Preservation of human corneas in organ culture: technical protocol

Using organ-culture preservation human corneas were preserved up to 5 weeks in a modified tissue culture medium at 37 degrees C. The endothelial viability was examined after staining with trypan-blue 0.3%, after determination of endothelial cell density and cell loss and by light microscopy after staining with trypan-blue 0.3% and alizarin red 1%. The biochemical analysis of the medium (pH, glucose, lactate) has allowed the evaluation of three kinds of storage. The influence of time preservation on the endothelial viability and the metabolic conditions are determined. At the end of this study a clinical trial is proposed.     

Pharmacological Characterization of Bradykinin Receptors Coupled to Phosphoinositide Turnover in SV40-Immortalized Human Trabecular Meshwork Cells

This study sought to pharmacologically characterize bradykinin receptors on SV40-immortalized human trabecular meshwork (HTM3) cells. Phosphoinositide (PI) turnover studies were conducted using [³H]myo-inositol-labeled HTM3 cells and anion exchange chromatography to quantify [³H]inositol phosphates generated in response to bradykinin (BK) and various BK analogs. The blockade of these responses was studied using two potent and receptor-subtype selective antagonists. BK and T-kinin (Ile-Ser-BK; TK) induced a 4.2–4.4 fold stimulation of PI turnover above base levels at 1–10 μM. Several other peptides unrelated to BK, including angiotensin II, endothelin, cholecystokinin, bombesin and peptide YY tested at 1–10 μMwere essentially inactive. The molar potencies (EC₅₀) of BK, TK and close analogs were: BK=4.5±0.5 nM(n=6), Lys-BK=6.5±0.7 nM(n=3), TK=38.8±6.6 nM(n=8), Met-Lys-BK=41.5±13.4 nM(n=4), Des-Arg⁹-BK=2093±626 nM(n=4). All the latter BK-related peptides>were full agonists. The actions of BK and TK were potently and competitively antagonized by Hoe-140 (molar potency=0.6–1 nM;pA₂n=8.97–9.21,n=3–4) and byD-Arg⁰[Hyp³,-Thi^5,8,-DPhe⁷]-BK (molar potency=251 nM;-log potency, pK_b=6.6), two selective B₂-type BK antagonists. In conclusion, rank order of potency of BK agonists and the blockade of BK- and TK-induced PI turnover by the selective antagonists are consistent with the classification of the BK receptors on HTM3 cells as the B₂-receptor subtype.     

Purification and chemical modification of porcine bone morphogenetic protein

Implantation of porcine bone morphogenetic protein (pBMP) in the muscle induces differentiation of mesenchymal-type cells and results in endochondral bone formation. pBMP was isolated from porcine demineralized bone matrix and purified by hydroxyapatite chromatography, Sephadex G75 gel filtration, preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), preparative isoelectric focusing (IEF), and chromatofocusing fast protein liquid chromatography (FPLC). Porcine BMP has an MW of 26 K and a range of pI from 4.65 to 4.73 determined by SDS-PAGE and IEF, respectively. Reconstitution with the citrate buffer supernatant fraction enables as little as 50 micrograms of the soluble pBMP fractions to induce osteogenesis in an in vivo assay. Chemical modification studies indicate that the osteoinductive potential of the pBMP molecule depends on tyrosine, carboxyl groups, and disulfide bonds and can be increased by modification of sulfhydryl groups. Modification of arginine and tryptophan has no effect on bioactivity. By pepsin-limited proteolysis, fragments of pBMP with an MW of 6-14 K show definite, although reduced, BMP activity.     

Microvascular corrosion casting of normal tissue, transitional mucosa and adenocarcinoma in the human colon.

The microcirculatory architecture of normal tissue, transitional mucosa and adenocarcinoma of the human colon was investigated with microvascular corrosion casting (MVCC) combined with scanning electron microscopy (SEM). The study showed that the capillaries within the normal mucosa were arranged in a regular hexagonal pattern around the mucosal glands and that the microvessels of transitional mucosa mostly had lost the typical hexagonal pattern and become slightly wider in diameter. The microvessels in the tumor periphery were increased in number and disorganized, and presented large variation in morphology with claw-like formations, widened sinuses, diverticula and appendixoid patterns. Microvessels were lacking in the central areas of tumors. These morphological alterations may serve as additional indicators of tumor development.     

The Intubation Difficulty Scale (IDS): Proposal and Evaluation of a New Score Characterizing the Complexity of Endotracheal Intubation

Background: A quantitative scale of intubation difficulty would be useful for objectively comparing the complexity of endotracheal intubations. The authors have developed a quantitative score that can be used to evaluate intubating conditions and techniques with the aim of determining the relative values predictive factors of intubation difficulty and of the techniques used to decrease such difficulties.

Methods: An Intubation Difficulty Scale (IDS) was developed, based on parameters known to be associated with difficult intubation. It was then evaluated prospectively in a group of 311 consecutive prehospital intubations and 315 intubations in an operating room. In the operating room, the IDS was compared with two other parameters: the time to completion of intubation and the visual analog scale (VAS). Time was measured by an independent observer. Operators in both groups completed a checklist regarding the conditions of intubation.

Results: There is a good correlation between the IDS scale and the VAS assessment of difficulty and time to completion of intubation. VAS and time to completion have a significant but lesser correlation to each other. Comparison of IDS with operator-assessed subjective categorical impression of difficulty by Kruskall-Wallis was statistically significant.