Sequential development of intercellular junctions in bioengineered human corneas.

We have carried out a sequential study of intercellular junction formation and differentiation on human corneal substitutes consisting of an artificial corneal stroma and a corneal epithelium, developed by tissue engineering. To generate these artificial human corneas, we developed a corneal stroma substitute, using fibrin and agarose scaffolds with human keratocytes immersed within, then cultured the human corneal epithelium on top. Electron microscopy and immunofluorescence analyses revealed that artificial corneas with one or two epithelial cell layers did not show any formation of intercellular junctions. In contrast, several types of cell-cell junction, especially desmosomes, were found in multilayered mature corneal substitutes. Concomitantly, the expression of genes encoding for plakoglobin 3 (PKG3), desmoglein 3 (DSG3) and desmoplakin (DSP), zonula occludens 1 (ZO-1) and 2 (ZO-2) and connexin 37 (Cx37) was higher in multilayered artificial corneas than in immature artificial corneas, as shown by both microarray and immunofluorescence. Although expression of ZO-1, ZO-2 and Cx37 proteins was homogeneous, PKG3, DSG3 and DSP expression was restricted to the most apical cell layers in artificial corneas submerged in culture medium at all times, whereas expression was higher in intermediate cell layers, similar to normal human control corneas, when corneal substitutes are submitted to air-liquid culture techniques. These results suggest that cultured corneal substitutes submitted to air-liquid culture technique tend to form a well-developed epithelium that is very similar to the epithelium of human native corneas, suggesting that these artificial corneas could eventually be used for clinical or in vitro purposes.

Volumetric and ionic regulation during the in vitro development of a corneal endothelial barrier.

Corneal endothelium is responsible for generating an ion flux between the corneal stroma and the anterior chamber of the eye that is necessary for the cornea to remain transparent. However, the ion transport regulatory mechanisms that develop during the formation of the endothelial barrier are not known. In this study, we determined the influence of cell confluence on cell volume and intracellular ionic content on the corneal endothelial cells of rabbits. Our results demonstrate that non-confluent endothelial cells display a hypertrophic volume increase, with higher intracellular contents of potassium and chlorine than those of confluent cells. In contrast, when cells reach confluence and the endothelial barrier forms, cell volume decreases and the intracellular contents of potassium and chlorine decrease. Our genetic analysis showed a higher expression of CFTR and CA2 genes in non-confluent cells, and of the gene KCNC3 in confluent cells. These results suggest that the normal ionic current that keeps the corneal stroma dehydrated and transparent is regulated by cell-cell contacts and endothelial cell confluence, and could explain why the loss of corneal endothelial cells is often associated with corneal edema and even blindness.

Evaluation of the viability of cultured corneal endothelial cells by quantitative electron probe X-ray microanalysis.

Construction of artificial organs and tissues by tissue engineering is strongly dependent on the availability of viable cells. For that reason, the viability and the physiological status of cells kept in culture must be evaluated before the cells can be used for clinical purposes. In this work, we determined the viability of isolated rabbit corneal endothelial cells by trypan blue staining and quantitative electron probe X-ray microanalysis. Our results showed that the ionic content of potassium in cultured corneal endothelial cells tended to rise initially, but significantly decreased in cells in the fifth (and final) subculture, especially in comparison to cells in the fourth subculture (P < 0.001). However, the concentration of sulfur was higher in the fifth subculture than in the fourth subculture (P < 0.001), with a nonsignificant increase in sodium in the fifth subculture (P = 0.031). These data imply a remarkable decrease in the K/Na ratio from the fourth to the fifth subculture. Our microanalytical results, along with the morphological differences between cells in the last two subcultures, are compatible with an early phase of the preapoptotic process in the fifth subculture, and suggest that cells of the first four subcultures would be better candidates for tissue engineering.

[Increase of blood levels of creatine kinase following intramuscular injection]. / Elevación de las concentraciones séricas de creatincinasa tras inyección intramuscular.

BACKGROUND: To analyze the effect of intramuscular injections of drugs on serum creatine kinase (CK) and to evaluate the possible existence of factors that lead to predict the probability of appearance of serum CK increase after injection. PATIENTS, MATERIAL AND METHODS: One hundred and seventy six consecutive patients admitted to a short-term medical ward for non-cardiac reasons who had normal serum CK levels, were selected for the study. 120 of them, selected for random allocation, received a single 0 multiple injections of drugs (with broad use in emergency medicine) in the gluteal muscle (group IM). The remaining 56 patients were the group control, in all the cases serum CK was measured at 0, 6, 12, 24 and 48 h since the admission. RESULTS: A significant elevation of serum CK levels occurred in 58.3% cases of group IM. The higher ratio of cases with increment of CK levels was observed after the injection of chlorpromazine (100%), followed of diclofenac (74.2%), metamizol (60%) and multiple drugs (60%). In most cases the highest increment was observed at 12 h after injection. A tenfold increase in serum CK level was found after intramuscular injection of diclofenac. There were significant differences between the elevation or not of serum CK and the type of drug administered (p < 0.05), sex (p < 0.05), and the solvent of the drug injected (p < 0.05). A higher probability of serum CK elevation was observed in men (p = 0.009) and when the solvent of the drug injected was ethanol (p = 0.0499). CONCLUSIONS: There is a high probability of serum CK increment after intramuscular injection of drugs. The sex and the solvent of the drug injected have influence on this probability. The magnitude of this enzymatic increment depends on the kind of the drug injected, being non steroidal anti-inflammatory drugs those higher serum CK increment have been produced in the present study.

[Acute rhabdomyolysis and tetraparesis secondary to hypokalemia due to ingested licorice]. / Rabdomiólisis aguda y tetraparesia secundarias a hipocaliemia por ingesta de regaliz.

The glycirrinic acid, a common component of the natural licorice, has a potent mineralacorticoid effect (primary pseudohyperaldosteronism) which may cause severe hypokalemia and acute rhabdomyolysis. We present the case of a 36-years-old patient who, as the result of the intake of five daily licorice sticks (25 gr/day) for one month, developed analytical and clinical signs of acute rhabdomyolysis associated to the typical disorders of mineralcorticoid excess, that is, severe hypokalemia, arterial hypertension and metabolic alkalosis. The relevance of this clinical case lies on the low frequency of this finding and on the need that physicians working at emergency care centers must be aware of the onset of acute tetraparesis related to hypokalemia secondary to licorice ingesta. The early detection of this pathology is essential, since it will result in the beginning of an specific treatment, avoiding thus, as far as possible, the severe complications that might appear.

Cyproheptadine-induced remission of Cushing's disease due to pituitary basophil adenoma.

Serotonin is involved in the control of ACTH secretion, possibly by stimulating corticotropin releasing factor secretion from the hypothalamus. Cushing's disease seems to be due to defective hypothalamic regulation of ACTH release from the pituitary gland. Cyproheptadine is a potent antagonist of serotonin and has been used successfully in some patients with Cushing's disease, although, generally, in women without radiological evidence of pituitary tumors. We report the successful use of cyproheptadine in a 54-year-old man with Cushing's disease due to pituitary basophil adenoma. Significant clinical and biochemical improvement was noted 45 days after treatment began. The results in this patient support our findings that cyproheptadine can be effective in patients with Cushing's disease due to pituitary tumors, as well as in preparing very ill patients for surgery or managing such patients until radiotherapy takes effect.