María Sancho-Tello

Astrocytes in culture express the full-length Trk-B receptor and respond to brain derived neurotrophic factor by changing intracellular calcium levels: effect of ethanol exposure in rats.

Although cultured astroglial cells were reported to express exclusively the truncated non-catalytic Trk B receptor for brain-derived neurotrophic factor (BDNF), we detect here, using a sensitive ribonuclease protection assay, mRNAs for both truncated (TrkB-T) and the full length catalytic (TrkB-fl) form of BDNF receptor in developing cortical astrocytes and neurons in culture. Cortical neurons and immature astroglia, such as radial glia and proliferating astrocytes, express both the protein and mRNAs for TrkB-fl and TrkB-T, whereas the differentiation of astrocytes leads to a decrease in the trkB-fl mRNA, being the truncated TrkB the predominant receptor in differentiating and confluent astrocytes. The levels of TrkB-fl expression in proliferating and differentiating astrocytes and neurons correlates with the cell response to BDNF, monitored by the rise in intracellular [Ca(2+)](i). Foetal exposure to ethanol alters astroglial development and delays the reduction in trkB-fl mRNA levels observed with differentiation of astrocytes. These results demonstrate that immature astrocytes are able to express the catalytic Trk B receptors and to respond to BDNF with the activation of conventional signal transduction pathways. The results suggest that this signalling pathway is more activated in ethanol-exposed cells.

Early lipoic acid intake protects retina of diabetic mice.

The aim of this study was to test the effect of lipoic acid treatment on the retina after a short diabetic insult. Diabetes was induced by alloxan and mice were divided into sub-groups; control, diabetic, diabetic+insulin and all groups received±lipoic acid (100 mg/kg body weight) for 3 weeks. GSH content, MDA concentration, GPx activity were measured and electroretinograms (ERG) were recorded. Early administration of lipoic acid to diabetic mice prevented the statistically significant decreases of GSH content and GPx activity and normalized MDA concentration. Moreover, lipoic acid restored electroretinogram b-wave amplitude of diabetic animals to control values. Lipoic acid has a protective effect on the diabetic retina.

Intracellular Location, Temporal Expression, and Polysialylation Of Neural Cell Adhesion Molecule in Astrocytes in Primary Culture

Neural cell adhesion molecules (NCAMs) constitute a group of cell surface glycoproteins that control cell‐cell interactions and play important morphoregulatory roles in the developing and regenerating nervous system. NCAMs exist in a variety of isoforms differing in the cytoplasmic domain and/ or their content in sialic acid. The highly sialylated form (PSA‐NCAM) is expressed by neurons, whereas it is believed that the less sialylated NCAM forms are synthesised by astrocytes. Moreover, little is known about the molecular sequence of the events that contribute to its expression at the cell surface. Here we report that during the proliferation of cortical astrocytes, at 4 days in primary culture, these cells expressed PSA‐NCAM as well as NCAM 180. Then, during cell differentiation these isoforms progressively disappeared and the NCAM 140 became predominant. By immunofluorescence and immunocytochemistry studies we also show that PSA‐NCAM and NCAM are first observed in small cytoplasmic spots or vesicles, located in or near the Golgi apparatus, as demonstrated by their co‐localization with labelled wheat germ agglutinin (WGA) in this cell organelle. Thereafter, immunostained cytoplasmic NCAM gradually disappeared and became detectable at the cell surface of differentiating astrocytes. We also describe for the first time sialyltransferase activity in these cells and report that the levels of this activity correlated with the decrease in PSA‐NCAM expression during the differentiation of astrocytes. These results will contribute to our understanding of the PSA and NCAM intracellular transport pathways and their expression at the cell surface. Moreover, the presence of PSA‐NCAM in astrocytes suggests their possible role in nerve branching, fasciculation, and synaptic plasticity. GLIA 24:415–427, 1998.

CHRONIC ALCOHOL FEEDING INDUCES BIOCHEMICAL, HISTOLOGICAL, AND FUNCTIONAL ALTERATIONS IN RAT RETINA

AIMS Ethanol consumption originates a wide spectrum of disorders, including alteration of visual function. Oxidative stress is included among the mechanisms by which alcohol predisposes nervous tissue to injury. Retina, which is the neurosensorial eye tissue, is particularly sensitive to oxidative stress. METHODS In this study we analyze the effect of long-term alcohol consumption on oxidative stress parameters of the rat retina, and its correlation to retinal function, as well as to the expression of the antiapoptotic protein Bcl-2. We also study the protective effect of ebselen, a synthetic selenoorganic antioxidant. RESULTS Herein we show that ethanol has a toxic effect on rat retina associated with oxidative stress. Decreases in retina glutathione concentration and increases in malondialdehyde content in whole eye homogenate significantly correlate with ERG b-wave decrease and Bcl-2 overexpression. We also show how ebselen is able to prevent all the alterations observed. CONCLUSION Chronic ethanol consumption induces oxidative stress in rat retina associated with an impairment of ERG and Bcl-2 overexpression, suggesting a role for glial cells. All these alterations in the rat allow the proposal of an alcoholic retinopathy in this species.

Biomaterials coated by dental pulp cells as substrate for neural stem cell differentiation

This study is focused on the development of an in vitro hybrid system, consisting in a polymeric biomaterial covered by a dental pulp cellular stroma that acts as a scaffold offering a neurotrophic support for the subsequent survival and differentiation of neural stem cells. In the first place, the behavior of dental pulp stroma on the polymeric biomaterial based on ethyl acrylate and hydroxy ethyl acrylate copolymer was studied. For this purpose, cells from normal human third molars were grown onto 0.5-mm-diameter biomaterial discs. After cell culture, quantification of neurotrophic factors generated by the stromal cells was performed by means of an ELISA assay. In the second place, survival and differentiation of adult murine neural stem cells on the polymeric biomaterials covered by dental pulp stromal cells was studied. The results show the capacity of dental pulp cells to uniformly coat the majority of the materials surface and to secrete neurotrophic factors that become crucial for a subsequent differentiation of neural stem cells. The use of stromal cells cultured on scaffolding biomaterials provides neurotrophic pumps that may suggest new criteria for the design of cell therapy experiments in animal models to assist the repair of lesions in Central Nervous System.

Aqueous Humor Levels of Vascular Endothelial Growth Factor in Retinitis Pigmentosa

PURPOSE To determine the level of vascular endothelial growth factor A (VEGF-A) in aqueous humors of patients with retinitis pigmentosa (RP). METHODS A prospective, comparative control study. Aqueous humor was collected from 16 eyes of 16 patients with RP. The level of VEGF-A was determined with a commercially available enzyme-linked immunosorbent assay kit. The control group comprised 16 aqueous samples from 16 patients about to undergo cataract surgery and without any other ocular or systemic diseases. RESULTS The concentration of VEGF-A in aqueous humor was markedly lower in patients with RP than in control subjects (Mann-Whitney U test, P < 0.001). The level of VEGF-A was 94.9 +/- 99.8 (mean +/- SD) pg/mL in eyes with RP and 336.5 +/- 116.8 pg/mL in the eyes of the control group. CONCLUSIONS In patients with RP, the concentration of VEGF-A in aqueous humors is lower than in non-RP subjects. The lack of angiogenic actions attributed to VEGF-A may explain some of the clinical manifestations of this disease, such as narrowing and fibrotic degeneration of retinal blood vessels.

Low glutathione peroxidase in rdl mouse retina increases oxidative stress and proteases

Malondialdehyde, reduced glutathione, glutathione peroxidase, glutathione reductase and cysteine protease cathepsins at postnatal (PN) days 2, 7, 14, 21 and 28 in controls (wt) and the retinal degeneration 1 (rd1) mouse model for retinitis pigmentosa retinas were measured to determine oxidative stress. In PN28 wt and PN2 rd1 retinas, elevated malondialdehyde and low glutathione peroxidase activity indicate higher oxidative load, despite higher reduced glutathione in PN2 rd1 retinas. This is due to physiological exposure to light and retinal vascular/neural restructuring, respectively. Compared with wt retinas, relatively high malondialdehyde at PN2 and cathepsin levels at PN14, 21 and 28 in rd1 retinas indicate that cells of the residual inner retina also contribute to the oxidative stress and retinal degeneration.

Time evolution of in vivo articular cartilage repair induced by bone marrow stimulation and scaffold implantation in rabbits.

Purpose Tissue engineering techniques were used to study cartilage repair over a 12-month period in a rabbit model. Methods A full-depth chondral defect along with subchondral bone injury were originated in the knee joint, where a biostable porous scaffold was implanted, synthesized of poly(ethyl acrylate-co-hydroxyethyl acrylate) copolymer. Morphological evolution of cartilage repair was studied 1 and 2 weeks, and 1, 3, and 12 months after implantation by histological techniques. The 3-month group was chosen to compare cartilage repair to an additional group where scaffolds were preseeded with allogeneic chondrocytes before implantation, and also to controls, who underwent the same surgery procedure, with no scaffold implantation. Results Neotissue growth was first observed in the deepest scaffold pores 1 week after implantation, which spread thereafter; 3 months later scaffold pores were filled mostly with cartilaginous tissue in superficial and middle zones, and with bone tissue adjacent to subchondral bone. Simultaneously, native chondrocytes at the edges of the defect started to proliferate 1 week after implantation; within a month those edges had grown centripetally and seemed to embed the scaffold, and after 3 months, hyaline-like cartilage was observed on the condylar surface. Preseeded scaffolds slightly improved tissue growth, although the quality of repair tissue was similar to non-preseeded scaffolds. Controls showed that fibrous cartilage was mainly filling the repair area 3 months after surgery. In the 12-month group, articular cartilage resembled the untreated surface. Conclusions Scaffolds guided cartilaginous tissue growth in vivo, suggesting their importance in stress transmission to the cells for cartilage repair.

Comparison of the Acute Effects of anti-TNF-alpha Drugs on a Uveitis Experimental Model

Purpose: To compare histopathological and biochemical effects of the anti-TNF-alpha drugs adalimumab and infliximab in a uveitis experimental model. Methods: Histopathological evaluation was performed 24 h after endotoxin (200 μg into the footpad) and drug administration, as well as biochemical analysis of oxidative stress-related markers in the aqueous humor. Results: Severe inflammation was found in rat anterior chamber of the eye 24 h after endotoxin. Only infliximab administration partially prevented the endotoxin-induced disruption of the blood–aqueous barrier, as well as the increase in Rantes and MCP-1 concentration in aqueous humor. Both drugs ameliorated the histopathological score after endotoxin. Biochemical analysis revealed that both drugs protected against endotoxin-induced oxidative stress, restoring all markers to control levels, except infliximab, which failed to restore GSH concentration. Conclusions: Both anti-TNF-alpha drugs were effective in reducing histopathological inflammation but their mechanism of action appears to be different.

Implantation of a polycaprolactone scaffold with subchondral bone anchoring ameliorates nodules formation and other tissue alterations.

Purpose Articular cartilage has limited repair capacity. Two different implant devices for articular cartilage regeneration were tested in vivo in a sheep model to evaluate the effect of subchondral bone anchoring for tissue repair. Methods The implants were placed with press-fit technique in a cartilage defect after microfracture surgery in the femoral condyle of the knee joint of the sheep and histologic and mechanical evaluation was done 4.5 months later. The first group consisted of a biodegradable polycaprolactone (PCL) scaffold with double porosity. The second test group consisted of a PCL scaffold attached to a poly(L-lactic acid) (PLLA) pin anchored to the subchondral bone. Results For both groups most of the defects (75%) showed an articular surface that was completely or almost completely repaired with a neotissue. Nevertheless, the surface had a rougher appearance than controls and the repair tissue was immature. In the trials with solely scaffold implantation, severe subchondral bone alterations were seen with many large nodular formations. These alterations were ameliorated when implanting the scaffold with a subchondral bone anchoring pin. Discussions The results show that tissue repair is improved by implanting a PCL scaffold compared to solely microfracture surgery, and most importantly, that subchondral bone alterations, normally seen after microfracture surgery, were partially prevented when implanting the PCL scaffold with a fixation system to the subchondral bone.