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A general technique is described for using slide-mounted unfixed tissue sections to characterize and visualize drug and neurotransmitter receptors in brain or other tissues. The preparation of material, from fresh frozen, unfixed brain to dried sections securely attached to slides, is described in detail. The tissue can be kept intact during incubation at varying temperatures in solutions containing radiolabeled ligand, ions, buffers, and allosteric effectors. Strategies are described for determining optimal stereospecific binding with highest signal-to-noise ratios and for determining that a "meaningful" receptor is being studied. Dry formaldehyde fixation by vapors from heated paraformaldehyde preserves the tissue quality and traps the ligand near its site on the receptor, permitting subsequent histological processing through alcohols, solvents, and aqueous media, including liquid nuclear track emulsion. Visualization of [3H]naloxone- or [3H]enkephalin-labeled opiate receptor distributions in rat and human brains is achieved by tritium-sensitive film or by classical "wet" emulsion autoradiography. The advantages of the film include its ease of use and the ability to quantify receptor density by densitometry which can be computer-assisted. The advantage of the emulsion is the greater resolution and the concomitant appearance of morphology in cell-stained sections. Examples of correlations of opiate receptor distributions which underlying cytoarchitecture illustrate the potential for receptor localization studies.
The isolated rat hindquarter preparation often used for physiological studies was subjected to morphological examination. A two-hour perfusion with balanced oxygenated dextran solution was followed by perfusion fixation with aldehydes. Fixation was performed at isogravimetry when an isotonic fixative was used (2.5% glutaraldehyde in 0.05 M Na cacodylate + 4% dextran 70). Muscle specimens were prepared for light and electron microscopy. Most muscle tissue was well perfused but a limited tissue volume could retain blood after the perfusion period. Within perfused tissue the perfusion result varied macroscopically with some regions apparently not being reached by fixative. Non-fixed tissue was estimated to amount to maximally one fourth of the total tissue mass. Also within macroscopically well-fixed tissue the microscopy indicated varying patency of perfused capillaries. Thus a marked perfusion flow heterogeneity prevailed in the fixed specimens which is compatible with earlier functional findings of a 50% reduction of capillary surface area during isogravimetric fixation. The possible relation with prefixation flow heterogeneity in the vasodilated preparation is discussed. There were no obvious alterations of endothelial ultrastructure after prolonged artificial perfusion. Isogravimetrically fixed muscle had narrow extracellular spaces which were evidently compressed further after use of absorbing hyperosmolar fixative. Extracellular expansion and intracellular oedema followed after hypo-osmolar fixation. Muscle cells often exhibited insufficient mitochondrial fixation, probably as a result of tissue hypoxia.
Today, most tooth replacement in the esthetic zone is done using implants placed in a delayed surgical protocol. Unfortunately, this delay can result in loss of both hard and soft tissue during the healing period, necessitating guided tissue regeneration techniques at the time of implant placement. Recent developments with tapered implants have facilitated predictable immediate implant placement, preserving the osseous structure surrounding the socket. Further developments with custom healing abutments can preserve the crestal soft tissues, including the papillae. This article reviews techniques that provide for the preservation of both bone and soft tissue while enhancing the esthetic results around implants.
It is well recognized that the clarity of the cornea is a function of its hydration, and that this hydration is controlled by a "pump-and-leak" mechanism operating across the posterior monolayer of cells called the endothelium. A breakdown of the endothelium through disease or injury causes a marked increase in corneal thickness as the stroma imbibes fluid from the aqueous humor in the anterior chamber of the eye. This thickened, edematous condition of the stroma results in a cloudy cornea with an associated marked decrease in visual acuity. Treatment for this condition is usually by full-thickness corneal transplantation (penetrating keratoplasty), the success of which is dependent upon the donor cornea having an intact and healthy endothelium. It is essential, therefore, that any method of corneal storage for penetrating keratoplasty should protect and preserve the endothelium in a viable state. Current clinical practice relies upon short-term methods of preservation by two principal methods. Moist Chamber Storage is the time-honored corneal preservation method; it consists of keeping enucleated eyes at 0-4 degrees C in a sealed jar containing a pad of cotton gauze soaked in saline to provide a humid environment. The time limit placed upon this method of storage is 24-48 hr after which the viability of the endothelium deteriorates rapidly. Storage in M-K (McCarey-Kaufman) Medium involves excision of the corneoscleral segment from the donor eye and immersing it, endothelial side uppermost, in a medium consisting of tissue culture medium, 5% Dextran 40, and antibiotics. Laboratory and clinical studies indicate that storage in M-K medium at 4 degrees C preserves human endothelial cells for up to 4 days when the eye has been removed from the cadaver in less than 10 hr postmortem. Long-term preservation of corneas by freezing has long been a major goal in eye banking because indefinite storage by cryopreservation offers significant advantages for the quality and the quantity of material for use in keratoplasty, as well as for its distribution. However, procedures that have been developed for the cryopreservation of corneas have not been widely used, and a number of studies have shown that these procedures are inadequate for maintaining the integrity of the corneal endothelium. Not surprisingly, clinicians are now reluctant to accept corneas that have been frozen by these methods, though the clinical need is now greater than ever.(ABSTRACT TRUNCATED AT 400 WORDS)
Decompressive craniotomies with removal of the bone flap are still frequently performed in brain injured patients. Usually these bone flaps are refrigerated until reimplantation. In approximately 60% of cranioplasties performed in our clinic by this method we have observed considerable osteolysis especially in the marginal regions. This resulted in a instability which disturbed many patients and required an additional cranioplasty. Other methods for cranioplasty are briefly described. - For the last 3 years we have implanted the removed bone flaps immediately after craniotomy subcutaneously in the abdominal wall instead of preserving them in the refrigerator. By these means we were able to reimplant vital bone during the cranioplasty procedure weeks to months after the initial craniotomy. Our experience with this method shows a significant decrease of resorption in autologous cranioplasties. Out of 43 cases with a follow-up control after one and two years only 2 patients showed signs of resorption. One other bone flap was lost due to infection.
The requirement for unfixed tissue is a major drawback in the use of immunohistochemistry for the diagnosis of inflammatory and neoplastic disease. We describe the use of a novel gel transport medium (U.K. Patent No. WO 90/07703, international patent applications pending) to preserve unfixed skin biopsies from allergic contact dermatitis reactions for 1 week prior to frozen section and immunohistochemistry for leucocyte antigens. The medium can be used with biopsies up to 6 mm3 and does not require any alteration in the usual frozen section or immunohistochemical staining procedures. The results show a subjective improvement in both morphology and staining quality. We believe this to be due to improved cutting of the sections and reduced background staining of collagen. This transport medium should be of considerable benefit to the provision of a clinical service, because it allows tissue for immunohistochemical examination to be taken at locations distant from the pathology laboratory.
To evaluate the optimum conditions for osteogenesis during limb lengthening and to study the changes in soft tissues undergoing elongation, a series of experiments were performed on the canine tibia. The experiments used the transfixion-wire, Ilizarov circular external skeletal fixator in configurations of differing stability of fixation in combination with a second variable, i.e., preservation of the periosteum, bone marrow, and medullary blood supply. Both increased fixator stability, and maximum preservation of the periosseous and intraosseous soft tissues enhanced bone formation during limb lengthening. To assess the role that the direction of the elongation vector plays in osteogenesis, canine tibiae were widened rather than lengthened in a second series of experiments using an Ilizarov apparatus modified for lateral distraction. The new bone formed parallel to the tension vector even when perpendicular to the bone's mechanical axis. As in longitudinal lengthening, damage to the bone marrow inhibits osteogenesis occurring by the influence of a lateral tension-stress vector. In a third series of experiments, half- and full-circumference cortical defects were created in canine tibiae to study the osteogenic potential of the marrow. New bone formed rapidly, even when the marrow was separated from the surrounding periosseous soft tissues by a sheet of polyvinyl chloride, attesting to the importance of marrow element preservation during osteotomy for limb lengthening.
Some primary and secondary liver tumours are not absolutely irresectable, but cannot be resected using a conventional approach because of the limited warm ischaemia tolerance of the liver or poor accessibility of the tumour region. In such situations, the techniques of ex vivo liver surgery, pioneered by Rudolf Pichlmayr some 10 years ago, offer new chances for R0 resection. All the three different approaches, namely "in situ"-, "ante situm"-, and "ex situ" resection, require the use of measures originally developed for transplantation, such as hypothermic liver perfusion and veno-venous bypass. They differ mainly in the extent to which major vessels are divided in order to achieve optimal mobility of the organ. The results show that radical resection can be achieved accomplished in many cases. If necessary, complex vascular reconstructions can be performed. Although perioperative morbidity and mortality are high, there are a number of long-term survivors. Tumour recurrence, however, remains the main problem over the long term. In conclusion, ex vivo liver surgery is an important extension of surgical treatment possibilities. However, the procedure is suitable only for a small number of carefully selected patients and should be reserved for use in specialised centres. Furthermore, in view of the fact that the results are not yet optimal, additive and adjuvant treatment modalities are needed.
'Articulated' rhynchonelliformean brachiopods are abundant shelly fossils, but the direct fossil record of their soft parts was hitherto confined to a single pyritized trace possibly representing a lophophore. Anatomical knowledge of extinct rhynchonelliformeans relies heavily on analogies to extant species; these analogies are untested for stem-group clades. The Silurian Herefordshire (UK) Konservat-Lagerstätte (about 425 Myr bp) yields exceptionally preserved three-dimensional fossils that provide unrivalled insights into the palaeobiology of a variety of invertebrates. The fossils are preserved as calcitic void in-fills in carbonate concretions within a volcaniclastic horizon, and are reconstructed digitally. Here we describe a stem-group rhynchonelliformean specimen from this deposit; it most probably belongs in the order Orthida. A robust ridged pedicle with distal rootlets is preserved, together with a lophophore and other soft-tissue structures. The pedicle morphology is novel, urging caution in inferring stem-group rhynchonelliformean anatomy from that of crown-group species. Smaller brachiopods are attached to the specimen; these include a probable atrypide, with pedicle and marginal setae preserved.
A technique is described for extraction of bulky tissue during laparoscopic surgery using a dedicated non-disposable instrument specifically designed for the purpose. The technique was used on seven patients with acute cholecystitis, three patients with big gallstones in elective cholecystectomy and two patients with acute appendicitis. The procedure took about 1 min in all patients. No patient suffered from complications related to the procedure.
In the present study the functional and morphologic effects of two pulmoplegic solutions are evaluated. Single left-lung allotransplantation with ligation of the right pulmonary artery was performed in 15 piglets (13-20 kg). The lungs were preserved after donor prostaglandin E-1 treatment with single pulmonary artery flush with either modified Euro-Collins solution (mECS) (9 pigs) or oxygenated fluorocarbon emulsion (FC-43) (6 pigs) and transplanted after 6-hr storage in cold Physiosol solution. Tidal volumes of 15 ml/kg x fr (18) with 40% inspired oxygen were used for ventilation during reperfusion. Function of the transplanted lung was monitored for 4 hr postoperatively by determining pa CO2 and pa O2 levels from arterial samples and by noninvasive monitoring of end-tidal CO2 values and arterial oxygen saturations. Sequential morphologic changes in pulmonary artery flow surface and lung tissue were studied after 6-hr storage and 4-hr reperfusion, using light, scanning, and transmission electron microscopy (LM, SEM, TEM). There was no mortality. After transplantation the mECS group experienced significant hypoxia and hypercarbia and had low end-tidal CO2 values as signs of defective oxygenation and gas exchange, whereas the FC-43 group was normoxic and normoventilated without disturbed elimination of carbon dioxide. After storage and reperfusion, LM showed signs of increased vascular permeability and reperfusion damage--more evident in the mECS group compared with the FC-43 group--while the lymphoid cell population was more intensely activated in the latter group. Electron microscopy after storage showed good overall preservation of structures in both groups. After reperfusion preservation of pulmonary artery flow surface and lung tissue was estimated to be moderate in the mECS group, whereas it was good-to-moderate in the FC-43 group by SEM (NS). TEM of lung tissue, however, showed significantly better-preserved alveolar epithelial lining in the FC-43 group compared with the mECS group. In conclusion, oxygenated fluorocarbon (FC-43) pulmoplegia gave better functional and morphologic preservation of lung grafts compared with modified Euro-Collins solution.
It was established that plant tissues killed by various aldehydes in low concentrations, which evoked general enzyme inactivation for a long time retain permeability and lipoprotein bonds in the cell membranes. Such preparations from Elodea Canadensis leaves are used for: Cu, Zn, Pb, Hg toxical effect investigation. It was stated by chemoluminescence methods that these ions induce slow developed oxidative radical chain reaction in lipid cell membranes and break the lipoprotein bonds.
OBJECTIVE: To compare the bone graft cryopreservation method (at -80 degrees C) with a preservation method using a 98% glycerol solution at room temperature (10 degrees C-35 degrees C), by testing the antibacterial and fungal effects of 98% glycerol and comparatively analyzing the observed histological changes resulting from the use of both methods. METHOD: This study was of 30 samples of trabecular bone tissue from 10 patients undergoing total hip arthroplasty. Each femoral head provided 3 samples that were randomized into 3 groups, namely, the control group, the cryopreserved group, and the group preserved in a 98% glycerol at room temperature for 1 year. The samples were submitted to histomorphologic, cell feasibility, and microbiologic analyses. The results were statistically analyzed using the McNemar test, with a statistical significance index of 0.05. RESULTS: Values obtained using the McNemar test to compare probability distributions of histomorphologic variables (mature or lamellar bone, immature bone, and necrosis) and cell feasibility (osteoblasts and osteoclasts) indicated that there is no difference between the distributions of variables under the 3 experimental conditions. Microbiological analysis of the 98% glycerol solution and bone fragments from samples stored for 1 year at room temperature did not show bacterial or fungal growth. The histological and microbiological investigation were performed at 2 different time points: immediately after the sample processing and after 1 year. CONCLUSION: The method used to preserve bone grafts kept in 98% glycerol at room temperature (10 degrees C-35 degrees C) was similar to cryopreservation in terms of bone matrix preservation; no bacteria or fungi were found in the samples.