Majority of cerebrospinal fluid-contacting neurons in the spinal cord of C57Bl/6N mice is present in ectopic position unlike in other studied experimental mice strains and mammalian species.

Cerebrospinal fluid contacting neurons (CSF-cNs) represent a specific class of neurons located in close vicinity of brain ventricles and central canal. In contrast with knowledge gained from other vertebrate species, we found that vast majority of CSF-cNs in the spinal cord of C57Bl/6N mice is located in ectopic distal ventral position. However, we found that small number of ectopic CSF-cNs is present also in spinal cord of other investigated experimental mice strains (C57Bl/6J, Balb/C) and mammalian species (Wistar rats, New Zealand White rabbits). Similarly, as the proximal populations, ectopic CSF-cNs retain PKD2L1-immunoreactivity and synaptic contacts with other neurons. On the other side, they show rather multipolar morphology lacking thick dendrite contacting central canal lumen. Ectopic CSF-cNs in the spinal cord of C57Bl/6N mice emerge during whole period devoted to production of CSF-cNs and reach their ventral destinations during first postnatal weeks. In order to identify major gene, whose impairment could trigger translocation of CSF-cNs outside the central canal area, we took advantage of close consanguinity of C57Bl/6J substrain with normal CSF-cN distribution and C57Bl/6N substrain with majority of CSF-cNs in ectopic position. Employing in silico analyses, we ranked polymorphisms in C57Bl/6N substrain and selected genes Crb1, Cyfip2, Adamts12, Plk1, and Herpud2 as the most probable candidates, whose product dysfunction might be responsible for the ectopic distribution of CSF-cNs. Furthermore, segregation analysis of F2 progeny of parental C57Bl/6N and Balb/C mice revealed that polymorphic loci of Crb1 and Cyfip2 underlie the ectopic position of CSF-cNs in the spinal cord of C57Bl/6N mice.

Novel Insights into the Effect of Hyperforin and Photodynamic Therapy with Hypericin on Chosen Angiogenic Factors in Colorectal Micro-Tumors Created on Chorioallantoic Membrane.

Photodynamic therapy with hypericin (HY-PDT) and hyperforin (HP) could be treatment modalities for colorectal cancer (CRC), but evidence of their effect on angiogenic factors in CRC is missing. Convenient experimental model utilization is essential for angiogenesis research. Therefore, not only 2D cell models, but also 3D cell models and micro-tumors were used and compared. The micro-tumor extent and interconnection with the chorioallantoic membrane (CAM) was determined by histological analyses. The presence of proliferating cells and HY penetration into the tumor mass were detected by fluorescence microscopy. The metabolic activity status was assessed by an colorimetric assay for assessing cell metabolic activity (MTT assay) and HY accumulation was determined by flow cytometry. Pro-angiogenic factor expression was determined by Western blot and quantitative real-time polymerase chain reaction (RT-qPCR). We confirmed the cytotoxic effect of HY-PDT and HP and showed that their effect is influenced by structural characteristics of the experimental model. We have pioneered a method for analyzing the effect of HP and cellular targeted HY-PDT on pro-angiogenic factor expression in CRC micro-tumors. Despite the inhibitory effect of HY-PDT and HP on CRC, the increased expression of some pro-angiogenic factors was observed. We also showed that CRC experimental micro-tumors created on quail CAM could be utilized for analyses of gene and protein expression.