus. Our data revealed that pre- and neonatal exposure to low dose of Pb promotes pathological changes in synapses, including nerve endings swelling, blurred and thickened synaptic cleft structure as well as enhanced density of synaptic vesicles in the presynaptic area. Moreover, synaptic mitochondria were elongated, swollen or shrunken in Pb-treated animals. These structural abnormalities were accompanied by decrease in the level of key synaptic proteins: synaptotagmin-1 in cerebellum, SNAP25 in hippocampus and syntaxin-1 in cerebellum and hippocampus. In turn, increased level of synaptophysin was noticed in the cerebellum, while the expression of postsynaptic PSD-95 was significantly decreased in forebrain cortex and cerebellum, and raised in hippocampus. Additionally, we observed the lower level of BDNF in all brain structures in comparison to control animals. In conclusion, perinatal exposure to low doses of Pb caused pathological changes in nerve endings associated with the alterations in the level of key synaptic proteins. All these changes can lead to synaptic dysfunction, expressed by the impairment of the secretory mechanism and thereby to the abnormalities in neurotransmission as well as to the neuronal dysfunction.Lead (Pb), environmentally abundant heavy-metal pollutant, is a strong toxicant for the developing central nervous system. Pb intoxication in children, even at low doses, is found to affect learning and memorizing, with devastating effects on cognitive function and intellectual development. However, the precise mechanism by which Pb impairs synaptic plasticity is not fully elucidated. The purpose of this study was to investigate the effect of pre- and neonatal exposure to low dose of Pb (with Pb concentrations in whole blood below 10μg/dL) on the synaptic structure and the pre- and postsynaptic proteins expression in the developing rat brain. Furthermore, the level of brain-derived neurotrophic factor (BDNF) was analyzed. Pregnant female Wistar rats received 0.1% lead acetate (PbAc) in drinking water from the first day of gestation until weaning of the offspring, while the control animals received drinking water. During the feeding of pups, mothers from the Pb-group were continuously receiving PbAc. Pups of both groups were weaned at postnatal day 21 and then until postnatal day 28 received only drinking water. 28-day old pups were sacrificed and the ultrastructural changes as well as expression of presynaptic (VAMP1/2, synaptophysin, synaptotagmin-1, SNAP25, syntaxin-1) and postsynaptic (PSD-95) proteins were analyzed in: forebrain cortex, cerebellum and hippocampus. Our data revealed that pre- and neonatal exposure to low dose of Pb promotes pathological changes in synapses, including nerve endings swelling, blurred and thickened synaptic cleft structure as well as enhanced density of synaptic vesicles in the presynaptic area. Moreover, synaptic mitochondria were elongated, swollen or shrunken in Pb-treated animals. These structural abnormalities were accompanied by decrease in the level of key synaptic proteins: synaptotagmin-1 in cerebellum, SNAP25 in hippocampus and syntaxin-1 in cerebellum and hippocampus. In turn, increased level of synaptophysin was noticed in the cerebellum, while the expression of postsynaptic PSD-95 was significantly decreased in forebrain cortex and cerebellum, and raised in hippocampus. Additionally, we observed the lower level of BDNF in all brain structures in comparison to control animals. In conclusion, perinatal exposure to low doses of Pb caused pathological changes in nerve endings associated with the alterations in the level of key synaptic proteins. All these changes can lead to synaptic dysfunction, expressed by the impairment of the secretory mechanism and thereby to the abnormalities in neurotransmission as well as to the neuronal dysfunction.

Cancer seeding contributes to intestinal anastomotic dehiscence

BackgroundSurgical wounds in cancer patients have a relatively high dehiscence rate. Although colon cancer resections are performed so as to include macroscopically non-involved tissues, some cancer cells can be present in the line of transection. The local healing process may facilitate proliferation of these localized cancer cells and the high cytokine concentration within the healing wound may also attract cancer cells from distant sites to migrate into the wound area. The growing tumor cells may then stretch the wound, hampering its contraction process.MethodsThe aim of the study was to monitor and compare, using immunohistochemical methods, the healing process of intestinal anastomosis in both normal rats and in rats with disseminated cancer (the CC531 colon cancer model).ResultsThere was a significantly higher rate of anastomotic dehiscence in the group of rats with disseminated cancer, than in the group of normal rats. There were no significant differences between the two groups in the levels of mononuclear wound infiltration or of formation of connective tissue or new vessels. All anastomotic wounds in animals with disseminated cancer had abundant infiltrates of both migrating and proliferating cancer cells.ConclusionsWe confirmed that the environment of a healing wound attracts cancer cells. Migration of cancer cells to the wound and centrifugal cancer proliferation may adversely affect the healing process and cause wound disruption.

Spleen migrating dendritic cells primed with CC531 colon cancer antigen and LPS - is it a method to compromise liver metastases?

The anti-tumor vaccination is burdened by low recruitment rate of intravenously administered in vitro primed DC in liver metastases and lack of supplying them continuously in large numbers. Therefore, it seemed rational to create a model of in vivo vaccination with specifically primed splenic DC and cytotoxic T lymphocytes being continuously supplied to the liver vascular bed. The question we raised was whether anti-tumor immunized splenic DC flowing to liver metastases could adhere to and be cytotoxic to tumor cells. We immunized rats with CC531 tumor cells and stimulated them with Escherichia coli LPS. Subsequently, spleen DC-enriched population was isolated, its activation by LPS, adherence to CC531 cells and cytotoxicity were measured. Spleen cells home to the liver reaching it via splenic vein. These cells can be retrieved by simple washout of liver sinusoids (liver sinusoidal washout cells - LSWC). Their adherence to and cytotoxicity against CC531 cells were evaluated. Moreover, in vitro adherence of splenic DC-enriched cells and LSWC to CC531 liver tumor sections was measured. We found that in vivo immunization of splenic population containing DC, NK cells and lymphocytes with CC531 cells and stimulation with LPS activated these cells but did not significantly increase the cytotoxicity against CC531 cells. There was also no increase in cytotoxicity of LSWC. Adhesion of splenic DC and LWSC to liver CC531 metastases on cryosections was higher than to the adjacent liver tissue. However, it was more expressed on tumor stromal than neoplastic cells. The level of splenic Treg cells down-regulating immune response was found only slightly increased after immunization. Taken together, in the model of in vivo immunization against CC531 cells, low level of spleen DC and spleen-derived LSWC cytotoxicity as well as adherence rate to tumor cells were observed. More effective methods of immunizing splenic DC overcoming the suppressive mechanisms should be looked for.