Comparison of preserved vascular allografts using glycerol and University of Wisconsin solution in a goat carotid artery transplantation model.

BACKGROUND: Prosthetic grafts have poor patency rates in peripheral arterial reconstructions. Glycerol (GL)-preserved grafts are an alternative. The aim of this study was to examine patency, graft morphology and function of GL-preserved allografts in a goat carotid artery animal model. METHODS: The first group (n = 7) underwent bilateral replacement of the carotid artery by a carotid allograft that was preserved in GL for 1 week. In the second group (n = 5), a carotid artery allograft that was preserved in University of Wisconsin solution (UW) for 48 h was used. In the third group (n = 5), the jugular vein (autologous vein, AU) was used. The follow-up was 3 months. RESULTS: One UW graft and 1 GL graft occluded in the first 24 h postoperatively. Three-month primary patency rates for GL, UW and AU grafts were 93, 100 and 80%, respectively (p = 0.39). Graft diameter was increased in UW allografts (p < 0.005), whereas GL allografts remained unchanged. After explantation, GL allografts demonstrated contraction and relaxation capacity and lower intimal thickness (p < 0.001). CONCLUSION: GL preservation has proven to be a feasible method for arterial allograft transplantation in a large animal model with decreased intimal hyperplasia and renewed functional capability.

Repair of abdominal aortic aneurysms with aorto-uni-iliac stentgraft and femoro-femoral bypass.

Endovascular repair (EVAR) is accepted as effective treatment for abdominal aortic aneurysms (AAAs) and has become the standard of care in many instances. The standard bifurcated stentgraft (BFG) is often not possible in patients with unfavourable aneurysm morphology. The aorto-uni-iliac (AUI) graft configuration with femoro-femoral bypass (FFBP) is a promising alternative which may extend the scope of EVAR for AAAs. The aim of this study was to evaluate the feasibility, efficacy and durability of AUI with FFBP. Design. The results of a single institution and a single surgeon were prospectively collected from January 2002 to August 2010. All patients were followed up at 1, 3, 6 and 12 months and then annually. Results. There were 33 patients (27 males) with a mean age of 71.7 years (range 46 - 84). Open surgery posed an unacceptably high risk to all patients owing to advanced age and/or American Society of Anesthesiologists (ASA) classification 3/4. Ineligibility for BFG was due to unfavourable anatomy or a combination of factors in most cases (31 patients). Two patients had anastomotic aneurysms after previous open surgery. The technical success rate was 100%. One severe intra-operative complication occurred (perforated iliac artery). Two patients (ASA 4) died within 30 days (peri-operative mortality rate 6.1%). Seven patients (21.1%) developed postoperative wound complications. Eight patients died during follow-up of non-aneurysm-related conditions. Twenty-three patients are alive, with mean follow-up of 24.4 months and a survival rate of 69.7%. Two complications occurred during long-term follow-up, namely 1 case of graft sepsis and 1 of FFBP occlusion. Conclusion. AUI with FFBP is a safe, effective and durable alternative in high-risk patients with AAAs where standard open repair is contraindicated and BFG repair is not possible owing to unfavourable aneurysm morphology.

Determination of the scattering anisotropy with optical coherence tomography.

In this work we demonstrate measurements with optical coherence tomography (OCT) of the scattering phase function in the backward direction and the scattering anisotropy parameter g. Measurements of the OCT attenuation coefficient and the backscattering amplitude are performed on calibrated polystyrene microspheres with a time-domain OCT system. From these measurements the phase function in the backward direction is determined. The measurements are described by the single scattering model and match Mie calculations very well. Measurements on Intralipid demonstrate the ability to determine the g of polydisperse samples and, for Intralipid, g = 0.35 ± 0.03 is measured, which is well in agreement with g from literature. These measurements are validated using the Intralipid particle size distribution determined from TEM measurements. Measurements of g and the scattering phase function in the backward direction can be used to monitor changes in backscattering, which can indicate morphological changes of the sample or act as contrast enhancement mechanism.

The effects of hyperosmosis or high pH on a dual-species biofilm of Enterococcus faecalis and Pseudomonas aeruginosa: an in vitro study.

AIM: To investigate the effect of hyperosmotic hyperosmosis or alkaline stress on a dual-species biofilm of Enterococcus faecalis and Pseudomonas aeruginosa. METHODOLOGY: Biofilms were grown on glass cover slips suspended in bacterial inoculate for 96 h, after which the cover slips with attached biofilms were immersed in brain heart infusion broth (BHI-broth) with 6 mol L(-1) sodium chloride (NaCl) representing the hyperosmotic group or Ca(OH)(2), pH 12.1, representing the alkaline group. Two per cent sodium hypochlorite and BHI- broth served as positive and negative controls, respectively. After treatment, the biofilms were washed, harvested and plated on blood-agar plates after serial dilution. The bactericidal effect was assessed by determining the colony-forming units (CFU). The effect on the biofilm mass was imaged with confocal laser scanning microscopy (CLSM). RESULTS: Hyperosmosis reduced the CFU of both species significantly after 72 h (P < 0.0001). After 168 h, P. aeruginosa was eradicated and the E. faecalis reduction was more than 99%. High pH could not induce a significant bacterial reduction. CLSM revealed dense flocculation of the biofilms incubated in alkaline broth. CONCLUSION: Hyperosmosis effectively reduced a dual-species biofilm of E. faecalis and P. aeruginosa, whilst high pH had limited bactericidal effect in this model.

[Myocarditis and cardiomyopathy: underestimated complications resulting from clozapine therapy]. / Myocarditis en cardiomyopathie; ernstige complicaties van clozapinetherapie.

BACKGROUND: Treatment with clozapine can affect the heart, leading to serious complications such as myocarditis and cardiomyopathy. When in their early stages both illnesses are difficult to diagnose; this can have serious consequences. Recent analyses of clozapine data suggest that particularly myocarditis is possibly more common than has been assumed hitherto. AIM: To determine the frequency of these complications and to find out what diagnostic tests are available and whether it is necessary or possible to adjust current guidelines on these complications. METHOD: The relevant literature was consulted via PubMed, Embase Psychiatry and Psycinfo on the basis of the keywords 'clozapine' and 'myocarditis', 'cardiomyopathy' and 'heart failure'. RESULTS: Studies showed that the incidence of myocarditis varied from 0.015 to 1.3%. Cardiomyopathy was the subject of fewer studies, one study reported an incidence of 0.022%. More than 50% of the cases of myocarditis developed during the first few weeks of treatment, the average time being about 15 days. For an early diagnosis it is important to monitor the patient's symptoms carefully, especially during the first four weeks following the start of medication. Monitoring should include laboratory tests and electrocardiography. Echocardiography and MRI can be useful additions to the diagnostic process. CONCLUSIONS: Early diagnosis of myocarditis is important because it is a serious condition. Timely recognition of subclinical myocarditis could possibly prevent later complications such as cardiomyopathy. Clinical guidelines are proposed on the basis of the literature.

HIV-occlusive vascular disease.

OBJECTIVES: To evaluate peripheral arterial occlusive disease in HIV-infected patients regarding clinical presentation and outcome of surgical intervention. DESIGN: Prospective clinical survey. PATIENTS AND METHODS: Routine voluntary testing for HIV/AIDS was performed in all patients presenting to our vascular unit. HIV+ patients were enrolled in a registry and followed up prospectively. RESULTS: We identified 154 HIV+ patients, of whom 91 (59%) presented with occlusive disease. There were 71 males and 20 females with a mean age of 44.2 years. The usual risk factors for atherosclerosis were present, but the incidence was less than reported in the classic atherosclerosis population. More than 90% of the patients presented with advanced stage vascular disease (Fontaine III/IV), which explains the high rate (31.9%) of primary amputation. Eighty-seven patients presented with lower-limb ischaemia, 2 patients with upper-limb ischaemia and 2 patients with symptomatic carotid artery stenosis. Seventy-eight procedures were performed on 72 patients, with a perioperative mortality of 6.95%. The limb salvage rate for femoro-popliteal bypass procedures was poor (36.1%), resulting in a high incidence of secondary amputations and prolonged hospital stay. Long-term mortality for the operated patients was 20% over a mean follow-up period of 15.4 months. Hypo-albuminaemia was found to be an important predictor of outcome. CONCLUSION: Patients presenting with HIV-associated peripheral arterial disease should be carefully selected for intervention, taking into consideration nutritional and immune status, stage of the vascular disease and selecting the appropriate procedure.

Autosomal recessive ichthyosis with hypotrichosis syndrome: further delineation of the phenotype.

Autosomal recessive ichthyosis with hypotrichosis (ARIH) syndrome, which is characterized by congenital ichthyosis, abnormal hair and corneal involvement, has recently been shown in one consanguineous Israeli Arab family to be caused by a mutation in the ST14 gene, which encodes serine protease matriptase. No other families have so far been described since the original report. In this current report we describe a female patient from a second family with ARIH syndrome who carries a homozygous novel mutation, p.M1I. The patient has congenital ichthyosis, light brown, curly, sparse hair, improving with age, and sparse body hair, eyebrows and eyelashes. She does not suffer from photophobia, but has blepharitis. The phenotype of this patient closely resembles that of the affected individuals in the previously reported family, although she does not have tooth abnormalities and the ichthyosis is milder.

Class III P-glycoproteins mediate the formation of lipoprotein X in the mouse.

Cholestasis is associated with hypercholesterolemia and appearance of the abnormal lipoprotein X (LpX) in plasma. Using mice with a disrupted Mdr2 gene, we tested the hypothesis that LpX originates as a biliary lipid vesicle. Mdr2-deficient mice lack Mdr2 P-glycoprotein, the canalicular translocator for phosphatidylcholine, and secrete virtually no phospholipid and cholesterol in bile. Bile duct ligation of Mdr2(+)/+ mice induced a dramatic increase in the plasma cholesterol and phospholipid concentration. Agarose electrophoresis, density gradient ultracentrifugation, gel permeation, and electron microscopy revealed that the majority of phospholipid and cholesterol was present as LpX, a 40-100 nm vesicle with an aqueous lumen. In contrast, the plasma cholesterol and phospholipid concentration in Mdr2(-)/- mice decreased upon bile duct ligation, and plasma fractionation revealed a complete absence of LpX. In mice with various expression levels of Mdr2 or MDR3, the human homolog of Mdr2, we observed that the plasma level of cholesterol and phospholipid during cholestasis correlated very closely with the expression level of these canalicular P-glycoproteins. These data demonstrate that during cholestasis there is a quantitative shift of lipid secretion from bile to the plasma compartment in the form of LpX. The concentration of this lipoprotein is determined by the activity of the canalicular phospholipid translocator.

The human kinesin-like protein RB6K is under tight cell cycle control and is essential for cytokinesis.

Several members of the kinesin superfamily are known to play a prominent role in the motor-driven transport processes that occur in mitotic cells. Here we describe a new mitotic human kinesin-like protein, RB6K (Rabkinesin 6), distantly related to MKLP-1. Expression of RB6K is regulated during the cell cycle at both the mRNA and protein level and, similar to cyclin B, shows a maximum during M phase. Isolation of the RB6K promoter allowed identification of a CDE-CHR element and promoter activity was shown to be maximal during M phase. Immunofluorescence microscopy using antibodies raised against RB6K showed a weak signal in interphase Golgi but a 10-fold higher signal in prophase nuclei. During M phase, the newly synthesized RB6K does not colocalise with Rab6. In later stages of mitosis RB6K localized to the spindle midzone and appeared on the midbodies during cytokinesis. The functional significance of this localization during M phase was revealed by antibody microinjection studies which resulted exclusively in binucleate cells, showing a complete failure of cytokinesis. These results substantiate a crucial role for RB6K in late anaphase B and/or cytokinesis, clearly distinct from the role of MKLP-1.

Emergency endovascular repair for ruptured abdominal aneurysms--a feasibility study and report of 10 cases.

BACKGROUND: Endovascular aneurysm repair (EVAR) has been proved to be effective and safe in the elective management of abdominal aortic aneurysms (AAAs). Initial reports concerning endovascular management of ruptured aneurysms have been promising. OBJECTIVE: To determine the outcome of endovascular repair of ruptured aneurysms in the local setting. Materials and methods. Patients who presented with ruptured AAAs were considered for endovascular repair if they were haemodynamically stable and had suitable aneurysm morphology for EVAR. RESULTS: Ten patients (9 males, 1 female) with a mean age of 74.9 years were treated. All aneurysms were successfully excluded using aorta uni-iliac stent grafts in 7 patients and bifurcated stent grafts in 2 patients. In 1 patient who had had a previous EVAR, a proximal extension device was used. Two patients died in the peri-operative period (30-day mortality of 20%) and 1 patient died after 2 months. Seven patients are still alive. No endo-leaks occurred in any of the survivors. CONCLUSION: Endovascular repair of ruptured AAAs is feasible with acceptable peri-operative mortality and short- to medium-term results.

A prospective comparison of secondary interventions and mortality in open and endovascular infrarenal abdominal aortic aneurysm repair.

Endovascular aneurysm repair (EVAR) has provided a safe and effective alternative to the standard open repair of abdominal aortic aneurysms (AAAs). It has, however, been associated with a high requirement for secondary interventions. This prompted us to compare the two procedures with regard to secondary interventions and mortalities. The sample size was 278 patients, of whom 156 had undergone the open operation and 122 had undergone EVAR. The perioperative morbidity and mortality, as well as the major and minor secondary intervention rates, were obtained for these patients. The results suggest that there is no significant difference in secondary interventions and mortality between the two groups, despite the EVAR group being at significantly higher risk.

Cerebral monitoring during carotid endarterectomy--a comparison between electroencephalography, transcranial cerebral oximetry and carotid stump pressure.

OBJECTIVE: Various modalities are used for cerebral monitoring during carotid endarterectomy (CEA). The aim of this study was to evaluate whether transcranial cerebral oximetry (TCO) and carotid stump pressure (SP) are as accurate as electroencephalography (EEG) for monitoring cerebral ischaemia during carotid cross-clamping. METHODS: One hundred consecutive patients who underwent CEA were studied with continuous and simultaneous EEG and TCO. SP was measured for each patient. The percentage decrease of oxygenation on TCO was calculated during cross-clamping and surgery. EEG findings were used as the benchmark to detect cerebral ischaemia and were the indication for insertion of a temporary shunt. The relationship with TCO was observed in terms of percentage decrease in oxygenation. RESULTS: A total of 6 patients were shunted on the basis of their EEG changes. TCO changed more than 20% in these 6 patients, but an additional 12 patients had TCO changes with a normal EEG. This correlated with a decrease in blood pressure (BP) and was corrected by increasing the BP. The positive predictive values (PPVs) and negative predictive values (NPVs) for shunting based on TCO (as compared with EEG) were 33% and 100% respectively. Thirty-four patients had SP <50 mmHg, of whom 4 were shunted based on EEG changes. Two of 66 patients with SP >50 mmHg were shunted based on EEG changes. If a shunting policy had been based on a SP of 50 mmHg, 30 patients would have been shunted unnecessarily (PPV 12%), whereas the non-requirement for a shunt was predicted correctly in 64 of 66 patients (NPV 97%). There were 2 major strokes: 1 contralateral on day 3 in a patient with bilateral severe stenoses, and 1 ipsilateral in a nonshunted patient with normal EEG, TCO and SP >50 mmHg. CONCLUSION: Compared with EEG, TCO is a practical and non-invasive monitoring system with a high sensitivity (100%) but a low specificity. TCO is more sensitive to a drop in BP and responds earlier to these changes than EEG. SP should not be used as the sole predictor for shunting during CEA.

The effect of the antimicrobial peptide, Dhvar-5, on gentamicin release from a polymethyl methacrylate bone cement.

The objective of this study was to investigate the release mechanism and kinetics of the antimicrobial peptide, Dhvar-5, both alone and in combination with gentamicin, from a standard commercial polymethyl methacrylate (PMMA) bone cement. Different amounts of Dhvar-5 were mixed with the bone cement powders of Osteopal and the gentamicin-containing Osteopal G bone cement and their release kinetics from the polymerized cement were investigated. Additionally, the internal structure of the bone cements were analysed by scanning electron microscopy (SEM) of the fracture surfaces. Secondly, porosity was investigated with the mercury intrusion method and related to the observed release profiles. In order to obtain an insight into the mechanical characteristics of the bone cement mixtures, the compressive strength of Osteopal and Osteopal G with Dhvar-5 was also investigated. The total Dhvar-5 release reached 96% in the 100 mg Dhvar-5/g Osteopal cement, whereas total gentamicin release from Osteopal G reached only 18%. Total gentamicin release increased significantly to 67% with the addition of 50mg Dhvar-5/g, but the Dhvar-5 release was not influenced. SEM showed an increase of dissolved gentamicin crystals with the addition of Dhvar-5. The mercury intrusion results suggested an increase of small pores (< 0.1 microm) with the addition of Dhvar-5. Compressive strength of Osteopal was reduced by the addition of Dhvar-5 and gentamicin, but still remained above the limit prescribed by the ISO standard for clinical bone cements. We therefore conclude that the antimicrobial peptide, Dhvar-5, was released in high amounts from PMMA bone cement. When used together with gentamicin sulphate, Dhvar-5 made the gentamicin crystals accessible for the release medium presumably through increased micro-porosity (< 0.1 microm) resulting in a fourfold increase of gentamicin release.

Redistribution of Ca2+, Mg2+-ATPase activity in relation to alterations of the cytoskeleton and tight junctions in hepatocytes of cholestatic rat liver.

Ca2+, Mg2+-ATPase is a membrane-bound enzyme localized at the bile canalicular membranes of hepatocytes. Cytoskeleton and tight junctions are important for maintenance of the polar distribution of plasma membrane proteins. In order to understand the mechanisms involved in the redistribution of Ca2+, Mg2+-ATPase due to cholestasis, the relationship between Ca2+, Mg2+-ATPase, microfilaments and tight junctions was examined. Cholestasis was induced in rat liver by common bile duct ligation (CBDL) for 2 weeks. Localization of Ca2+, Mg2+-ATPase activity was studied at the light and electron microscopic level. Double-staining of the enzyme and F-actin was performed using phase-contrast and fluorescence microscopy of 7-nitrobenzene-2-oxa-1,3-diazole phallacidin (NBD-ph), respectively. Immunofluorescence microscopy of ZO-1 was applied for the observation of tight junctions. Furthermore, cytoskeleton and junctional complexes were investigated electron microscopically in saponin-extracted tissues. The results showed that CBDL induced redistribution of Ca2+, Mg2+-ATPase activity from the apical to the entire plasma membrane of hepatocytes, which seemed to occur independently of F-actin. F-actin was present at all membrane domains of hepatocytes in control liver, whereas CBDL increased the amounts of F-actin mainly at the bile canalicular membranes. An inverse distribution pattern of Ca2+, Mg2+-ATPase activity and F-actin was found in epithelial cells of bile ducts in control and cholestatic livers. Marked alterations in microfilaments were observed at the bile canaliculi, which were defined as hypertrophy and atrophy and were in association with changes in tight junctions. Structural impairment of the tight junctions was proven by disordered immunofluorescence of ZO-1. It is concluded that changes in the distribution of Ca2+, Mg2+-ATPase and F-actin due to CBDL are independent of each other. CBDL-induced disorders of microfilaments are related to impairment of structural integrity of tight junctions that is suggested to be responsible for the redistribution of Ca2+, Mg2+-ATPase in hepatocytes.

Living skin substitutes: survival and function of fibroblasts seeded in a dermal substitute in experimental wounds.

The healing of full-thickness skin defects requires extensive synthesis and remodeling of dermal and epidermal components. Fibroblasts play an important role in this process and are being incorporated in the latest generation of artificial dermal substitutes. We studied the fate of fibroblasts seeded in our artificial elastin/collagen dermal substitute and the influence of the seeded fibroblasts on cell migration and dermal substitute degradation after transplantation to experimental full-thickness wounds in pigs. Wounds were treated with either dermal substitutes seeded with autologous fibroblasts or acellular substitutes. Seeded fibroblasts, labeled with a PKH-26 fluorescent cell marker, were detected in the wounds with fluorescence microscopy and quantitated with flow cytofluorometric analysis of single-cell suspensions of wound tissue. The cellular infiltrate was characterized for the presence of mesenchymal cells (vimentin), monocytes/macrophages, and vascular cells. Dermal substitute degradation was quantitated by image analysis of wound sections stained with Herovici's staining. In the wounds treated with the seeded dermal substitute, fluorescent PKH-26-labeled cells were detectable up to 6 d and were positive for vimentin but not for the macrophage antibody. After 5 d, flow cytofluorometry showed the presence of 3.1 (+/-0.9) x 10(6) (mean +/- SD, n = 7) PKH-26-positive cells in these wounds, whereas initially only 1 x 10(6) fluorescent fibroblasts had been seeded. In total, the percentage of mesenchymal cells minus the macrophages was similar after 5 d between wounds treated with the seeded and the acellular substitutes. In the wounds treated with the seeded substitute, however, 19.5% of the mesenchymal cells were of seeded origin. Furthermore, the rate of substitute degradation in the seeded wounds was significantly lower at 2-4 wk after wounding than in wounds treated with the acellular substitute. Vascular in-growth and the number of infiltrated macrophages were not different. In conclusion, cultured dermal fibroblasts seeded in an artificial dermal substitute and transplanted onto full-thickness wounds in pigs survived and proliferated. The observed effects of seeded fibroblasts on dermal regeneration appeared to be mediated by reducing subcutaneous fibroblastic cell migration and/or proliferation into the wounds without impairing migration of monocytes/macrophages and endothelial cells. Moreover, the degradation of the implanted dermal substitute was retarded, indicating a protective activity of the seeded fibroblasts.

Heterogeneous suppression of experimentally induced colon cancer metastasis in rat liver lobes by inhibition of extracellular cathepsin B.

Metastatic rat colon cancer cells but not normal rat hepatocytes showed activity of cathepsin B on their plasma membranes. Activity was visualized in living cells with a new fluorogenic substrate, [Z-Arg]2-cresyl violet, and confocal microscopy. When these cancer cells were injected into the portal vein of rats, the animals developed tumors in the liver in a heterogeneous fashion. Three- to four-fold more tumors were found in the small caudate lobe than in the other three large lobes of the liver. Oral treatment with a selective water-soluble inhibitor of extracellular cathepsin B, Mu-Phe-homoPhe-fluoromethylketone, resulted in 60% reduction of the number of tumors and 80% reduction of the volume of tumors in the three large lobes whereas tumor development was not affected in the small caudate lobe. This study supports the conclusions that (a) extracellular cathepsin B plays a crucial but complex role in liver colonisation by rat colon carcinoma cells in vivo, (b) its selective inhibition suppresses tumor growth heterogeneously in the liver and (c) the caudate lobe of the liver is a relatively large risk factor for tumor development.

Experimental arterial allografting under low and therapeutic dosages of cyclosporine for immunosuppression.

The aim of this study was to investigate performance of preserved arterial allografts under the protection of a high-dose and a low-dose immunosuppressive regimen, with cyclosporine (CsA). Dog carotid arteries were harvested and stored for 14 days at 4 degrees C in University of Wisconsin organ preservation solution. Segments (6 cm) of carotid artery were orthotopically and bilaterally implanted in mongrel dogs (n = 18). CsA was given in two dosage regimens: 25 mg/kg/day (group I, n = 7) and 10 mg/kg/day (group II, n = 7). The control group received no CsA (group III, n=4). After 3 months of implantation, patency was assessed by angiography. The grafts were excised for investigation of vessel wall and endothelial function and morphology. For assessment of function in vitro, slices of arterial segments were connected as ring preparations to an isometric force transducer and immersed in a 5 ml organ bath (37 degrees C) containing Tyrode's solution. The contractile response was examined by adding 40 mM KCl and phenylephrine (100 microM) to the organ bath; endothelium-dependent relaxation was examined by adding methacholine (100 microM). Morphology was assessed semiquantitatively. The functional responses to KCl, phenylephrine (Phe) and methacho- line (Met) after 14 days of storage in UW, were 30.2 +/- 1.2 mN, 26.9 +/- 1.0 and 45 +/- 1.2% (means +/- SEM, n=9), respectively. Patency after three months of implantation for group I was 100% (14/14), for group II 50% (7/14), and for group III 75% (6/8). In vitro functional responses of preserved arteries, after 3 months of implantation in group I were 58.5 +/- 10.6 mN (KCl), 36.5 +/- 5.8 mN (Phe), and 57.4 +/- 9.7% (Met), respectively. Functions in group II were 1.2 +/- 0.1 mN (KCl, 0.0 mN (Phe), and 0.0% (Met). Grafts in group III showed no function. Measurement of medial thickness showed significant thinning (P <0.05) in groups II and III. Patency and function of arterial allografts under a therapeutic dose of CsA were superior to grafts implanted under low-dose CsA or no immunosuppressive treatment.

Localization and quantification of the insulin-like growth factor-1 receptor in mouse articular cartilage by confocal laser scanning microscopy.

Insulin-like growth factor-1 (IGF-1) is an essential anabolic growth factor in the regulation of cartilage metabolism and exerts its effects by binding to the IGF-1 Type 1 receptor on the chondrocyte membrane. We have localized and quantified in situ IGF-1 receptor expression in intact articular cartilage of normal mice. The IGF-1 receptor was detected immunohistochemically with antibodies to the IGF-1 receptor and visualized with conventional light microscopy and confocal laser scanning microscopy (CLSM). CLSM analysis enabled us to distinguish IGF-1 receptor immunoreactivity on the chondrocyte cell membrane from intracellular staining. We have established two approaches to quantify in confocal images low levels of fluorescence intensity of the immunolocalized IGF-1 receptor at the chondrocyte membrane, i.e., mean pixel measurement and area measurement. The majority of IGF-1 receptor fluorescence intensity was localized on chondrocytes in the middle and deeper zones of cartilage, whereas chondrocytes in the surface zone exhibited negligible fluorescence. The variable distribution of IGF-1 receptor in chondrocytes of articular cartilage suggests that effects of IGF-1 on chondrocytes may be distinctly heterogeneous in the different mouse articular cartilage zones.

Square arrays in early cortical lens opacities.

A combined freeze-fracture and scanning electron microscopic study of early opaque spots in the aging human lens showed the absence of gap junctions and the presence of square arrays in the membranes of disturbed fibers and neighboring unaffected fibers. Square arrays, with membrane particles of 6-7 nm, are considered as rearranged gap junctions and/or intramembranous particles, with particle sizes between 8.5-9.5 nm; they are a sign of electric and metabolic uncoupling. These ultrastructural observations lend support to the idea of an uncoupling mechanism in the aging human lens, conserving the transparency of unaffected parts of the lens, as postulated previously.

Repair in the rat lens after threshold ultraviolet radiation injury.

PURPOSE: To investigate the development and recovery of lens damage after in vivo close-to-threshold exposure to ultraviolet B radiation. METHODS: One eye of young, female Sprague-Dawley rats was exposed to 5 kJ/m2 narrowband ultraviolet radiation (UVR) (lambda(max) = 302 nm) for 15 minutes. Groups of rats were killed 1, 7, and 56 days after exposure. The structure of the exposed and nonexposed lenses was examined with light microscopy, scanning electron microscopy, transmission electron microscopy, freeze-fracture, fluorescent membrane staining, and Fourier transform analysis. RESULTS: One day after UVR exposure the lens surface had flakelike opacities. Seven days after exposure, the lens surface appeared opaque and corrugated, and the equatorial cortex had small opacities. At 56 days postexposure, the surface and equator appeared clear, but the cortex had a subtle shell-shaped opacity. At 1 day postexposure, apoptotic cell death occurred in the lens epithelium, but the cortical fibers were normal. At 7 days postexposure, the epithelium and the fibers between the 10th and 40th growth shell below the capsule contained extracellular spaces of different sizes. After 56 days, the epithelial layer appeared normal, and the extracellular spaces had disappeared; but abnormal fibers were found between the 60th and 100th growth shell below the capsule. Fibers above and below the damaged growth shells appeared fully normal. CONCLUSIONS: A close-to-threshold dose of UVR causes cataract, which is largely reversible. The UVR exposure leads to apoptosis in the lens epithelium, and after a latency period of several days, lens fibers are abnormal. Extracellular spaces develop in the epithelium and fibers. Within several weeks after exposure, the epithelium fully recovers and new fibers develop normally. The originally affected fibers are repaired. However, this repair is incomplete, leaving a small zone of enhanced light scattering in the equatorial cortex.