Differential expression of disialic acids in the cerebellum of senile mice

It is known that disialic acids (diSia) are present in the mammalian brain. However, the precise anatomical distribution and the chronology of its expression along life are not well studied yet. It is accepted that the transfer of diSia in the brain is mediated mainly by the enzyme ST8Sia III (α2,8-sialyltransferase III). We studied the expression of diSia glycoepitopes and of the ST8Sia III gene in different structures of the mouse brain at different postnatal stages by immunohistochemistry and real-time polymerase chain reaction, respectively. C57BL/6 mice of different stages were used. Samples of hippocampus, olfactory bulb, cortex and cerebellum were processed for studies of molecular biology and immunohistochemistry. Histological analysis revealed an important decrease in diSia labeling in the senile cerebellum compared with other structures and stages (P ≪ 0.001). In concordance with these results, a significant decrease in ST8Sia III gene expression was found in the cerebellum of senile animals (P < 0.001). These results suggest that diSia are constantly expressed but with differential expression in various areas of the mouse central nervous system. On the other hand, the concordance in the decreased expression of ST8Sia III and the diSia epitope in the cerebellum of senile animals suggest a role of diSia in this structure or, inversely, an influence of aging on the expression of diSia in the cerebellum. Further research in that direction could elucidate the roles of diSia in brain function in health and disease.Fil: Rinflerch, Adriana Raquel. Hospital Italiano; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Burgos, Valeria Laura. Hospital Italiano; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Hidalgo, Alejandra Mabel. Hospital Italiano; ArgentinaFil: Loresi, Monica Alejandra. Hospital Italiano; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Argibay, Pablo F.. Hospital Italiano; Argentin

Inhibition of brain ST8SiaIII sialyltransferase leads to impairment of procedural memory in mice

Several glycoproteins in mammalian brains contain α2,8-linked disialic acid residues. We previously showed a constant expression of disialic acid (DiSia) in the hippocampus, olfactory bulb and cortex, and a gradual decrease of expression in the cerebellum from neonatal to senile mice. Previous publications indicate that neurite extension of neuroblastoma-derived Neuro2A cells is inhibited in the presence of DiSia antibody. Based on this, we treated Neuro2A cell cultures with RNA interference for ST8SiaIII mRNA, the enzyme responsible for DiSia formation. We observed that neurite extension was inhibited by this treatment. Taking this evidence into consideration and the relationship of the cerebellum with learning and memory, we studied the role of DiSia expression in a learning task. Through delivery of pST8SiaIII into the brains of C57BL/6 neonatal mice, we inhibited the expression of ST8SiaIII. ST8SiaIII mRNA and protein expressions were analyzed by real-time PCR and western blot, respectively. In this work, we showed that pST8SiaIII-treated mice presented a significantly reduced level of ST8SiaIII mRNA in the cerebellum (p < 0.01) in comparison to control mice at 8 days after treatment. It is also noted that these levels returned to baseline values in the adulthood. Then, we evaluated behavioural performance in the T-Maze, a learning task that estimates procedural memory. At all ages, pST8SiaIII-treated mice showed a lower performance in the test session, being most evident at older ages (p < 0.001). Taken all together, we conclude that gene expression of ST8SiaIII is necessary for some cognitive tasks at early postnatal ages, since reduced levels impaired procedural memory in adult mice.Fil: Rinflerch, Adriana Raquel. Hospital Italiano. Instituto de Cs.basicas y Medicina Experimental; ArgentinaFil: Burgos, Valeria Laura. Hospital Italiano. Instituto de Ciencias Básicas y Medicina Experimental; ArgentinaFil: Ielpi, Marcelo. Hospital Italiano. Instituto de Ciencias Básicas y Medicina Experimental; ArgentinaFil: Ojea Quintana, Ignacio María. Hospital Italiano. Instituto de Ciencias Básicas y Medicina Experimental; ArgentinaFil: Hidalgo, Alejandra M.. Hospital Italiano. Instituto de Ciencias Básicas y Medicina Experimental; ArgentinaFil: Loresi, Monica Alejandra. Hospital Italiano. Instituto de Ciencias Básicas y Medicina Experimental; ArgentinaFil: Argibay, Pablo. Hospital Italiano. Instituto de Ciencias Básicas y Medicina Experimental; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Histologic Changes After Urethroplasty Using Small Intestinal Submucosa Unseeded With Cells in Rabbits With Injured Urethra

OBJECTIVE To determine whether small intestine submucosa has the same regenerative capacity when urethroplasty is performed in injured urethras. METHODS Our experiment was conducted in 30 New Zealand male rabbits, all of which had urethral injury. One month after the injury, the animals were randomized into a control group or a group with onlay urethroplasty with small intestine submucosa. The animals were euthanized at 2, 4, 12, 24, and 36 weeks after urethroplasty, and their urethras were removed for histologic and immunohistochemical examination. Before the scheduled euthanasia, urethrography and cystoscopy were performed. RESULTS After 2 weeks, there was evidence of a continuous monolayer of stratified epithelial cells and absence of smooth muscle fibers. One month later, the epithelium showed no changes from the previously observed features, but some smooth muscle fibers (representing newly formed vessels) became apparent. After 3 months, the graft showed increased concentration of smooth muscle fibers. After 6 and 9 months, the density of smooth muscle cells remained unchanged. Fiber arrangement was irregular, particularly at the anastomosis site. Epithelial and smooth muscle phenotypes were confirmed by immunohistochemistry using anti-pan-citokeratin (AE1/AE3) antibodies and anti-a-smooth muscle actin, respectively. CONCLUSION Small intestine submucosa promotes regeneration in traumatized urethras, with slightly delayed epithelialization and abnormal distribution of smooth muscle. Urethral damage caused by trauma interferes with the normal healing process.Fil: Villoldo, Gustavo Martin. Hospital Italiano. Instituto de Ciencias Básicas y Medicina Experimental; ArgentinaFil: Loresi, Monica Alejandra. Hospital Italiano. Instituto de Ciencias Básicas y Medicina Experimental; ArgentinaFil: Giudice, Carlos. Hospital Italiano. Instituto de Ciencias Básicas y Medicina Experimental; ArgentinaFil: Damia, Oscar. Hospital Italiano. Instituto de Ciencias Básicas y Medicina Experimental; ArgentinaFil: Moldes, Juan Manuel. Hospital Italiano. Instituto de Ciencias Básicas y Medicina Experimental; ArgentinaFil: Debadiola, Francisco. Hospital Italiano. Instituto de Ciencias Básicas y Medicina Experimental; ArgentinaFil: Barbich, Mariana. Hospital Italiano. Instituto de Ciencias Básicas y Medicina Experimental; ArgentinaFil: Argibay, Pablo. Hospital Italiano. Instituto Universitario - Escuela de Medicina. Departamento de Ciencias Básicas. Area Inmunología de Transplantes; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Assessing the role of selected growth factors and cytostatic agents in an in vitro model of human dura mater healing

OBJECTIVES: Cerebrospinal fluid (CSF) leaks are a common concern in skull base surgery. Appropriate dural healing is crucial to prevent CSF leaks but the entire process has been barely understood so far. Here, we review the impact of growth factors and chemotherapeutic agents on an explant culture of human dural fibroblasts and a 3D subculture grown in a collagen mesh scaffold. METHODS: Human dural specimens were harvested during surgical procedures where they would not be further used therapeutically or diagnostically. Explant cultures were grown in Petri dishes, and subcultures were grown in collagen mesh scaffolds. Insulin, fibroblast growth factor type 2 (FGF-2), and human serum were analyzed for their effect as growth factors, whereas mitomycin C, vincristine, and colchicine were analyzed for their role as inhibitors. Cell count was used as a parameter to assess the effects of these factors. In addition, the effects of human serum were assessed using collagen mesh scaffolds. RESULTS: Insulin, FGF-2, and human serum increased culture cell count; human serum also achieved an increased number of viable fibroblasts embedded in a collagen mesh. Mitomycin C, which is a mitosis inhibitor, showed no significant effect on cell count, whereas colchicine and vincristine, which inhibit both mitosis and migration, resulted in cell growth suppression. DISCUSSION: In our model, dural defect closure is achieved through cell migration rather than through cell growth. Adding growth factors to the dural suture line or into a collagen mesh might prove useful to stimulate dural closure.Fil: Goldschmidt, Ezequiel Darío. Hospital Italiano; ArgentinaFil: Ielpi, Marcelo. Hospital Italiano. Instituto de Ciencias Básicas y Medicina Experimental; ArgentinaFil: Loresi, Monica Alejandra. Hospital Italiano. Instituto de Ciencias Básicas y Medicina Experimental; ArgentinaFil: D'adamo, Maximiliano. Hospital Italiano. Instituto de Ciencias Básicas y Medicina Experimental; ArgentinaFil: Giunta, Diego Hernan. Hospital Italiano; ArgentinaFil: Carrizo, Antonio. Hospital Italiano; ArgentinaFil: Ajler, Pablo. Hospital Italiano; ArgentinaFil: Yampolsky, Claudio. Hospital Italiano; ArgentinaFil: Argibay, Pablo. Hospital Italiano. Instituto de Ciencias Básicas y Medicina Experimental; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Skin Fibroblasts from Patients with Type 1 Diabetes (T1D) Can Be Chemically Transdifferentiated into Insulin-Expressing Clusters: A Transgene-Free Approach

The conversion of differentiated cells into insulin-producing cells is a promising approach for the autologous replacement of pancreatic cells in patients with type 1 diabetes (T1D). At present, cellular reprogramming strategies encompass ethical problems, epigenetic failure or teratoma formation, which has prompted the development of new approaches. Here, we report a novel technique for the conversion of skin fibroblasts from T1D patients into insulin-expressing clusters using only drug-based induction. Our results demonstrate that skin fibroblasts from diabetic patients have pancreatic differentiation capacities and avoid the necessity of using transgenic strategies, stem cell sources or global demethylation steps. These findings open new possibilities for studying diabetes mechanisms, drug screenings and ultimately autologous transgenic-free regenerative medicine therapies in patients with T1D.Fil: Pereyra Bonnet, Federico Alberto. Hospital Italiano. Instituto de Ciencias Básicas y Medicina Experimental; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Gimeno, Maria Laura. Hospital Italiano. Instituto de Ciencias Básicas y Medicina Experimental; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Argumedo, Nelson R.. Hospital Italiano. Instituto de Ciencias Básicas y Medicina Experimental; ArgentinaFil: Ielpi, Marcelo. Hospital Italiano. Instituto de Ciencias Básicas y Medicina Experimental; ArgentinaFil: Cardozo, Johana A.. Hospital Italiano. Instituto de Ciencias Básicas y Medicina Experimental; ArgentinaFil: Giménez, Carla Alejandra. Hospital Italiano. Instituto de Ciencias Básicas y Medicina Experimental; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Hyon, Sung Ho. Hospital Italiano; ArgentinaFil: Balzaretti, Vilma Teresa. Hospital Italiano; ArgentinaFil: Loresi, Monica Alejandra. Hospital Italiano; ArgentinaFil: Fainstein Day, Patricia. Hospital Italiano; ArgentinaFil: Litwak, Sara Alejandra. Hospital Italiano; ArgentinaFil: Argibay, Pablo. Hospital Italiano. Instituto de Ciencias Básicas y Medicina Experimental; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin