Effects of hypothermic storage on the vascular endothelium: a scanning electron microscope study of morphological change in human vein.

OBJECTIVE: The aims of this study were: i) to identify morphological changes occurring in the endothelium of human umbilical veins subjected to the typical storage procedures used in transplantation and ii) to determine the relative efficacy of preservation solutions containing intra and extracellular levels of sodium and potassium. EXPERIMENTAL DESIGN: Prospective. PROCEDURE: Scanning electron micrographs were taken pre and post cold hypoxic storage of human umbilical veins for 3 or 16 hours. RESULTS: Cold preservation resulted in severe cell detachment with subsequent loss of monolayer continuity and exposure of thrombogenic basal membrane components (highly significant after only 3 hours of cold storage, Kruskal-Wallis, p < 0.01). The morphological alterations culminated in EC with spherical shapes. Cytoplasmic membranes presented an increased number of microvilli and intercellular processes, followed by microvillous swelling and surface blebbing as damage increased. Bleb detachment was seen in severely damaged specimens. However, morphological preservation was not significantly affected by the duration of hypoxia or the ionic balance of the solution tested. CONCLUSIONS: The present results demonstrate that even short periods (3 hours) of cold storage without revascularization cause significant morphological damage to the endothelium. The ionic composition of the preservation solution did not significantly affect the process. The morphological changes seen in this study could explain storage-related problems such as loss of normal vascular permeability and increased thrombogenicity, problems often associated with transplantation procedures.

Scanning electron microscopic changes in morphology of pulmonary endothelium in rat lung isografts following hypothermic ischaemic storage and transplantation.

Endothelial monolayer integrity is a critical factor limiting vascular permeability of solid organs in transplantation. Several in vitro, ex vivo and in vivo studies suggest that damage to endothelial cells (EC) due to hypothermia and ischaemia-reperfusion injury causes morphological and functional damage to the endothelium leading to parenchymal oedema and haemorrhage. Aiming to study morphological changes to arterial pulmonary EC subjected to transplantation procedures, random scanning electron micrographs of vascular endothelium of rat lungs were taken. Forty-eight rat lungs were hypothermically stored for 48 or 72 hours in two different preservation solutions and studied either at the end of the cold storage period, or 5 min, 24 h or 4 weeks following transplantation. After 5 minutes of revascularization, micrographs showed EC shape variations, bleb formation and cell retraction with intercellular gap formation. Twenty-four hours after transplantation loss of monolayer continuity was widely extended. Four weeks of revascularization resulted in either well preserved specimens with nearly normal endothelium, or badly preserved arteries with fibrotic degeneration of the luminal vessel wall. The morphological disruptions found in this study help to explain the alterations in permeability control and vascular dysfunction observed in lung transplantation.

Dantrolene sodium protects against experimental ischemia and reperfusion damage to skeletal muscle.

The effect of 4 hours of ischemia followed by reperfusion for 1 hour has been studied in fully anesthetized rabbits. Muscles from the limb subjected to ischemia and reperfusion showed considerable ultrastructural damage, although the distribution of damage between muscles was not uniform (anterior tibialis > soleus > quadriceps). Damage to the muscle was associated with a significant increase in the concentration of some indicators of free radical-mediated processes (thiobarbituric acid-reactive substances and diene conjugates), although others (glutathione and protein sulfhydryl groups) were unchanged. Reperfused muscles also showed considerable changes in their calcium and sodium contents. Treatment of animals with dantrolene sodium (4 mg/hr) throughout the periods of ischemia and reperfusion was found to preserve the ultrastructural appearance of quadriceps, soleus and anterior tibialis muscles. No effect of dantrolene sodium on indicators of free radical activity or muscle cation content was seen.

Morphological changes in rat single lung isografts after long-term survival.

A high priority in organ transplantation research is to increase the number of hours that an organ can be successfully preserved. Transplant programmes rely on hypothermia and flush solutions to maintain organ viability during the storage period. We studied long-term morphology in lungs stored for 24 or 48 hours using two modified versions of University of Wisconsin solution, one mimicking the extracellular medium and the other the intracellular medium. Four weeks after transplantation, X-ray and angiograms were used to assess the proportion of ventilating tissue, and light and electron microscopy to analyse morphology. Pulmonary tissue presented near-normal histological appearance in well preserved areas while fibrosis and chronic inflammation were found in scarring processes. Electron microscopy studies revealed some damage-related changes in tissue which appeared histologically normal. Four weeks after transplantation, quality and quantity of recovery were uniform for both solutions tested after 24 hours of storage. However, without reaching significance, after 48 hours the quantity of successfully preserved pulmonary tissue was greater in the group stored in the intracellular solution.

Cold (0 degree C) ischaemic tolerance of latissimus dorsi free flaps in rats: a macroscopic and morphological study.

The purpose of this study was to define the cold (0 degree C) ischaemic tolerance (CIT) of the latissimus dorsi free flap in inbred rats. Flaps were isolated, stored at 0 degree C for periods of time ranging from 0 to 120 hr and then revascularised and reperfused for 14 days. Thereafter the CIT was assessed macroscopically, and by light microscopy. Electron microscopy was performed in flaps after 0 and 24 hr of reperfusion following a cold ischaemic interval ranging from 0 to 120 hr. From this preliminary study, it was concluded that the CIT was 48-72 hr.

A porcine model using skin graft chambers for studies on cultured keratinocytes.

In wound healing research, animal models permit an extensive tissue analysis which is not normally possible in clinical studies. A morphological comparison of human and porcine skin was made in order to identify those aspects of the wound healing process where a porcine model may help our understanding of clinical problems. We describe a porcine model for evaluating the growth of cultured keratinocytes on a variety of wound beds. Polytetrafluoroethylene skin graft chambers were used to isolate wounds and prevent epidermal healing from the skin edge. The chambers remained in situ for 5-7 weeks. We detail the surgical technique, the method of porcine keratinocyte culture and highlight some practical measures that were taken to optimise the "take" of the cultured keratinocyte grafts.

Kerato-dermal grafts: the importance of dermis for the in vivo growth of cultured keratinocytes.

In a porcine model, we studied the benefit of dermis for the growth of cultured autologous keratinocytes (CAK) on full-thickness wounds isolated within skin graft chambers. Kerato-dermal grafts were prepared in a two stage process using autologous de-epidermalised dermis (DED) and CAK (Group 1). Control wounds were prepared by grafting either CAK only (Group 2) or DED only (Group 3). The median epidermal cover of 34 wounds in Group 1 was 47% and was significantly greater (p < 0.001) than the epidermal cover of 12 wounds in Group 2 (4%) and 14 wounds in Group 3 (12%). The epidermis in Group 1 was durable whereas it was fragile in the control wounds. Histologically rête ridges were present at 2 weeks in Group 1, but not in the control wounds. These data indicate that a dermal wound bed significantly improves the in vivo growth of cultured keratinocytes.

Ultrastructural changes in rat adipomusculocutaneous flaps during warm ischaemic storage and reperfusion.

Electron microscopy (EM) was used to evaluate ultrastructural changes in adipomusculocutaneous rat flaps which had been stored for 0, 2, 4, 6 and 8 hours under normothermic conditions (37 degrees C). The effects of treatment with desferrioxamine (DFX) or hypertonic citrate flush (HCA), prior to 4 hours of storage, were compared to untreated flaps which had been stored for 4 hours. Ultrastructural changes caused by 30 minutes of reperfusion, were also studied. Most ultrastructural alterations occurred between 2 and 4 hours of warm storage and there were further changes in some cells after short periods of reperfusion. DFX decreased smooth muscle damage and HCA protected adipocytes but neither of these agents preserved endothelial cells. These studies indicated that free radical-dependent and independent mechanisms were both involved in events which led to flap necrosis after periods of warm storage and reperfusion.

Ultrastructural changes and lipid peroxidation in rat adipomusculocutaneous flap isotransplants after normothermic storage and reperfusion.

Parallel in vivo, histological, and ultrastructural studies were carried out and markers of lipid peroxidation (Schiff's bases [SB] and thiobarbituric-acid-reactive material [TBAR]) were measured in rat adipomusculocutaneous flap isotransplants that had been stored for 0, 2, 4, 6, and 8 hr under normothermic (37 degrees C) conditions and reperfused for specific periods. Flaps stored for 4 hr and treated with intravenous desferrioxamine (DFX) or hypertonic citrate flush (HCA) were also evaluated. In vivo assessment was made after 7 days of reperfusion. Flaps stored for 4 hr eventually exhibited partial necrosis in vivo, and neither DFX or HCA flush increased the area of surviving skin. Electron microscopy revealed extensive storage damage in epidermal, follicle, fat, and smooth muscle cells and in endothelium. HCA significantly preserved fat cells (P = 0.0035) and DFX diminished smooth muscle damage. Reperfusion injury was seen in endothelial cells in the form of swelling that was not prevented by HCA or DFX. Ultrastructural alterations correlated with changes in susceptibility to lipid peroxidation in fat but not in skin. The results of these parallel studies indicate that both free radical-dependent and independent mechanisms operate in ischemia and reperfusion injury in flap tissue and that fat has a greater predisposition to free radical damage than skin.

Immunoelectron microscopic studies reveal differences in distribution of sialo-oligosaccharide receptors for Mycoplasma pneumoniae on the epithelium of human and hamster bronchi.

Long-chain sialo-oligosaccharides with poly-N-acetyllactosamine backbones (Ii antigen type) are major host cell receptors for the human pathogen Mycoplasma pneumoniae. Previous immunofluorescence studies of the human bronchial epithelium, using sequence-specific monoclonal antibodies to the branched I-type and linear i-type backbones, have indicated that sialylated and nonsialylated long-chain sequences of both types are richly expressed on the ciliated cells, where they are polarized at the apical aspects. These sequences are lacking in the goblet cells. In the present study, the display of these oligosaccharides has been investigated by electron microscopy (immunogold labelling) in the human bronchial epithelium and in that of the hamster, an animal model commonly used for M. pneumoniae infection. In the human bronchial epithelium, the long-chain branched sequences have been detected along the entire length of the cilia and on microvilli, whereas the linear sequences are confined to the microvilli and the basal aspects of the cilia. On the ciliated epithelial cells of the hamster, by contrast, the branched and linear sequences (sialo- and asialo-) have been detected exclusively on microvilli. A further striking difference is that in the hamster these structures are expressed in abundance on the goblet cells and in the intracellular globules. We suggest that the latter finding may partly explain the relatively large doses of M. pneumoniae required to establish experimental infection in the hamster, as the receptor-bearing secreted mucus may have a protective role in binding to the microorganisms, leading to their clearance by bronchociliary action.

Origins of the parasitophorous vacuole membrane of the malaria parasite, Plasmodium falciparum, in human red blood cells.

We have attempted to determine whether the parasitophorous vacuole membrane, in which the malaria parasite (merozoite) encapsulates itself when it enters a red blood cell, is derived from the host cell plasma membrane, as the appearance of the invasion process in the electron microscope has been taken to suggest, or from lipid material stored in the merozoite. We have incorporated into the red cell membrane a haptenic phospholipid, phosphatidylethanolamine, containing an NBD (N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)) group, substituted in the acyl chain, and allowed it to translocate into the inner bilayer leaflet. After invasion of these labelled cells by the parasite, Plasmodium falciparum, immuno-gold electron microscopy was used to follow the distribution of the labelled lipid; this was found to be overwhelmingly in favour of the host cell membrane relative to the parasitophorous vacuole. Merozoites of P. knowlesi were allowed to attach irreversibly to red cells without invasion, using the method of pretreatment with cytochalasin. The region of contact between the merozoite and the host cell membrane was in all cases devoid of the labelled phosphatidylethanolamine. These results lead us to infer that the parasitophorous vacuole membrane is derived wholly or partly from lipid preexisting in the merozoite.

Accumulation of membrane-bound melanosomes occurs in Langerhans cells of patients with the Leopard syndrome.

The Langerhans cells in the lentigines of four patients with the Leopard syndrome contained large membrane bound accumulations of melanin granules. Giant melanosomes were only seen in two patients. The patients had no immune-based symptoms relating to their lentigines. The Leopard Syndrome, also known as multiple lentigines syndrome, progressive cardiomyopathic lentiginosis, lentiginosis profusa syndrome and the cardiocutaneous syndrome, refers to an inherited abnormality of the skin, often associated with cardiomyopathy. The aetiology of the condition is so far unknown and the penetrance is variable. Here we describe electron microscopical findings of large accumulations of melanin within Langerhans cells.

Reperfusion injury in rat adipomusculocutaneous flaps: an ultrastructural and microangiographic study.

Electron microscopy (EM), microangiography (MA) and histological studies were carried out to demonstrate the vascular basis of ischaemic and reperfusion injury in rat adipomusculocutaneous free flaps which had been stored under normothermic conditions (37 degrees C) for specific periods of time. MA revealed that areas destined to die reperfused for at least 6 h in this model. EM showed progressive endothelial damage as a result of both storage and reperfusion. The use of the iron chelator desferrioxamine (DFX) did not benefit the endothelium or improve salvage of ischaemic flaps.

Basal cell glycoprotein in pig epidermis closely resembles the beta 1 subunit of the integrin family of cell adhesion molecules.

A 135-kD conA-binding glycoprotein isolated from pig epidermis was previously localized to the surface of basal cells in stratified epithelia using affinity-purified antibodies. Preembedding immunoperoxidase electron microscopy has now shown that this glycoprotein is concentrated on the lateral surfaces of basal cells but is not detectable on those surfaces adjacent to the basement membrane indicating a role in cell-cell rather than cell-substrate interactions. The basal cell glycoprotein was shown to resemble the beta 1 subunit of the integrin family following the generation of a specific monoclonal antibody (M5.25). The epidermal glycoprotein recognized by M5.25 and by antibodies against the beta 1 fibronectin receptor from human placenta co-migrated on SDS gels under both reducing and non-reducing conditions. Its response to disulphide reducing agents was characteristic of beta 1 integrin subunits. In addition, the basal cell glycoprotein was shown to bind to the 120-kD cell-binding fragment of fibronectin in a RGD-dependent manner. It was readily detected by immunoblotting whole cell lysates of cultured pig keratinocytes suggesting increased expression in cultured cells compared to fresh epithelial tissue. The results suggest that beta 1 integrin subunits may be involved in cell-cell interactions between basal keratinocytes in pig epidermis and that these receptors are lost from the cell surface during terminal differentiation. Thus modulation of beta 1 integrin subunit expression may play an important role in regulating differentiation in pig epidermis.

Identification of hematopoietic progenitors of macrophages and dendritic Langerhans cells (DL-CFU) in human bone marrow and peripheral blood.

Colonies of cells with distinctive dendritic appearance were observed in methylcellulose cultures of human bone marrow and peripheral blood mononuclear cells (PBMC). Such cells appeared alone in colonies of less than 50 cells, together with macrophages in mixed colonies and also within clusters of T lymphocytes at high culture cell numbers. The morphologic resemblance to lymphoid dendritic cells was confirmed by electron microscopy and the cells were distinguished from macrophages by immunoenzymatic and immunogold labeling with monoclonal antibodies (MoAbs). Like macrophages they were HLA-DR+ and CD4+. However, they lacked nonspecific esterase and the macrophage cytoplasmic marker Y1/82A. Most strikingly, cells were strongly HLA-DQ+ and expressed CD1a (T6), which is characteristic of skin Langerhans cells. Their functional similarity to lymphoid dendritic cells was demonstrated by their ability to stimulate allogeneic mixed leukocyte reactions. Dendritic cell colony numbers were estimated in both bone marrow and peripheral blood of controls and in leukemia and lymphoma patients before and after chemotherapy. Colony numbers were low in control blood and in patients before treatment (less than 1.0 to 3.7/10(5) cells). However, during hematopoietic recovery the mean value increased to 37.5/10(5) cells and this increase correlated closely with the observed increase in circulating colony forming unit-granulocyte macrophage (CFU-GM) in individual patients. Autoradiographic studies demonstrated mitotic activity within CD1a+ colonies and a linear relationship between cultured cells and both pure and mixed colonies was consistent with their derivation from a single precursor. These data indicate that a novel hematopoietic progenitor of dendritic/Langerhans cells (DL-CFU) may now be identified in a clonal assay system and suggest a probable common progenitor for these cells and macrophages.

Lipid peroxidation and ultrastructural changes in rat lung isografts after single-passage organ flush and 48-hour cold storage with and without one-hour reperfusion in vivo.

Rat lung isografts were preserved for 48 hr at 0 degrees C using a simple organ flush technique. After storage alone, isotonic saline flush resulted in significantly raised indices of lipid peroxidation in vitro (Schiff bases and thiobarbituric-acid-reactive material [TBAR]). Lungs flushed with hypertonic citrate (HCA) had significantly less oxidative damage than saline-flushed lungs. The addition to the HCA flush of verapamil, a calcium channel blocker, or desferrioxamine, an iron chelator, significantly reduced TBA reactivity in stored lungs compared with HCA alone. After 1-hr reperfusion in vivo, lipid peroxidation was reduced in HCA-flushed lungs compared with saline flush (TBAR alone), but no additional protection from the use of desferrioxamine or verapamil was demonstrated. Electron microscopy after saline flush and storage alone showed gross endothelial swelling and fragmentation. Reperfusion with blood for 1 hr resolved cell swelling, but alveolar/capillary wall rupture occurred. HCA protected against cell swelling, but endothelial vesiculation and widening of the basement membrane were observed. After reperfusion, HCA-flushed lungs developed much endothelial loss that was considerably reduced by the use of desferrioxamine and verapamil. The lipid peroxidation results suggest that iron- and calcium-mediated free radical production may be important mechanisms in oxidative damage to stored rat lungs. Electron microscopy findings correlated with biochemical evidence of free-radical-mediated injury. Reduction of endothelial loss on reperfusion by the use of verapamil and desferrioxamine provides circumstantial evidence that ischemia and reperfusion damage of organs stored for transplantation is partly due to Fe++(+)- and Ca+(+)-dependent mechanisms that probably involve increased free radical production.

Ultrastructural changes in rat lungs after 48 h cold storage with and without reperfusion.

Using a left lung orthotopic isograft model in AS strain rats, we have investigated ultrastructural changes in lungs preserved for 48 h at 0 degrees C after a simple flush technique. Lungs were examined after storage alone and after storage followed by up to 1 h reperfusion with blood in vivo. Grafts were flushed with either isotonic saline (NaCl) or hypertonic citrate solution (HCA) alone, or with HCA containing either verapamil (a Ca2+ channel blocker), desferrioxamine (a Fe2+ antagonist), prostacyclin PGI2, nifedipine (a Ca2+ channel blocker) or allopurinol (a xanthine oxidase inhibitor). These agents were also given intravenously to both donor and recipient. Storage in NaCl produced gross cytoplasmic swelling and disruption with widespread nuclear injury. Reperfusion for 1 h resolved cell swelling but some endothelial loss and alveolar capillary wall rupture were seen. HCA with or without additional agents protected against cell swelling but endothelial blebbing and widening of the basement membrane occurred. Reperfusion for 1 h led to recovery of the basement membrane thickness but widespread endothelial loss was observed which was reduced by the addition of verapamil, desferrioxamine, nifedipine or allopurinol to the flush, but not by prostacyclin. Examination of lungs reperfused for shorter periods (5 and 15 min) identified three main types of damage to the vascular endothelium: (1) gross cell swelling, (2) detachment of intact endothelial cells from the underlying basal lamina, and (3) attenuation of cytoplasm due to blebbing. The results suggest that endothelial injury occurring on reperfusion is partly Fe2+ and Ca2(+)-mediated and that reactions catalysed by xanthine oxidase (which include oxygen free radical production) may also be important.

Red cell membrane protein distribution during malarial invasion.

Immuno-gold labelling electron microscopy of thin sections was used to determine the distribution of red cell membrane and membrane skeleton proteins in the vicinity of internalized malaria parasites. When examined immediately after invasion (young ring-stage parasites), the parasitophorous vacuole membranes of both Plasmodium falciparum and P. knowlesi were found to be characterized by the essentially complete absence of spectrin, ankyrin and the most abundant transmembrane protein, band 3. P. knowlesi merozoites were trapped in the attached but not internalized state by pretreatment with cytochalasin B. In this merozoite-red cell complex antibody labelling showed that band 3 had been eliminated from the region of the host cell membrane in contact with the parasite. Internal vesicles, originating apparently from the site of attachment, were often observed in the red cell. Opposite the attached parasite a cavity was also sometimes seen in the host cell, presumably representing an incipient internal vesicle. The membrane was intact, as judged by the absence of protein (haemoglobin) in the cavity, and, like the membranes surrounding the internal vesicles, was devoid of membrane proteins. A large multilamellar body was sometimes seen in the merozoite close to its point of attachment. The lamellar spacing was about 50 nm. The electron microscope images suggest a diffusion of electron-dense material from the lamellar body into the cavity in the host cell.

Ultrastructural changes accompany inhibition of proteoglycan synthesis in chondrocytes by Cyclofenil diphenol.

Cyclofenil diphenol, a weak non-steroidal oestrogen, profoundly inhibits [35S]proteoglycan synthesis in cultures of Swarm chondrosarcoma chondrocytes under conditions in which protein synthesis is only marginally reduced. In the present experiments it was shown that after a 40-min treatment with Cyclofenil diphenol (90 micrograms ml-1) most of the normally abundant Golgi stacks in these cells disappeared and after 60 min they were absent. After 2-3 h treatment the cisternae of the endoplasmic reticulum (ER) were grossly distended and transformed into large ribosome-studded vesicles containing flocculent and filamentous material. These changes were dependent on the concentration of Cyclofenil and were fully reversible within 21 h of withdrawing the drug. The ultrastructural changes differed in some aspects if protein synthesis was blocked with cycloheximide for 15 min or 180 min before and during treatment with Cyclofenil. The Golgi disappeared but the ER cisternae, though distended, formed a continuous network and swollen ribosome-studded vesicles did not develop. However, non-membrane-bounded structures containing lipid droplets and material of low electron density developed in the cytoplasm under these conditions. The ultrastructural changes induced by Cyclofenil differ from those induced by monensin and diethylcarbamazine, suggesting that the drug acts at a different point in the secretory pathway for macromolecules.

Desmoglein II-derived glycopeptides in human epidermis.

Antisera raised against a major 78 kD glycopeptide from pig epidermis were used to identify desmoglein II-derived glycopeptides in the conA-binding material isolated from human epidermis. In whole CaCl2-separated epidermis the antiserum recognized conA-binding components with apparent Mr of 115, 100, 82, 68, 50, 48, and 46 kD. The 82, 68, 48, and 46 kD immunoreactive bands were present in normal stratum corneum and plantar callus. Psoriatic scales contained significantly more of the 82 kD components and less of the 48 and 46 kD bands. Psoriatic scales also contained a major 50 kD conA-binding component unrelated to keratins or desmoglein II. Proteolytic peptide mapping showed that the major immunoreactive bands in normal stratum corneum and plantar callus were also chemically related. The 82 to 46 kD immunoreactive glycopeptides in plantar callus coincided with the major coomassie blue stained bands and were homogeneous on two-dimensional gels suggesting that this tissue may be a valuable source of human desmoglein II-derived glycopeptides. An antiserum directed against the electrophoretically co-purified 48/46 kD glycopeptides from plantar callus recognized the 82 to 46 kD bands in immunoblotting. In indirect immunofluorescence of frozen skin sections this antiserum stained the surface of epidermal cells in the spinous and granular layers of the tissue. In immunogold labeling of paraformaldehyde-fixed skin sections affinity-purified antibodies stained intact desmosomes in spinous and granular cells and desmosomal remnants in the stratum corneum. The results are consistent with our hypothesis that desmoglein II undergoes limited cleavage to stable fragments during terminal differentiation. Proteolytic degradation appears to be incomplete in psoriatic epidermis.