Vitrectomy with peeling of the inner limiting membrane for treating diabetic macular edema.

PURPOSE: to evaluate the results of pars plana vitrectomy with peeling of the inner limiting membrane (ILM stained with infracyanine green (IfCG) in 26 patients with diabetic macular edema, and to identify which factors are associated with a better postoperative visual outcome. PATIENTS AND METHODS: 26 patients with diabetic macular edema were included in the study. A pars plana vitrectomy with ILM peeling stained with IfCG was performed, in some cases combined with cataract surgery. In 22 patients the ILM was retained, examined with electron microscopy and compared with normal ILM's. Visual acuity and fundus examination were recorded several months after surgery. To determine which factors lead to the best postoperative results, patients were divided into different groups and compared. RESULTS: during surgery, a taut posterior hyaloid was found in 26 patients, which was successfully detached in all cases. ILM peeling within the vessel arcade succeeded in all patients. Postoperative examination showed improved visual acuity and decreased macular edema in 19 patients, unaltered visual acuity in 3 patients and decreased visual acuity in 4 patients. Comparison between different groups of patients revealed that young patients with recent vision loss and without previous macular laser treatment, had better postoperative results. Electron microscopical examination showed a more condensed ILM in diabetic patients, consisting of a layer of fine curled fibers. CONCLUSION: pars plana vitrectomy with peeling of the ILM stained with IfCG leads to good postoperative results in young diabetic patients with recent vision loss due to macular edema and without previous macular laser treatment.

Double vital staining using trypan blue and infracyanine green in macular pucker surgery.

AIMS: To study the clinical properties of double vital staining in premacular fibrosis, facilitating complete removal of all epiretinal tissue. METHODS: In a two step surgery, the epiretinal pucker was removed after staining with trypan blue, whereafter the inner limiting membrane was peeled after staining with infracyanine green. RESULTS: In all 30 patients, a separate epiretinal layer and inner limiting membrane were removed from the macular area. Pathological examination showed different histological properties of the removed layers. An increased visual acuity was measured in 26 patients, and a slightly decreased visual acuity in one patient. CONCLUSION: The described double staining technique could be a novel valuable tool that may help to achieve optimal anatomical and functional recovery after surgery for premacular fibrosis

Sulphated and sulphonated polymers inhibit the initial interaction of hepatitis B virus with hepatocytes.

The initial step during hepatitis B virus (HBV) infection is the specific attachment of the virus to the hepatocyte. Here we studied whether the binding of HBV to hepatocytes can, as is the case with most other enveloped viruses, be blocked by polyanionic compounds. Viral particles produced by HepAD38 cells were used as inoculum and HBV-negative HepG2 cells, as well as primary human hepatocytes, as target cells. Three sulphated polymers, that is, PAVAS (a co-polymer of acrylic acid with vinyl alcohol sulphate), heparin and dextran sulphate (DS) (MW 5000), and the sulphonated polymer PAMPS [poly(2-acryl-amido-2-methyl-1-propanesulfonic acid] (MW approximately 7000-12000), proved strong inhibitors of the binding of HBV to HepG2 cells and primary hepatocytes. The 50% effective concentration (EC50) for inhibition of HBV binding to HepG2 cells by PAVAS, heparin, DS and PAMPS was 1.3 microg/ml, 1.6 microg/ml, 1.8 microg/ml and 3.3 microg/ml, respectively, and to primary hepatocytes 1.6 microg/ml (PAVAS), 1.6 microg/ml (heparin), 2.6 microg/ml (DS) and 4.1 microg/ml (PAMPS). These values are in the same range as the concentrations required for these compounds to prevent such viruses as herpesviruses and HIV from binding to cells. These findings may be helpful in elucidating the mechanism of the initial interaction of HBV with hepatocytes.

Immunocytochemical localization of a diuretic hormone of the beetle Tenebrio molitor, Tenmo-DH(37), in nervous system and midgut.

Although the mealworm Tenebrio molitor inhabits very dry environments, it has at least two diuretic peptides, which increase fluid secretion by the free portions of the Malpighian tubules. Unlike other insect corticotropin-releasing factor (CRF)-related peptides isolated to date, these are non-amidated peptides. The immunocytochemical localization of Tenmo-DH(37) was investigated using antisera raised against this hormone. Immunoreactive neurosecretory cells were found in the brain and abdominal ganglia with immunoreactive processes projecting to the peripheral nervous system. Intense staining of the neurohaemal release site, the corpora cardiaca, was observed. In addition, neurosecretory cells immunoreactive to Tenmo-DH(37) were found in the posterior midgut and a network of immunoreactive nerve processes extended over the surface of the midgut. Tenmo-DH(37) is widely distributed and its staining pattern resembles that found for other, amidated CRF-related diuretic peptides.

Productive infection of primary macrophages with human herpesvirus 7.

Here we demonstrate replication of human herpesvirus 7 (HHV-7), a T-lymphotropic virus, in macrophages. Productive replication was lost after 2 weeks, but HHV-7 DNA was detected up to 1 month after infection. Thus, macrophages become infected by HHV-7 and might play an important role as a viral reservoir, as has been demonstrated for human immunodeficiency virus type 1.

Sleeve-related thrombosis: a new form of catheter-related thrombosis.

In a detailed study of central venous catheter-related sleeve and thrombosis in experimental animals, a new form of thrombosis was detected and termed sleeve-related thrombosis. A silastic catheter was placed in the jugular vein and the anterior vena cava of 22 rabbits and 54 rats. After intervals of 1, 3, 7 days, 2, 3, 4 weeks and 1, 2, 4, 6 months the veins were examined by light microscopy and by transmission electron microscopy. In about 50% of the rats a thrombus was observed at the end of the catheter sleeve. Consecutive cutting allowed the visualization of a transition from a sleeve via part of sleeve and part of thrombus to a pure thrombus. This thrombus was separated from the vein wall and could not be considered a mural thrombus. As the thrombus was only attached to the terminal part of the organized catheter sleeve we propose the name sleeve-related thrombosis.

Antimineralization treatments in stentless porcine bioprostheses: an experimental study.

BACKGROUND AND AIM OF THE STUDY: Photo-oxidation treatment of porcine stentless bioprostheses (Photofix) was compared with glutaraldehyde fixation, with either AOA (Freestyle valve) or Tween-80 (Edwards Prima Plus valve). METHODS: Six valves of each type were implanted in juvenile sheep, in the pulmonary position. Valves were explanted after three or six months and examined macroscopically, by X-radiography, and by light and transmission electron microscopy. Calcium content was determined by atomic absorption spectrometry. RESULTS: The cusps of all valves were free of calcification, and had normal histology and function. Calcium contents (median +/- IQR) were 0.63+/-0.45, 0.73+/-1.46 and 0.46+/-1.42 microg/mg for the Photofix, Freestyle and Prima Plus valves, respectively (p = NS). Calcium contents of the aortic wall portions were 0.71+/-1.27 (Photofix), 10.78+/-77.22 (Freestyle) and 28.70+/-66.53 (Prima Plus) (p <0.05 for Photofix versus Freestyle or Prima Plus). CONCLUSION: Photo-oxidation of a porcine stentless valve prevents calcification not only in the cusps, but also in the aortic wall portion.

Calcification characteristics of porcine stented valves in a juvenile sheep model.

BACKGROUND: Different antimineralization treatments of stented porcine bioprostheses were evaluated: ethanol (Epic), alpha-amino-oleic acid (AOA) (Mosaic), and sodium dodecyl sulfate (SDS) (Hancock II). A nontreated, glutaraldehyde-fixed valve (Labcor) served as control. METHODS: For each treatment, six valves were implanted in juvenile sheep in the pulmonary position. Valves were explanted after 3 and 6 months and examined macroscopically, by roentgenogram and light and transmission electron microscopy. Calcium content (microg/mg) was determined by atomic absorption spectrometry. RESULTS: The Labcor valves revealed small calcium deposits in the cusps, although calcium content remained low (median value 0.4+/-0.8 microg/mg). SDS did not prevent cusp calcification as assessed by histology and calcium content measurement, which was higher than in all other valves: 1.9+/-4.6 microg/mg (p < 0.05). Cusp retraction and rupture were occasionally found in the Hancock. The Mosaic and Epic valves showed no cusp calcification and had low calcium contents (0.3+/-2.4 microg/mg and 0.7+/-0.6 microg/mg, respectively). Epic showed less pannus formation, but had hematoma or iron staining in the cusps. CONCLUSIONS: SDS is inefficient as an antimineralization treatment, in contrast to ethanol or AOA. Cusp hematoma after ethanol treatment needs further investigation.

Pulmonary neuroendocrine cells (PNEC) and neuroepithelial bodies (NEB): chemoreceptors and regulators of lung development.

The airway and alveolar epithelia contain pulmonary neuroendocrine cells whose structure indicates an endocrine function. They are also in contact with sensory nerve fibres. These cells often aggregate into distinct corpuscles-neuroepithelial bodies-and carry membrane receptors sensitive to a number of stimuli, including hypoxia and nicotine. They synthesise, store and release a number of bioactive substances such as serotonin, calcitonin gene-related peptide and the mitogen bombesin. When these are released they contribute to redistribution of pulmonary blood flow, regulation of bronchomotor tone, modulation of the immune response, stimulation of sensory nerve fibres and regulation of lung growth and development. Pulmonary neuroendocrine cells and neuroepithelial bodies seem to be most important in the fetal and neonatal lung as regulators of airway development and hypoxia-sensitive chemoreceptors. There is a link between these cells and specific types of lung cancer and their involvement in lung and paediatric pathology may be profound.

Prominent axonopathy and disruption of axonal transport in transgenic mice expressing human apolipoprotein E4 in neurons of brain and spinal cord.

The epsilon 4 allele of the human apolipoprotein E gene (ApoE4) constitutes an important genetic risk factor for Alzheimer's disease. Recent experimental evidence suggests that human ApoE is expressed in neurons, in addition to being synthesized in glial cells. Moreover, brain regions in which neurons express ApoE seem to be most vulnerable to neurofibrillary pathology. The hypothesis that the expression pattern of human ApoE might be important for the pathogenesis of Alzheimer's disease was tested by generating transgenic mice that express human ApoE4 in neurons or in astrocytes of the central nervous system. Transgenic mice expressing human ApoE4 in neurons developed axonal degeneration and gliosis in brain and in spinal cord, resulting in reduced sensorimotor capacities. In these mice, axonal dilatations with accumulation of synaptophysin, neurofilaments, mitochondria, and vesicles were documented, suggesting impairment of axonal transport. In contrast, transgenic mice expressing human ApoE4 in astrocytes remained normal throughout life. These results suggest that expression of human ApoE in neurons of the central nervous system could contribute to impaired axonal transport and axonal degeneration. The possible contribution of hyperphosphorylation of protein Tau to the resulting phenotype is discussed.

Intimal hyperplasia after long-term venous catheterization.

OBJECTIVE: Intimal hyperplasia is a well-known consequence of arterial injury and arterialization in vein grafts. However, the subacute and chronic vein wall changes which occur after catheterization have not been well studied. In this animal study, intimal hyperplasia in the vein wall after catheterization was examined. METHODS: A silicon catheter was placed in the anterior caval vein of 54 rats. After in situ fixation at scheduled intervals (1 day to 6 months), the pathologic changes in the vein wall were studied on semi-serial histology sections by means of light microscopy. RESULTS: Three forms of intimal hyperplasia could be observed: plaque-like, papillary-like and incorporation of the mural part of the sleeve into the underlying vein wall. Although the appearance of each was different, their composition was identical. All were mainly composed of alpha-actin-positive cells and collagen localized above the internal elastin layer, and covered by endothelium if facing the lumen. The plaque-like and papillary-like forms were mainly localized in the anterior vena cava, while sleeve incorporation mainly occurred in the jugular vein. Plaque-like and papillary-like intimal hyperplasia could be seen together on the same slide, but these two forms were never seen together with sleeve incorporation. CONCLUSION: Intimal hyperplasia occurs after venous catheterization and is probably caused by chronic injury to the vein wall due to knocking and rubbing movements of the catheter against the wall.

The influence of stenting on the behavior of amino-oleic acid-treated, glutaraldehyde-fixed porcine aortic valves in a sheep model.

BACKGROUND AND AIM OF THE STUDY: The durability of freehand-sewn aortic valve homografts used for valve replacement in humans is greater than for stented aortic homografts. In analogy with this, it is expected that the durability of a stentless heterograft will be superior to that of its stented counterpart. Our objective was to investigate the influence of stenting on amino-oleic acid (AOA)-treated, glutaraldehyde-fixed porcine aortic valve bioprostheses. METHODS: Twelve young sheep underwent implantation of porcine aortic valves in the pulmonary artery: six porcine aortic stentless valves (Freestyle) and six porcine aortic stented valves (Mosaic). In each series, three valves were explanted after three months, and three after six months. Valves were analyzed by gross inspection, radiography, histology, and transmission electron microscopy. Quantitative determination of calcium content was made with atomic absorption spectrometry. RESULTS: The porcine aortic stentless valve showed extensive calcification of its aortic wall portion, but had perfectly functioning, pliable cusps without calcification up to six months. The cusps of porcine aortic stented valves were also pliable and functioning without calcification up to six months. Only minimal calcification was seen in the aortic wall of the stented valves. At six months after implantation the cusps of stentless valves contained significantly less calcium than those of stented valves (2.7+/-1.2 microg/mg and 7.9+/-2.3 microg/mg, respectively; p = 0.011). However, the aortic wall from stentless valves contained significantly more calcium than that of stented valves (three-month explants: 39.2+/-14.4 versus 7.2+/-2.8 microg/mg; p <0.05; six-month explants: 49.3+/-14.0 versus 14.1+/-5.9 microg/mg; p <0.05). CONCLUSION: These data suggest that stenting does influence cuspal calcification of AOA-treated, glutaraldehyde-fixed porcine aortic valves.

Postnatal maturation of neuroepithelial bodies and carotid body innervation: a quantitative investigation in the rabbit.

The intrapulmonary airways contain oxygen-sensitive chemoreceptors which may be analogous to the arterial chemoreceptors: the neuroepithelial bodies (NEB). While the NEB are prominent in the neonatal lung, physiological studies indicate that the carotid bodies are still relatively inactive at birth. This points to an unequal degree of development of both during the early neonatal period. As a reflexogenic chemoreceptor function depends on a well-developed innervation, we undertook a comparative investigation of the development of the NEB and the carotid body glomus cell innervation. Two morphological aspects of the innervation of NEB and carotid body glomus cells were quantified in rabbits of different age groups. The total sectional area of intracorpuscular and intraglomerular nerve endings per NEB or glomus cell group, respectively, was measured and the area percentage of mitochondria and synaptic vesicles was determined. In the NEB, no significant difference in total sectional area of the nerve endings between the age groups was observed, while in the carotid body there was a significant increase in the adult age group. In addition, the area percentage of mitochondria and synaptic vesicles of the nerve endings did not change significantly with age in the NEB, while in the carotid body these increased and decreased, respectively, with age. These observations point to a shift from morphologically efferent nerve endings, rich in synaptic vesicles, to morphologically afferent nerve endings, rich in mitochondria. Our interpretation of these findings is that, at birth, the NEB innervation is more mature than the carotid body glomus cell innervation and that the latter matures at a later time than the former. These findings support the theory that the NEB may act as complementary chemoreceptors to the carotid body during the early postnatal period.

Prominent axonopathy in the brain and spinal cord of transgenic mice overexpressing four-repeat human tau protein.

Mutations in the human tau gene cause frontotemporal dementia and parkinsonism linked to chromosome 17. Some mutations, including mutations in intron 10, induce increased levels of the functionally normal four-repeat tau protein isoform, leading to neurodegeneration. We generated transgenic mice that overexpress the four-repeat human tau protein isoform specifically in neurons. The transgenic mice developed axonal degeneration in brain and spinal cord. In the model, axonal dilations with accumulation of neurofilaments, mitochondria, and vesicles were documented. The axonopathy and the accompanying dysfunctional sensorimotor capacities were transgene-dosage related. These findings proved that merely increasing the concentration of the four-repeat tau protein isoform is sufficient to injure neurons in the central nervous system, without formation of intraneuronal neurofibrillary tangles. Evidence for astrogliosis and ubiquitination of accumulated proteins in the dilated part of the axon supported this conclusion. This transgenic model, overexpressing the longest isoform of human tau protein, recapitulates features of known neurodegenerative diseases, including Alzheimer's disease and other tauopathies. The model makes it possible to study the interaction with additional factors, to be incorporated genetically, or with other biological triggers that are implicated in neurodegeneration.

The pulmonary neuroendocrine system: the past decade.

The pulmonary neuroendocrine system consists of specialized airway endocrine epithelial cells, associated with nerve fibres. The epithelial cells, the pulmonary neuroendocrine cells (PNEC), can be solitary or clustered to form neuroepithelial bodies (NEB). During the last thirty years, the pulmonary neuroendocrine system has been intensively investigated and much knowledge of its function has been obtained. This text reviews work which dates from the last ten years. In this period, the picture of the pulmonary neuroendocrine system we previously had, has not fundamentally changed. The pulmonary neuroendocrine system is still regarded as an oxygen sensitive chemoreceptor with local and reflex-mediated regulatory functions, and as a regulator of airway growth and development. Continuing research has much more refined this picture. This text reviews several aspects of the pulmonary neuroendocrine system: phylogeny, the amine and peptide content of its epithelial cells, ontogeny and influence on lung development, the influence of hypoxia and nonhypoxic stimuli, immunomodulatory function, innervation and pathology. Among the discoveries of the past decade, three stand out prominently because of their great significance: additional proof that the neural component of the pulmonary neuroendocrine system is sensory, sound experimental evidence that PNEC stimulate airway epithelial cell differentiation and the discovery of a specific membrane oxygen receptor in the PNEC.

Calcification characteristics of porcine stentless valves in juvenile sheep.

OBJECTIVE: To compare calcification characteristics of two porcine stentless valves (Toronto SPV and Freestyle) with different designs, fixation and antimineralization techniques using a juvenile sheep model of valve implantation inside the circulation. METHODS: The stentless valves (n = 2 x 6) were implanted in juvenile sheep in the pulmonary artery as an interposition, while the circulation was maintained with a right ventricular assist device. The model was validated by the implantation of, clinically well-known, porcine (Hancock II) and pericardial (Pericarbon) valves. Half of the valves were explanted after 3 months, the rest after 6 months. Valves were examined macroscopically, by X-ray, light microscopy (HE, Masson, Von Giesson, Von Kossa, PTAH stains), and transmission electron microscopy. Quantitative determination of the calcium content of the cusps was performed with atomic absorption spectrometry. RESULTS: After 3 months, the Freestyle had an extensively calcified aortic wall, most prominent at the outflow side of the porcine valve. After 6 months, calcification increased transmurally, but the valve cusps were free of calcification, and the inflow side was only slightly calcified. The Toronto SPV valve also started to calcify at the inflow side of the valve after 3 months with increased calcification after 6 months. The base of the Toronto SPV valve cusps showed slight calcification after 6 months of implantation. CONCLUSIONS: The pattern of calcification of the porcine aortic wall differs between the two studied stentless valves, with calcification located predominantly at the outflow side in the Freestyle valve, but also at the inflow side in the Toronto SPV valve. The cusps of the Freestyle valve were less prone to calcification than those from the Toronto SPV valve.

Stereological estimation of number and volume of pulmonary neuroepithelial bodies (NEBs) in neonatal hamster lungs.

The number and volume of pulmonary neuroepithelial bodies (NEBs) of 1- and 4-week-old hamsters were estimated using unbiased stereological principles and systematic sampling techniques. For comparative purposes, volume estimations were also made in the carotid body, the parathyroid gland, and the adrenal medulla. A significant decrease was found in the total number of NEBs, immunoreactive for CGRP, between 1 and 4 weeks. Individual as well as cumulative NEB volume also decreased significantly. The cumulative NEB volume in 1-week-old hamsters was in the same range as the volumes of the carotids and parathyroids in the same animals. The postnatal decrease of the NEB number suggests that the NEBs are of primary potential importance in the neonatal stage, when they may complement the chemoreceptor function of the carotid bodies, which are relatively inactive at birth. Since the cumulative NEB volume (at least at the age of 1 week) is equal to that of the carotid bodies and the parathyroids, their physiological function may be of similar importance.

The No-React anticalcification treatment: a comparison of Biocor No-React II and Toronto SPV stentless bioprostheses implanted in sheep.

Calcification of stentless aortic heterografts still limits the use of these bioprostheses in young patients despite their superior hemodynamic profile. The No-React treatment is described as an anticalcification treatment for biomaterials. We compared the Biocor No-React treated stentless bioprosthesis with the routine glutaraldehyde-fixed Toronto SPV bioprosthesis in a juvenile sheep model. Toronto SPV or Biocor No-React valves were implanted in pulmonary position in juvenile sheep (n = 6). The valves were explanted after 3 months and analyzed by gross inspection, x-ray studies, histological examination, and transmission electron microscopy. The Toronto SPV valve showed calcification of the aortic wall portion at both the inflow and outflow sides of the valve. No significant calcification of the cusps was found by gross inspection or by radiographic or histological examinations. Calcification was visible with electron microscopy in cell remnants and between collagen fibers in the cusps. The Biocor No-React valve showed extensive calcification of the residual aortic wall portion that is contained in the valve. With x-ray and histological examinations, clear calcification of the pericardial wrap, largely replacing the aortic wall tissue, was seen. Calcification scattered throughout the cusp was seen by electron microscopy. We conclude that the Biocor No-React process did not prevent calcification of glutaraldehyde-fixed stentless bioprostheses in a juvenile sheep experimental model. Furthermore, replacement of a large part of the aortic wall by a pericardial wrap did not prevent calcification of the stentless valve "wall."

Are stentless aortic heterografts suitable for right ventricular outflow tract reconstruction? An experimental study in juvenile sheep.

Allograft valved conduits are used routinely for reconstruction of the right ventricular outflow tract in children with congenital heart disease; however, allografts are scarce. The purpose of this study was to evaluate two stentless aortic valves that might be suitable alternatives for right ventricular outflow tract reconstruction. Twelve juvenile sheep underwent implantation of stentless aortic heterografts as an interposition in the pulmonary artery: six porcine aortic valves (Freestyle) and six fully pericardial valves (Pericarbon stentless). In each series, three valves were explanted after 3 months, the other three after 6 months. Valves were analyzed by gross inspection, radiography, and light microscopy. The porcine aortic stentless valve (Freestyle) showed extensive calcification of its aortic wall portion, with perfectly functioning, pliable cusps without calcification or fibrous overgrowth up to 6 months. The pericardial valves (Pericarbon stentless) showed extensive fibrous sheathing, causing progressive retraction of the leaflets and severe regurgitation. After 3 months, minimal calcification was seen in the pericardial wall. Calcification was more pronounced after 6 months, sometimes causing complete calcification of pericardial wall and leaflets, leading to a significant stenosis. We conclude that the pericardial stentless valve becomes rapidly dysfunctional after right-sided implantation as a result of fibrous sheathing and severe calcification. The porcine aortic stentless valve remains functional, but severe calcification of the aortic wall portion is problematic.

Na+/H+ exchange inhibition improves long-term myocardial preservation.

BACKGROUND: Na+/H+ exchange plays an important role in the ionic changes observed during myocardial ischemia and reperfusion. We investigated the cardioprotective efficacy of a selective Na+/H+ exchange inhibitor, 4-isopropyl-3-methylsulfonyl-benzoylguanidin-methanesulfonate (HOE642), in a canine model of long-term heart preservation. METHODS: Canine donor hearts were stored for 24 hours in hyperkalemic crystalloid cardioplegic solution; in cardioplegic solution enriched with HOE642; in cardioplegic solution enriched with HOE642, with donor and recipient treated with HOE642; in standard cardioplegic solution, with donor and recipient treated with HOE642; or in standard cardioplegic solution, with only the recipient treated. After orthotopic transplantation, pressure-volume relationships were obtained and dogs were weaned from bypass. Morphology was studied. RESULTS: Myocardial compliance was well preserved when donor and recipient were treated. These groups had the lowest myocardial water content, and no morphologic signs of irreversible damage. In these groups, weaning from cardiopulmonary bypass was successful in 10 of 10 animals, with a cardiac index around 2 L x min(-1) x m(-2). Only 3 of 5 animals in each of the other three groups could be weaned, with significantly lower cardiac indices. CONCLUSIONS: Treatment with HOE642 in both donor and recipient improves myocardial compliance, postweaning cardiac index, and ultrastructure of donor hearts preserved for 24 hours and orthotopically transplanted.