Plasma-Based Scaffold Containing Bone-Marrow Mononuclear Cells Promotes Wound Healing in a Mouse Model of Pressure Injury.

Pressure injuries, or pressure ulcers, are a common problem that may lead to infections and major complications, besides being a social and economic burden due to the costs of treatment and hospitalization. While surgery is sometimes necessary, this also has complications such as recurrence or wound dehiscence. Among the newer methods of pressure injury treatment, advanced therapies are an interesting option. This study examines the healing properties of bone marrow mononuclear cells (BM-MNCs) embedded in a plasma-based scaffold in a mouse model. Pressure ulcers were created on the backs of mice (2 per mouse) using magnets and assigned to a group of ulcers that were left untreated (Control, n = 15), treated with plasma scaffold (Plasma, n = 15), or treated with plasma scaffold containing BM-MNC (Plasma + BM-MNC, n = 15). Each group was examined at three time points (3, 7, and 14 days) after the onset of treatment. At each time point, animals were subjected to biometric assessment, bioluminescence imaging, and tomography. Once treatment had finished, skin biopsies were processed for histological and wound healing reverse transcription polymerase chain reaction (RT-PCR) array studies. While wound closure percentages were higher in the Plasma and Plasma + BM-MNC groups, differences were not significant, and thus descriptive data are provided. In all individuals, the presence of donor cells was revealed by immunohistochemistry on posttreatment onset Days 3, 7, and 14. In the Plasma + BM-MNC group, less inflammation was observed by positron emission tomography-computed tomography (PET/CT) imaging of the mice at 7 days, and a complete morphometabolic response was produced at 14 days, in accordance with histological results. A much more pronounced inflammatory process was observed in controls than in the other two groups, and this persisted until Day 14 after treatment onset. RT-PCR array gene expression patterns were also found to vary significantly, with the greatest difference noted between both treatments at 14 days when 11 genes were differentially expressed.

Stem cell and tissue engineering approaches in pressure ulcer treatment.

CONTEXT: Pressure ulcers or injuries, arise from ischemic damage to soft tissues induced by unrelieved pressure over a bony prominence. They are usually difficult to treat with standard medical therapy and often they recur. In the search for better treatment options, promising alternative forms of treatment are today emerging. Within the field of regenerative medicine, ongoing research on advanced therapies seeks to develop medicinal products based on gene therapy, somatic cell therapy, tissue-engineering and combinations of these. OBJECTIVE: The main objective is to perform an overview of experimental and clinical developments in somatic cell therapy and tissue engineering targeting the treatment of pressure injuries. METHODS: Searching terms as "PRESSURE ULCER", "STEM CELL THERAPY", "TISSUE ENGINEERING" or "WOUND HEALING" were used in combination or alone, including publications refered to basic and clinical research and focusing on articles showing results obtained in a clinical context. A total of 80 references are cited, including 23 references published in the 3 last years. RESULTS: The results suggest that this form of treatment could be an interesting option in patients with difficult-to-treat ulcers as spinal cord injury patients. CONCLUSION: This field of regenerative medicine is very broad and further research is warranted.

Treatment of pressure ulcers in patients with spinal cord injury: Conventional surgery vs. cellular therapy.

CONTEXT: Relapse and recurrence rates of pressure injuries (PIs) are very high in spinal cord injured patients. That is the reason why alternative therapies, such the stem cells derived from bone marrow, have been developed. OBJECTIVE: To compare this new technique of infiltration-infusion of mononuclear cells from bone marrow with conventional surgery. DESIGN: A retrospective study was carried out in patients with spinal cord injuries who had PIs, category III/IV, in the pelvic area, during a 14-year follow-up period. SETTING: One group was treated with conventional surgery and, in the other group, mononuclear cells were infused. PARTICIPANTS: One hundred and forty-nine patients were registered, 63 (42.3%) in the conventional surgery group and 86 (57.7%) in the mononuclear cell group. RESULTS: A comparative study between these 2 groups was carried out. There were no significant differences in ulcer healing in the first 6 months, but 6 months and one-year post-treatment, they were found. At 6 months, no patient in the conventional surgery group showed dehiscence or fistulization of the wound and, one year after surgery, only 3.17% recurred in the conventional group. In addition, there was a statistically significant relationship between days of hospitalization and the type of bacterial contamination and the intervention group. CONCLUSION: Bone marrow mononuclear cell infusion-infiltration is an alternative treatment for PIs and fistula during the first 6 months, instead of conventional surgery. However, in the medium-long term, conventional surgery is more effective.

Mesenchymal stem cells from different sources and their derived exosomes: A pre-clinical perspective.

Since the introduction of cell therapy as a strategy for the treatment of many diseases, mesenchymal stem cells have emerged as ideal candidates, yet the underlying mechanisms of their beneficial effects are only partially understood. At the start of the 21st century, a paracrine effect was proposed as a mechanism of tissue repair by these cells. In addition, a role was suggested for a heterogeneous population of extracellular vesicles in cell-to-cell communication. Some of these vesicles including exosomes have been isolated from most fluids and cells, as well as from supernatants of in vitro cell cultures. Recent research in the field of regenerative medicine suggests that exosomes derived from mesenchymal stem cells could be a powerful new therapeutic tool. This review examines the therapeutic potential of these exosomes obtained from the sources most used in cell therapy: bone marrow, adipose tissue, and umbilical cord.

The role of bone marrow mononuclear cell-conditioned medium in the proliferation and migration of human dermal fibroblasts.

BACKGROUND: Several recent studies have demonstrated the great potential of bone marrow cells in regenerative medicine, not only for their ability to differentiate to match a damaged cell type, but also because they synthesize and release various growth factors and cytokines.We examined the effect of bone marrow cell-conditioned medium in the healing process, especially in terms of fibroblast proliferation and migration. METHODS: These in vitro studies consisted of co-culture (without direct contact) of dermal fibroblasts with mononuclear bone marrow cells and the use of conditioned medium obtained from these cultures in a scratch wound model. RESULTS: Mononuclear cells were found to increase the proliferation of fibroblasts, and the conditioned medium showed a stimulatory effect on the migration of fibroblasts. CONCLUSION: When considered together with the observed increase in growth factor levels in conditioned medium, it appears that these cells act through a paracrine mechanism.

Platelet-Derived Mitochondria Display Embryonic Stem Cell Markers and Improve Pancreatic Islet ß-cell Function in Humans.

Diabetes is a major global health issue and the number of individuals with type 1 diabetes (T1D) and type 2 diabetes (T2D) increases annually across multiple populations. Research to develop a cure must overcome multiple immune dysfunctions and the shortage of pancreatic islet ß cells, but these challenges have proven intractable despite intensive research effort more than the past decades. Stem Cell Educator (SCE) therapy-which uses only autologous blood immune cells that are externally exposed to cord blood stem cells adhering to the SCE device, has previously been proven safe and effective in Chinese and Spanish subjects for the improvement of T1D, T2D, and other autoimmune diseases. Here, 4-year follow-up studies demonstrated the long-term safety and clinical efficacy of SCE therapy for the treatment of T1D and T2D. Mechanistic studies found that the nature of platelets was modulated in diabetic subjects after receiving SCE therapy. Platelets and their released mitochondria display immune tolerance-associated markers that can modulate the proliferation and function of immune cells. Notably, platelets also expressed embryonic stem cell- and pancreatic islet ß-cell-associated markers that are encoded by mitochondrial DNA. Using freshly-isolated human pancreatic islets, ex vivo studies established that platelet-releasing mitochondria can migrate to pancreatic islets and be taken up by islet ß cells, leading to the proliferation and enhancement of islet ß-cell functions. These findings reveal new mechanisms underlying SCE therapy and open up new avenues to improve the treatment of diabetes in clinics. Stem Cells Translational Medicine 2017;6:1684-1697.

Fibroblasts accelerate islet revascularization and improve long-term graft survival in a mouse model of subcutaneous islet transplantation.

Pancreatic islet transplantation has been considered for many years a promising therapy for beta-cell replacement in patients with type-1 diabetes despite that long-term clinical results are not as satisfactory. This fact points to the necessity of designing strategies to improve and accelerate islets engraftment, paying special attention to events assuring their revascularization. Fibroblasts constitute a cell population that collaborates on tissue homeostasis, keeping the equilibrium between production and degradation of structural components as well as maintaining the required amount of survival factors. Our group has developed a model for subcutaneous islet transplantation using a plasma-based scaffold containing fibroblasts as accessory cells that allowed achieving glycemic control in diabetic mice. Transplanted tissue engraftment is critical during the first days after transplantation, thus we have gone in depth into the graft-supporting role of fibroblasts during the first ten days after islet transplantation. All mice transplanted with islets embedded in the plasma-based scaffold reversed hyperglycemia, although long-term glycemic control was maintained only in the group transplanted with the fibroblasts-containing scaffold. By gene expression analysis and histology examination during the first days we could conclude that these differences might be explained by overexpression of genes involved in vessel development as well as in ß-cell regeneration that were detected when fibroblasts were present in the graft. Furthermore, fibroblasts presence correlated with a faster graft re-vascularization, a higher insulin-positive area and a lower cell death. Therefore, this work underlines the importance of fibroblasts as accessory cells in islet transplantation, and suggests its possible use in other graft-supporting strategies.

Ectopic bone formation during tissue-engineered cartilage repair using autologous chondrocytes and novel plasma-derived albumin scaffolds.

PURPOSE: The aims of this study were twofold: first, to evaluate the production of cartilaginous tissue in vitro and in vivo using a novel plasma-derived scaffold, and second, to test the repair of experimental defects made on ears of New Zealand rabbits (NZr) using this approach. MATERIALS AND METHODS: Scaffolds were seeded with chondrocytes and cultured in vitro for 3 months to check in vitro cartilage production. To evaluate in vivo cartilage production, a chondrocyte-seeded scaffold was transplanted subcutaneously to a nude mouse. To check in vivo repair, experimental defects made in the ears of five New Zealand rabbits (NZr) were filled with chondrocyte-seeded scaffolds. RESULTS: In vitro culture produced mature chondrocytes with no extracellular matrix (ECM). Histological examination of redifferentiated in vitro cultures showed differentiated chondrocytes adhered to scaffold pores. Subcutaneous transplantation of these constructs to a nude mouse produced cartilage, confirmed by histological study. Experimental cartilage repair in five NZr showed cartilaginous tissue repairing the defects, mixed with calcified areas of bone formation. CONCLUSION: It is possible to produce cartilaginous tissue in vivo and to repair experimental auricular defects by means of chondrocyte cultures and the novel plasma-derived scaffold. Further studies are needed to determine the significance of bone formation in the samples.

Regeneration of mandibular defects using adipose tissue mesenchymal stromal cells in combination with human serum-derived scaffolds.

Bone regeneration is a challenging issue. Traditional solutions bring risks, potential complications, and morbidity. The aim of the present study was to regenerate critical-sized mandible defects in athymic rats with adipose tissue mesenchymal stromal cells (AT-MSCs) in combination with human serum-derived scaffolds. Two approaches to treatment were performed. The first approach used differentiated stromal cells that became osteogenic cell lines. The second approach used no pre-differentiation. Follow-up periods were 45 days and 90 days. Both cell types were combined with human serum-derived scaffolds. Afterward, histological (haematoxylin-eosin and Masson's Trichrome stain modified by Goldner), immunohistochemical (human vimentin and Stro-1), and radiological (microCT) studies were performed. The level of calcification between the groups was compared by analysis of variance, and statistical significance was set at p < 0.05. The results demonstrate that bone regeneration can be achieved with both undifferentiated and pre-differentiated cells, but that the structure and level of calcification were better achieved with pre-differentiated cells (p < 0.05). The scaffold is suitable for this cell type, is osteoconductive and simple to perform. This article highlights the possible application of adipose tissue mesenchymal stromal cells in combination with a non-mineralized scaffold in bone regeneration.

Repair of segmental mandibular bone defects in sheep using bone marrow stromal cells and autologous serum scaffold: a pilot study.

AIM: The aim of this study was to evaluate effective bone regeneration using an autologous serum scaffold (alone or seeded with autologous bone marrow-mesenchymal stem cells, BM-MSCs), when implanted in a 30 mm length segmental mandibular defect in sheep. MATERIALS AND METHODS: The bone defect was filled either with serum scaffold alone (control group; n = 5) or combined with BM-MSCs (experimental group; n = 10). Bone regeneration was determined at 12 (T12; 2 control sheep and 4 experimental sheep) and 32 weeks (T32; 3 control and 6 experimental sheep), as measured by computed and microcomputed tomography and histological examination. RESULTS: Two sheep of the Experimental group died after surgery. While complete bone union in the control group was only observed at T32, it was observed both at T12 (1/4 sheep) and T32 (3/4 sheep) in the experimental group. When properties/characteristics of new bone where compared, a better bone quality, similar to native bone, was observed in the scaffold combined with BM-MSCs. CONCLUSIONS: Based on these results, we conclude that the serum scaffold can promote efficient repair of large bone defects, but the combination with BM-MSCs accelerates this process, increasing significantly the amount and quality of bone formed.

CD271 as a marker to identify mesenchymal stem cells from diverse sources before culture.

Mesenchymal stem cells, due to their characteristics are ideal candidates for cellular therapy. Currently, in culture these cells are defined by their adherence to plastic, specific surface antigen expression and multipotent differentiation potential. However, the in vivo identification of mesenchymal stem cells, before culture, is not so well established. Pre-culture identification markers would ensure higher purity than that obtained with selection based on adherence to plastic. Up until now, CD271 has been described as the most specific marker for the characterization and purification of human bone marrow mesenchymal stem cells. This marker has been shown to be specifically expressed by these cells. Thus, CD271 has been proposed as a versatile marker to selectively isolated and expand multipotent mesenchymal stem cells with both immunosuppressive and lymphohematopoietic engraftment-promoting properties. This review focuses on this marker, specifically on identification of mesenchymal stem cells from different tissues. Literature revision suggests that CD271 should not be defined as a universal marker to identify mesenchymal stem cells before culture from different sources. In the case of bone marrow or adipose tissue, CD271 could be considered a quite suitable marker; however this marker seems to be inadequate for the isolation of mesenchymal stem cells from other tissues such as umbilical cord blood or wharton's jelly among others.

Modulation of Autoimmune T-Cell Memory by Stem Cell Educator Therapy: Phase 1/2 Clinical Trial.

BACKGROUND: Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease that causes a deficit of pancreatic islet ß cells. The complexities of overcoming autoimmunity in T1D have contributed to the challenges the research community faces when devising successful treatments with conventional immune therapies. Overcoming autoimmune T cell memory represents one of the key hurdles. METHODS: In this open-label, phase 1/phase 2 study, Caucasian T1D patients (N = 15) received two treatments with the Stem Cell Educator (SCE) therapy, an approach that uses human multipotent cord blood-derived multipotent stem cells (CB-SCs). SCE therapy involves a closed-loop system that briefly treats the patient's lymphocytes with CB-SCs in vitro and returns the "educated" lymphocytes (but not the CB-SCs) into the patient's blood circulation. This study is registered with ClinicalTrials.gov, NCT01350219. FINDINGS: Clinical data demonstrated that SCE therapy was well tolerated in all subjects. The percentage of naïve CD4(+) T cells was significantly increased at 26 weeks and maintained through the final follow-up at 56 weeks. The percentage of CD4(+) central memory T cells (TCM) was markedly and constantly increased at 18 weeks. Both CD4(+) effector memory T cells (TEM) and CD8(+) TEM cells were considerably decreased at 18 weeks and 26 weeks respectively. Additional clinical data demonstrated the modulation of C-C chemokine receptor 7 (CCR7) expressions on naïve T, TCM, and TEM cells. Following two treatments with SCE therapy, islet ß-cell function was improved and maintained in individuals with residual ß-cell function, but not in those without residual ß-cell function. INTERPRETATION: Current clinical data demonstrated the safety and efficacy of SCE therapy in immune modulation. SCE therapy provides lasting reversal of autoimmune memory that could improve islet ß-cell function in Caucasian subjects. FUNDING: Obra Social "La Caixa", Instituto de Salud Carlos III, Red de Investigación Renal, European Union FEDER Funds, Principado de Asturias, FICYT, and Hackensack University Medical Center Foundation.

Repair of long-bone pseudoarthrosis with autologous bone marrow mononuclear cells combined with allogenic bone graft.

BACKGROUND AIMS: Long-bone pseudoarthrosis is a major orthopedic concern because of numerous factors such as difficulty of the treatment, high recurrence, high costs and the devastating effects on the patients' quality of life, which sometimes ends in amputation. Although the "gold standard" for the treatment of this pathology is autologous bone grafting, which has high osteogenic, osteoconductive and osteoinductive properties, this treatment presents some restrictions such as the limited amount of bone that can be taken from the patient and donor site morbidity. Bone marrow mononuclear cells (BM-MNCs) comprise progenitor and stem cells with pro-angiogenic and pro-osteogenic properties. Allogenic cancellous bone graft is a natural and biodegradable osteoconductive and osteoinductive scaffold. Combination of these two components could mimic the advantages of autologous bone grafting while avoiding its main limitations. METHODS: Long-bone pseudoarthrosis was treated in seven patients with autologous BM-MNCs from iliac crest combined with frozen allogenic cancellous bone graft obtained from the tissue bank. RESULTS: All patients showed complete bone consolidation 5.3 ± 0.9 months (range, 2-9 months) after cell transplantation. Moreover, limb pain disappeared in all of them. The mean follow-up was 35.8 ± 4.6 months after transplantation (range, 24-51 months) without pseudoarthrosis recurrence or pain reappearing. CONCLUSIONS: Combination of autologous BM-MNCs and allogenic bone graft could constitute an easy, safe, inexpensive and efficacious attempt to treat long-bone pseudoarthrosis and non-union by reproducing the beneficial properties of autologous bone grafting while restricting its disadvantages.

Repair of rat mandibular bone defects by alveolar osteoblasts in a novel plasma-derived albumin scaffold.

Repair of bone deficiencies in the craniofacial skeleton remains a difficult clinical problem. The aim of this study was to evaluate a novel albumin scaffold seeded with human alveolar osteoblasts and implanted into experimental mandibular defects. An experimental solid protein scaffold was prepared with human plasmatic albumin crossed with a glutaraldehyde-type agent. Microstructure of scaffold and mechanical properties were examined using scanning electron microscopy and a stress-controlled rheometer. Bilateral critical mandibular defects were created in eight immunodeficient rats. Defects of the right side of the mandibles received the cell-scaffold construct in all animals. All left mandibular defects were left untreated as blank controls. Sections of the defects were collected at 5, 8, and 11 weeks postsurgery and processed for histological and immunohistochemical observation, computed tomography examination, and computed tomography digital analysis. Histologically, bone formation was observed in both groups at 5 weeks postsurgery, and the engineered bone became more mature after 8 and 11 weeks, which was similar to normal bone. The origin of bone-forming cells within the defects and the localization of implanted human osteoblasts were confirmed by human vimentin expression. No bone formation could be observed at any control defect. Bone density after 8 weeks was significantly higher than that of the 5-week group (p = 0.02), and significant differences were also observed between 8 and 11 weeks (p < 0.01). The results indicate the clinical feasibility of albumin scaffold loaded with human alveolar cells and that it can be used as a good alternative for bone regeneration.

Ectopic bone formation from mandibular osteoblasts cultured in a novel human serum-derived albumin scaffold.

The aim of this study was to evaluate the ectopic bone formation using a novel serum-derived albumin scaffold and cultured human mandibular osteoblasts in nude mice. Osteoblasts were cultured with 10% human serum and plated in a novel spongy noncalcified protein scaffold prepared with plasmatic albumin crossed with a glutaraldehyde type agent. Hematoxylin-eosin staining revealed a bone-like extracellular matrix and in vitro mineralization was confirmed by von Kossa staining. Histological and immunohistochemical evaluation showed progression of mineralization in vivo. These results suggest the clinical feasibility of alveolar cells and albumin scaffold as a good alternative for bone regeneration.

Biological differences between in vitro produced bovine embryos and parthenotes.

Parthenotes may represent an alternate ethical source of stem cells, once biological differences between parthenotes and embryos can be understood. In this study, we analyzed development, trophectoderm (TE) differentiation, apoptosis/necrosis, and ploidy in parthenotes and in vitro produced bovine embryos. Subsequently, using real-time PCR, we analyzed the expression of genes expected to underlie the observed differences at the blastocyst stage. In vitro matured oocytes were either fertilized or activated with ionomycin +6-DMAP and cultured in simple medium. Parthenotes showed enhanced blastocyst development and diploidy and reduced TE cell counts. Apoptotic and necrotic indexes did not vary, but parthenotes evidenced a higher relative proportion of apoptotic cells between inner cell mass and TE. The pluripotence-related POU5F1 and the methylation DNMT3A genes were downregulated in parthenotes. Among pregnancy recognition genes, TP-1 was upregulated in parthenotes, while PGRMC1 and PLAC8 did not change. Expression of p66(shc) and BAX/BCL2 ratio were higher, and p53 lower, in parthenotes. Among metabolism genes, SLC2A1 was downregulated, while AKR1B1, PTGS2, H6PD, and TXN were upregulated in parthenotes, and SLC2A5 did not differ. Among genes involved in compaction/blastulation, GJA1 was downregulated in parthenotes, but no differences were detected within ATP1A1 and CDH1. Within parthenotes, the expression levels of SLC2A1, TP-1, and H6PD, and possibly AKR1B1, resemble patterns described in female embryos. The pro-apoptotic profile is more pronounced in parthenotes than in embryos, which may differ in their way to channel apoptotic stimuli, through p66(shc) and p53 respectively, and in their mechanisms to control pluripotency and de novo methylation.

Plasma-fibroblast gel as scaffold for islet transplantation.

The transplant of pancreatic islets into the liver can restore normal blood glucose levels in patients with type I diabetes. However, long-term results have indicated that the site and method of transplantation still need to be optimized to improve islet engraftment. This study was designed to assess the efficiency of the use of clotted blood plasma containing fibroblasts ("plasma-fibroblast gel") as a scaffold for subcutaneous islet transplantation in diabetic athymic mice. Islets embedded in the plasma-fibroblast gel were able to resolve hyperglycemia in transplanted mice, restoring normoglycemia over a 60-day period and allowing gradual body weight recovery. Glucose clearances were significantly improved when compared to those recorded in diabetic animals and similar to those observed in the control group (free islets transplanted beneath the kidney capsule). Histological evaluation revealed functional islets within a subcutaneous tissue rich in collagen fibers that was well vascularized, with blood vessels observed around and inside the islets. These findings suggest that this approach could be used as an alternative option for the treatment of type I diabetes in human clinical practice.

Altered endogenous activation of CREB in the suprachiasmatic nucleus of mice with retinal degeneration.

The effect of cone- and rod-cell loss on the activation of transcription factor CREB (by phosphorylation at Ser133) was examined in the pacemaker of mammals, the suprachiasmatic nucleus (SCN). For this purpose, brain sections of rd/rd and wild-type C3H mice were immunolabeled with a polyclonal antibody that recognises p-CREB, i.e., the activated form of the protein. Both rd/rd and wild-type mice maintained in constant darkness showed a circadian variation of p-CREB nuclear staining: the number of immunopositive nuclear pixels at the subjective night was higher than the one observed at the subjective day. However, some differences were detected between both groups: (1) p-CREB immunolabelling in the SCN of rd/rd mice was significantly reduced throughout the 24-h cycle; (2) the time in which the activation of CREB begins to increase at the subjective night in these mice is delayed with regard to wild-type mice. When a light stimulus was given at the subjective night p-CREB immunostaining significantly increased in the SCN of both rd/rd and wild-type mice when compared to basal levels, while no significant effect was found when the stimulus was given at the subjective day. Taken together, our results suggest that despite lower levels of p-CREB, indicating that something is altered in the SCN of rd/rd mice, the main mechanisms of the clock (e.g., circadian oscillation, readjustment by light) are still fully functional in these mice. The present study supports the idea that the CREB/CRE pathway is a component of the circadian clock molecular mechanism.

The transcription factor CREB is phosphorylated in neurons of the piriform cortex of blind mice in response to illumination of the retina.

The vertebrate retina is known to mediate both visual and non-image-forming photic responses. With the use of statistical analyses of sections immunohistochemically labelled with a polyclonal antiserum against the activated form of protein CREB (p-CREB), a transcription factor which participates in some neural responses to stimuli, we have observed that the piriform cortex of both wild-type and retinally degenerate (rd) mice respond to light stimulation independently of the circadian time in which the stimulus was given. Responses in visually blind (rd/rd) mice corroborate the hypothesis that there must be neural connections between the retina and cortical brain areas other than those involved in image processing, and strongly support the idea that since these mice lack rods and cones, the melanopsin retinal ganglion cells could mediate this non-visual light input.

Co-localization of mesotocin and opsin immunoreactivity in the hypothalamic preoptic nucleus of Xenopus laevis.

The purpose of the present investigation was to provide a detailed description of the encephalic photoreceptors of Xenopus laevis at the light microscopic level and to determine their relationship with the neurosecretory cells of the hypothalamus in order to further our understanding of photoperiodic regulation of seasonal rhythms. Numerous opsin-positive neurons were found in the hypothalamic magnocellular preoptic nucleus and their axonal processes were seen to run laterally towards the basal regions of the brain, reaching the neural lobe of the hypophysis. Analysis of labelling with different antisera in adjacent sections, as well as double-immunolabelling carried out on the same section, revealed that mesotocin immunoreactivity was present in most of the opsin-positive neurons; however, no evidence for opsin and vasotocin coexpression was found in any of the sections analysed. The close localization of LHRH and opsin/mesotocin fibers in some regions of the brain, such as the median eminence, suggests that some interaction between these two systems might exist. In conclusion, in this study we provide the first strong evidence that the hypothalamic mesotocinergic neurons, which have been proved to be connected to the GnRH system in other species, are directly involved in photoreception in Xenopus laevis. These findings represent a novel contribution to our understanding of how light influences the seasonal reproductive cycles in lower vertebrates.