Javier S. Recio

CD45 expression on rat acinar cells: involvement in pro-inflammatory cytokine production.

CD45 transduces activation signals in inflammatory cells. We investigate CD45 expression on pancreatic acinar cells and examine its role in the inflammatory response which these cells have also shown under certain circumstances. Similar CD45 mRNA levels were found in acinar cells and leukocytes (positive control). Flowcytometric and immunohistochemical analysis showed a heterogeneous CD45 distribution on acinar cells. Activation of acinar cells by incubation with pancreatitis‐associated ascitic fluid as evidencied by TNF‐〈alpha〉 production resulted in a decreased CD45 expression, suggesting that CD45 acts as a negative regulator of cytokine production. As a validation of this finding in vivo, a decrease in the acinar CD45 expression in parallel with an increased ability to produce TNF‐〈alpha〉 was found in rats with acute pancreatitis. Our data show that CD45 is constitutively expressed in acinar cells and suggest that it plays an important role in negatively regulating cytokine production.

Long-lasting changes in the anatomy of the olfactory bulb after ionizing irradiation and bone marrow transplantation.

The adult brain is considered to be a radioresistant organ since it is mainly composed of non-dividing cells. However, in adult animals there are a few neurogenic brain areas that are affected by ionizing radiation whose plasticity and capacity for recovery are still unclear. Here, mice were irradiated with a minimal lethal dose of radiation in order to determine its effects on the subventricular zone (SVZ), the rostral migratory stream (RMS), and the olfactory bulb (OB). These regions underwent a dramatic reduction in cell proliferation and ensuing morphological alterations, accompanied by a patent reactive gliosis. Bone marrow stem cell (BMSC) transplants were also performed after the radiation treatment to allow the mouse survival with a view to analyzing long-term effects. Normal proliferation rates were not recovered over time and although bone marrow-derived cells reached the brain, they were not incorporated into the SVZ-RMS-OB pathway in an attempt to rescue the damaged regions. Since neurogenesis produces new interneurones in the OB, thus feeding cell turnover, the volume and lamination of the OB were analyzed. The volume of the OB proved to be dramatically reduced at postnatal day 300 (P300), and this shrinkage affected the periependymal white matter, the granule cell layer, the external plexiform layer, and the glomerular layer. These results should be taken into account in cell therapies employing BMSC, since such cells reach the encephalon, although they cannot restore the damage produced in neurogenic areas. This study thus provides new insight into the long-term effects of ionizing radiation, widely employed in animal experimentation and even in clinical therapies for human beings.

Bone Marrow Contributes Simultaneously to Different Neural Types in the Central Nervous System Through Different Mechanisms of Plasticity

Many studies have reported the contribution of bone marrow-derived cells (BMDC) to the CNS, raising the possibility of using them as a new source to repair damaged brain tissue or restore neuronal function. This process has mainly been investigated in the cerebellum, in which a degenerative microenvironment has been suggested to be responsible for its modulation. The present study further analyzes the contribution of BMDC to different neural types in other adult brain areas, under both physiological and neurodegenerative conditions, together with the mechanisms of plasticity involved. We grafted genetically marked green fluorescent protein/Cre bone marrow in irradiated recipients: a) the PCD (Purkinje Cell Degeneration) mutant mice, suffering a degeneration of specific neuronal populations at different ages, and b) their corresponding healthy controls. These mice carried the conditional lacZ reporter gene to allow the identification of cell fusion events. Our results demonstrate that BMDC mainly generate microglial cells, although to a lesser extent a clear formation of neuronal types also exists. This neuronal recruitment was not increased by the neurodegenerative processes occurring in PCD mice, where BMDC did not contribute to rescuing the degenerated neuronal populations either. However, an increase in the number of bone marrow-derived microglia was found along the life span in both experimental groups. Six weeks after transplantation more bone marrow-derived microglial cells were observed in the olfactory bulb of the PCD mice compared to the control animals, where the degeneration of mitral cells was in process. In contrast, this difference was not observed in the cerebellum, where Purkinje cell degeneration had been completed. These findings demonstrated that the degree of neurodegenerative environment can foster the recruitment of neural elements derived from bone marrow, but also provide the first evidence that BMDC can contribute simultaneously to different encephalic areas through different mechanisms of plasticity: cell fusion for Purkinje cells and differentiation for olfactory bulb interneurons.

Albumin attenuates DNA damage in primary-cultured neurons.

Human serum albumin (HSA) is an effective therapeutic agent that protects neurons after cerebral ischemia or related injuries by means of its antioxidant capacity. Our aim was to test whether bovine serum albumin (BSA) might also provide protection, especially against DNA damage. Rat cortical neurons were cultured in both the presence and absence of BSA. To test the neuroprotective role of BSA against DNA damage and neuronal death, primary cultures were investigated using both gamma-H2AX and pATM immunocytochemistry, and the TUNEL assay, respectively. Quantitative analyses revealed that the cultures in the absence of BSA had a higher number of apoptotic neurons. Additionally, neurons showing DNA strand breaks were fewer when BSA was added to the medium. BSA acts as a neuroprotective molecule, reducing both the DNA damage and apoptosis rates. This effect is similar to that described for HSA, probably due to its antioxidant activity. Hence, we have demonstrated that BSA provides a neuroprotective role when DNA damage occurs. Additionally, we suggest that BSA probably shares similarities with HSA in its antioxidant activity, opening new ways in the study of stroke and related brain diseases.

Mild cerebellar neurodegeneration of aged heterozygous PCD mice increases cell fusion of Purkinje and bone marrow-derived cells.

Bone marrow-derived cells have different plastic properties, especially regarding cell fusion, which increases with time and is prompted by tissue injury. Several recessive mutations, including Purkinje Cell Degeneration, affect the number of Purkinje cells in homozygosis; heterozygous young animals have an apparently normal phenotype but they undergo Purkinje cell loss as they age. Our findings demonstrate that heterozygous pcd mice undergo Purkinje cell loss at postnatal day 300, this slow but steadily progressing cell death starting sooner than has been reported previously and without massive reactive gliosis or inflammation. Here, transplantation of bone marrow stem cells was performed to assess the arrival of bone marrow-derived cells in the cerebellum in these heterozygous mice. Our results reveal that a higher number of cell fusion events occurs in heterozygous animals than in the controls, on days 150 and 300 postnatally. In sum, this study indicates that mild cell death promotes the fusion of bone marrow-derived cells with surviving Purkinje neurons. This phenomenon suggests new therapies for long-lasting neurodegenerative disorders.

Sex differences in catechol contents in the olfactory bulb of control and unilaterally deprived rats

The dopaminergic system plays important roles in the modulation of olfactory transmission. The present study examines the distribution of dopaminergic cells and the content of dopamine (DA) and its metabolites in control and deprived olfactory bulbs (OB), focusing on the differences between sexes. The content of DA and of its metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), were measured by HPLC. The morphology and distribution of dopaminergic neurons were studied using tyrosine hydroxylase (TH) immunohistochemistry. Cells were typified with TH–parvalbumin, TH–cholecystokinin or TH–neurocalcin double‐immunofluorescence assays. Biochemical analyses revealed sex differences in the content of DA and of its metabolites. In normal conditions, the OBs of male rats had higher concentrations of DA, DOPAC and HVA than the OBs of females. The immunohistochemical data pointed to sex differences in the number of TH‐immunopositive cells (higher in male than in female rats). Colocalization analyses revealed that dopaminergic cells constitute a different cell subpopulation from those labelled after parvalbumin, cholecystokinin or neurocalcin immunostaining. Unilateral olfactory deprivation caused dramatic alterations in the dopaminergic system. The DA content and the density of dopaminergic cells decreased, the contents of DA and DOPAC as well as TH immunoreactivity were similar in deprived males and females and, finally, the metabolite/neurotransmitter ratio increased. Our results show that the dopaminergic modulation of olfactory transmission seems to differ between males and females and that it is regulated by peripheral olfactory activity. A possible role of the dopaminergic system in the sexually different olfactory sensitivity, discrimination and memory is discussed.

Changes in the connections of the main olfactory bulb after mitral cell selective neurodegeneration

The connections of the main olfactory bulb (OB) of the mouse were studied with iontophoretic injections of biotinylated dextran amine. To sort efferences from mitral cells and tufted cells, the Purkinje cell degeneration (PCD) mouse was used. This mutant animal undergoes a specific neurodegeneration of mitral cells, whereas tufted cells do not degenerate. The unilateral tracer injections used were small and confined largely to the OB of both PCD and control mice at P120. Seven days after tracer injection, the efferences from the OB and the centrifugal afferences from secondary olfactory structures to it were studied. Although there is a large overlap of their target fields, mitral cell axons innervated more caudal regions of the olfactory cortex than tufted cell axons, thus providing definitive evidence of the differential projections of olfactory output neurons. Additionally, an important increase in retrogradely‐labeled neurons was detected in the ipsilateral anterior olfactory nucleus of the mutant animals. This was not observed in any other secondary olfactory structure, suggesting a strengthening of the centrifugal input to the OB from that central area after mitral cell loss. Moreover, we recorded a complete loss of bilaterality in the olfactory connections of the PCD mice due to degeneration of the anterior commissure. These results point to an important reorganization of this essential olfactory circuit between the anterior olfactory nucleus and the OB, and hint at a transsynaptic level of plasticity not considered previously in literature.

Effect of dexamethasone on peripheral blood leukocyte immune response in bile-pancreatic duct obstruction-induced acute pancreatitis

Our aim was to analyze the effects of dexamethasone (Dx) (1mg/kg), prophylactically or therapeutically administered, on the inflammatory response triggered by peripheral blood leukocytes during acute pancreatitis (AP) induced in rats by bile-pancreatic duct obstruction (BPDO) and their consequences in the progress of the disease. Flow cytometry was used to analyze the distribution of the major leukocyte populations, the CD45 expression and the activated state of monocytes as reflected by the membrane-bound intercellular adhesion molecule-1 (ICAM-1) and the production of tumor necrosis factor-alpha (TNF-alpha) and monocyte chemoattract protein-1 (MCP-1) in response to lipopolysaccaride (LPS). Interleukin-6 (IL-6) plasma levels, pancreatic fluid content and histology of pancreas sections were also evaluated. Dx, given either before or after AP, blunted the monocyte increase induced by BPDO-induced AP, but did not change lymphocyte and neutrophil counts. Membrane-bound ICAM-1 expression did not vary in circulating monocytes during BPDO, either in Dx-treated or non-treated rats. Both Dx treatments inhibited TNF-alpha and MCP-1 production in non-stimulated and LPS-stimulated monocytes, whose response was found to be higher than in controls from early AP. Leukocyte CD45 expression was found to be reduced in rats with AP and shifted to control values in Dx-post-treated rats. Cytokinemia as well as pancreatic edema and leukocyte infiltration found in BPDO rats were reduced by Dx given either before or after AP. We conclude that prophylactic and therapeutic Dx treatments inhibited the inflammatory response triggered by circulating leukocytes in rats with BPDO-induced AP, thus contributing to reducing the severity of the disease.

Zincergic innervation from the anterior olfactory nucleus to the olfactory bulb displays plastic responses after mitral cell loss.

Zinc ions are selectively accumulated in certain neurons (zinc-enriched neurons). The mouse olfactory bulb is richly innervated by zinc-enriched terminals. Here, the plasticity of the zincergic system was studied in the olfactory bulb of the Purkinje Cell Degeneration mutant mouse, an animal with specific postnatal neurodegeneration of the main projection neurons of the olfactory bulb. The analysis focused particularly on the anterior olfactory nucleus since most centrifugal afferents coming to the olfactory bulb arise from this structure. Zinc-enriched terminals in the olfactory bulb and zinc-enriched somata in the anterior olfactory nucleus were visualized after selenite injections. Immunohistochemistry against the vesicular zinc transporter was also carried out to confirm the distribution pattern of zinc-enriched terminals in the olfactory bulb. The mutant mice showed a clear reorganization of zincergic centrifugal projections from the anterior olfactory nucleus to the olfactory bulb. First, all zincergic contralateral neurons projecting to the olfactory bulb were absent in the mutant mice. Second, a significant increase in the number of stained somata was detected in the ipsilateral anterior olfactory nucleus. Since no noticeable changes were observed in the zinc-enriched terminals in the olfactory bulb, it is conceivable that mitral cell loss could induce a reorganization of zinc-enriched projections coming from the anterior olfactory nucleus, probably directed at balancing the global zincergic centrifugal modulation. These results show that zincergic anterior olfactory nucleus cells projecting to the olfactory bulb undergo plastic changes to adapt to the loss of mitral cells in the olfactory bulb of Purkinje Cell Degeneration mutant mice.

Bone marrow transplantation improves motor activity in a mouse model of ataxia

Ataxias are locomotor disorders that can have an origin both neural and muscular, although both impairments are related. Unfortunately, ataxia has no cure, and the current therapies are aimed at motor re‐education or muscular reinforcement. Nevertheless, cell therapy is becoming a promising approach to deal with incurable neural diseases, including neuromuscular ataxias. Here, we have used a model of ataxia, the Purkinje Cell Degeneration (PCD) mutant mouse, to study the effect of healthy (wild‐type) bone marrow transplantation on the restoration of defective mobility. Bone marrow transplants (from both mutant and healthy donors) were performed in wild‐type and PCD mice. Then, a wide battery of behavioural tests was employed to determine possible motor amelioration in mutants. Finally, cerebellum, spinal cord, and muscle were analysed to study the integration of the transplant‐derived cells and the origin of the behavioural changes. Our results demonstrated that the transplant of wild‐type bone marrow restores the mobility of PCD mice, increasing their capabilities of movement (52–100% of recovery), exploration (20–71% of recovery), speed (35% of recovery), and motor coordination (25% of recovery). Surprisingly, our results showed that bone marrow transplant notably improves the skeletal muscle structure, which is severely damaged in the mutants, rather than ameliorating the central nervous system. Although a multimodal effect of the transplant is not discarded, muscular improvements appear to be the basis of this motor recovery. Furthermore, the results from our study indicate that bone marrow stem cell therapy can be a safe and effective alternative for dealing with movement disorders such as ataxias.