Isolated brain microvessels: a purified, metabolically active preparation from bovine cerebral cortex.

A purified, metabolically active preparation of brain microvessels was isolated from bovine cerebral cortex by using a simple procedure involving mild disruption of the tissue by homogenization and trapping of the vessels on nylon sieves. This preparation permits in vitro metabolic and structural studies of small blood vessels.

Immunoelectron microscopic analyses of Maillard reaction products in bovine anterior lens capsule and Descemet's membrane.

It has been hypothesized that Maillard reaction products form in basement membranes during aging and may affect protein turnover. The purpose of this study was to localize Maillard reaction products in intact lens capsules and Descemet's membranes by immunoelectron microscopy to determine whether Maillard products accumulated with age and whether basement membrane thickness increased to a similar degree. The monoclonal antibodies antiglucitollysine and antipyrraline were employed to detect the products in native and glucose-treated bovine basement membranes. The content of basic amino acids, furosine, and fluorophores (370/440), as well as resistance to trypsin digestion showed that the basement membranes formed significant quantities of Maillard products when incubated with 200 mM glucose in vitro (P < 0.05). Likewise, incubation in 200 mM glucose resulted in at least a 4-fold increase in immunoreactivity (P < 0.001). Native basement membranes increased in thickness more than 2-fold with age (P < 0.001). Immunoreactivity varied similarly in that bound antiglucitollysine increased approx. 2-fold and antipyrraline approx. 3-fold in old vs. young basement membranes, but these differences were significant only in pyrraline immunoreactivity in the lens capsule (P < 0.01). Advanced products other than pyrraline may accumulate in Descemet's membrane since significant increases in fluorescence and resistance to trypsin were noted. These data suggest that the Maillard reaction may, to a small degree, contribute to basement membrane thickening.

Overexpression of metallothionein in pancreatic beta-cells reduces streptozotocin-induced DNA damage and diabetes.

The release of reactive oxygen species (ROS) has been proposed as a cause of streptozotocin (STZ)-induced beta-cell damage. This initiates a destructive cascade, consisting of DNA damage, excess activation of the DNA repair enzyme poly(ADP-ribose) polymerase, and depletion of cellular NAD+. Metallothionein (MT) is an inducible antioxidant protein that has been shown to protect DNA from chemical damage in several cell types. Therefore, we examined whether overexpression of MT could protect beta-cell DNA and thereby prevent STZ-induced diabetes. Two lines of transgenic mice were produced with up to a 30-fold elevation in beta-cell MT. Cultured islets from control mice and MT transgenic mice were exposed to STZ. MT was found to decrease STZ-induced islet disruption, DNA breakage, and depletion of NAD+. To assess in vivo protection, transgenic and control mice were injected with STZ. Transgenic mice had significantly reduced hyperglycemia. Ultrastructural examination of islets from STZ-treated mice showed that MT prevented degranulation and cell death. These results demonstrate that MT can reduce diabetes and confirm the DNA damage mechanism of STZ-induced beta-cell death.

Early changes in the arterial wall of chickens fed a cholesterol diet.

A total of 160 1-2 day old chickens were fed a 2% cholesterol diet for a period of 8 to 42 days and compared with an equal number of controls. Aortas were analyzed for various indexes of reactivity of connective tissue, cholesterol content and scanning electron microscopy (SEM) characteristics of the endothelial lining. Cholesterol feeding for a period up to 6 weeks resulted in doubling the level of serum cholesterol. It was, however, without effect on the activity of prolyl hydroxylase, lysyl oxidase, collagenase and collagen content in the aortic wall. As early as 3 weeks of feeding significant changes occurred in total and esterified cholesterol content. At the same time endothelial cells were characteristically contracted with several long cytoplasmic elongations and protrusions. A significant decrease of activity of the above enzymes was found in aortic tissue with increased age of the chicken. Collagen content in aortas increased with age of chickens. It is concluded that cholesterol as an atherogenic agent induces marked changes in endothelial cells and lipids of chicken aorta at earlier periods, prior to the activation of connective tissue.

Pharmacological blockade or genetic deletion of substance P (NK(1)) receptors attenuates neonatal vocalisation in guinea-pigs and mice.

The regulation of stress-induced vocalisations by central NK(1) receptors was investigated using pharmacological antagonists in guinea-pigs, a species with human-like NK(1) receptors, and transgenic NK1R-/- mice. In guinea-pigs, i.c.v. infusion of the selective substance P agonist GR73632 (0.1 nmol) elicited a pronounced vocalisation response that was blocked enantioselectively by the NK(1) receptor antagonists CP-99,994 and L-733,060 (0.1-10 mg/kg). GR73632-induced vocalisations were also markedly attenuated by the antidepressant drugs imipramine and fluoxetine (30 mg/kg), but not by the benzodiazepine anxiolytic diazepam (3 mg/kg) or the 5-HT(1A) agonist buspirone (10 mg/kg). Similarly, vocalisations in guinea-pig pups separated from their mothers were blocked enantioselectively by the highly brain-penetrant NK(1) receptor antagonists L-733,060 and GR205171 (ID(50) 3 mg/kg), but not by the poorly brain-penetrant compounds LY303870 and CGP49823 (30 mg/kg). Separation-induced vocalisations were also blocked by the anxiolytic drugs diazepam, chlordiazepoxide and buspirone (ID(50) 0.5-1 mg/kg), and by the antidepressant drugs phenelzine, imipramine, fluoxetine and venlafaxine (ID(50) 3-8 mg/kg). In normal mouse pups, GR205171 attenuated neonatal vocalisations when administered at a high dose (30 mg/kg) only, consistent with its lower affinity for the rat than the guinea-pig NK(1) receptor. Ultrasound calls in NK1R-/- mouse pups were markedly reduced compared with those in WT pups, confirming the specific involvement of NK(1) receptors in the regulation of vocalisation. These observations suggest that centrally-acting NK(1) receptor antagonists may have clinical utility in the treatment of a range of anxiety and mood disorders.

Fenestrated subendothelial basement membranes in human retinal capillaries.

A correlated TEM and SEM study of human retinal capillaries and their associated basement membranes (BMs) was carried out. Control tissues show that these vessels are comprised of a continuous layer of endothelial cells separated from overlying intramural pericytes by a discontinuous subendothelial BM (EBM) which accommodates endothelial cell-pericyte (periendothelial) junctions. Three types of junctions exist, including: (1) "peg-and-socket" arrangements where cytoplasmic processes of the two cell layers interdigitate; (2) adhering plaques similar to desmosomes; and (3) cell/cell contacts where adjacent cell membranes appear to fuse or remain separated by a approximately 2 nm space. Following detergent solubilization, acellular retinal capillaries maintain their cylindrical histoarchitectures and all BM components are imaged by TEM and SEM. Topographical (SEM) studies of cryofractured samples show EBM surfaces with numerous (approximately 1.5/microns 2) oval fenestrations (100-450 nm diameter) that correlate well with EBM discontinuities occupied by periendothelial junctions in control tissues. It seems possible that these structures may play an important role in diabetic retinal neovascularization where pericytes are known to degenerate selectively. In this condition, preformed EBM deficiencies could facilitate endothelial cell migration and sprout formation, leading ultimately to the sequelae of proliferative diabetic retinopathy.

Ultrastructural analyses of enzyme-treated microfibrils in rabbit corneal stroma.

Microfibrils have been identified within and between corneal collagen lamellae in a number of vertebrate species in a variety of developmental and pathological conditions, but they are relatively rare in normal adult animals. The present study was undertaken to analyze corneal microfibrils in adult rabbits using enzymatic digestion techniques. Transmission electron microscopy (TEM) showed clusters of 10-15 nm microfibrils arranged in quasi-parallel bundles within or between orthogonally arranged stromal collagen lamellae. When corneas were fixed with tannic acid/glutaraldehyde, the entire stroma showed increased electron density and microfibrillar bundles were heterogeneously stained. Peripheral fibrils were more electron-dense than those located more centrally. Following sequential detergent solubilization of unfixed corneas, all cellular elements were removed and collagen lamellae were distorted. Microfibrillar bundles remained intact, however, and resembled untreated controls. Subsequent treatment with pepsin, trypsin or elastase resulted in swollen corneal tissues in which collagen lamellae were no longer distinguishable but individual collagen fibrils maintained their morphological integrity. In these tissues microfibrillar bundles were rarely identifiable and were reduced to randomly oriented fragments or clusters of filamentous material. Testicular hyaluronidase or chondroitinase ABC did not affect the fibrils. These data indicate that rabbit corneal microfibrils are proteinaceous and that the tannic acid-staining component of the bundles is not glycosaminoglycan. The fibrils are indistinguishable from those identified as oxytalan in cornea and other ocular tissues. Moreover, their sensitivity to elastase and preferential staining with tannic acid/glutaraldehyde strongly suggest they may be related to the elastic system of fibrils.

Ascorbic acid uptake and metabolism by corneal endothelium.

Ascorbic acid is concentrated in various ocular compartments where it is thought to protect diurnal animal species against damaging effects of ultraviolet radiation. The authors evaluated the possibility that corneal endothelial cells have specific transport and/or metabolic properties that deliver ascorbic acid to the stroma. Bovine corneal endothelial cells were grown to confluence in multiple-well plates. Individual groups of cells (approximately 10(4)) were then incubated at various times at 34 degrees C in a physiologic buffer that contained a 10 microM level of 14C-labeled ascorbic acid or the oxidized product, dehydro-L-ascorbic acid. Endothelial cells take up dehydro-L-ascorbic acid at least seven times as rapidly as they take up ascorbic acid. After 30 sec of incubation with 14C-dehydro-L-ascorbic acid, most of the label accumulated in the cell is in the reduced form. Uptake is inhibited by cyanide and iodoacetamide but is unaffected by ouabain. Exposure of cultured cells to various intermediates in the energy metabolism pathways reduced uptake of ascorbic acid but had a minor effect on uptake of the oxidized molecule. These results suggest that the cornea has transport and metabolic capacity to extract dehydro-L-ascorbic acid from aqueous humor and reduce it, thus providing a source of ascorbic acid for corneal protection. This also would maintain "total" ascorbic acid of aqueous humor in the reduced state.

Altered proliferation of retinal microvascular cells on glycated matrix.

PURPOSE: To investigate the effect of nonenzymatic glycosylation (glycation) of basement membranes (BM) and isolated BM proteins on the growth of retinal pericytes and retinal endothelial cells. METHODS: Type IV collagen, laminin, Engelbreth-Holm-Swarm tumor basement membrane (EHS-BM) and bovine retinal basement membrane (RBM), after incubation in the presence of reducing sugars to induce glucose-mediated modifications, or in the absence of any sugar (control), were used as a substrate to culture bovine retinal microvascular cells. Cell growth on the nonenzymatically glycosylated and the corresponding control substrates was measured daily, using an automated cell counter. RESULTS: Retinal pericytes seeded on glycated type IV collagen proliferated consistently more slowly than on control type IV collagen (P = 0.02), showing a 20% to 33% decrease throughout most of the growth curve, whereas on glycated laminin the difference from control was not significant. In contrast, proliferation increased by 16% to 25% for retinal endothelial cells on glycated laminin compared with control substrate (P = 0.025), whereas on glycated type IV collagen the growth curve was not significantly different from the curve for the control. When seeded on whole glycated EHS-BM or RBM, proliferation of pericytes decreased by 20% to 30% (P = 0.04); the endothelial cells showed no difference on glycated EHS-BM, however, the growth rate increased on glycated RBM by 25% to 30% more than it did for the control (P = 0.01). CONCLUSIONS: Nonenzymatic glycosylation of intact BM or individual BM macromolecules resulted in reduced proliferation of retinal pericytes and increased proliferation of retinal endothelial cells. These in vitro observations resemble some of the pathologic changes of the retinal microvascular cells observed in situ, when diabetic retinopathy develops.

Alveolar bone of BB/W rats: a morphometric and histochemical study.

The present study reported histochemical changes in alveolar bone glycosaminoglycans (GAG) (using Safranin O) and in interdental bone height in three groups of BB/W rats: diabetic, diabetes prone, and diabetes resistant. Safranin O staining intensity suggested that total GAG levels were highest in diabetic bone (p less than 0.05 compared to diabetes resistant, p less than 0.005 compared to diabetes prone) but not significantly different between diabetes prone and resistant groups. Following chondroitinase AC and ABC digestion, staining reactions suggested that the highest levels of dermatan sulfate were in the diabetes resistant group (p less than 0.001 compared to diabetic, p less than 0.001 compared to diabetes prone) and the highest levels of chondroitin sulfates were in the diabetes prone group (p less than 0.001). Coincidently the mean height of diabetes prone interdental septum was significantly less than that of diabetes resistant or diabetic groups (p less than 0.05). The study suggested that 1) diabetes and "prediabetes" produce significant changes in levels of chondroitin 4, 6, and dermatan sulfates within alveolar bone, 2) in "prediabetic" animals, interdental bone loss occurs prior to the onset of clinical symptoms and in the absence of local irritating factors, the bone height appears to return to normal levels, and 3) there may be a correlation between alveolar bone height and relative levels of dermatan sulfate.

Electron microscopic histochemical and immunochemical analyses of heparan sulfate proteoglycan distribution in renal glomerular basement membranes.

Renal glomerular basement membranes (GBMs) exhibit a charge-selective barrier, comprised of anionic sites, that restrict the passage of anionic molecules into the urine. These sites are located primarily in the laminae rarae interna (LRI) and externa (LRE) of the GBM and consist of heparan sulfate proteoglycan (HSPG). Previous efforts to localize HSPG core protein within various layers of the GBM have been contradictory. In the present study when rat renal cortex blocks were treated by immersion with the cationic probe, polyethyleneimine (PEI), GBMs exhibited anionic sites concentrated primarily in the LRE and more irregularly within the LRI and lamina densa. All sites were heparitinase sensitive indicating that PEI positive sites represent negatively charged groups associated with heparan sulfate. In order to gain information on the distribution of the HSPG protein core, antibodies to HSPG from the EHS tumor matrix [anti-(EHS) HSPG] and GBMs [anti-(GBM) HSPG] were used together with immunogold to label thin sections of Lowicryl embedded kidney cortex. Depending upon the antisera used, markedly different distributions of HSPG were obtained. Immunolabelling with anti-(GBM) HSPG suggested a distribution of HSPG which was restricted to the laminae rarae, whereas labelling with anti-(EHS) HSPG indicated that the protein core penetrates through all layers of the GBM.

High resolution SEM analysis of acellular glomerular basement membrane following pepsin digestion: intrinsic fibrillar structures.

Microdissection of acellular rat renal cortex with pepsin was carried out to investigate the morphological substructure of glomerular basement membrane (GBM) by high resolution SEM. Renal cortical blocks (less than 5 mm3) from adult male Sprague Dawley rats were rendered acellular by sequential detergent extraction and digested up to 184 hrs with 5 mg/ml pepsin (185 U/mg) in 0.5 M acetic acid (pH 2) at 10-15 degrees C. Samples were conventionally prepared for SEM, and observed at original magnifications of 500-100,000 diameters. At low magnifications (500-5,000x), acellular GBM surfaces appeared smooth at all digestion times. At higher magnifications (50,000-100,000x), control GBM surfaces were finely granular. Granule diameter ranged from 20-80 nm, with most between 30-40 nm. Pepsin digestion did not affect average granule size. Beginning at 44 hrs of digestion, intrinsic fibrillar structures comprised of linear arrays of 20-40 nm granules were observed on/in GBM surfaces. At later incubation times, this component of GBM became more extensive. At 160 hrs, the fibrillar arrays frequently bifurcated and showed distinctive "forked" termini, some of which comprised two sides of a triangle (120-150 nm on a side). Fork "handles" (310-350 nm in length) radiated from each angle of the triangle. These sometimes terminated in large granules (approximately 100 nm in diameter), two of which appeared to connect fibrillar arrays end-to-end. Together with other arrays, the interconnected triangles appeared to comprise a three-dimensional meshwork extending into the GBM and possibly providing support for, its granular components.

Scanning and transmission electron microscopic studies of normal and diabetic acellular glomerular and retinal microvessel basement membranes.

Basement membranes (BMs) were first described in the mid-19th century, but they were not isolated and prepared for compositional studies until nearly 100 years later. Early methods of isolation were carried out on renal glomeruli, which were first sub-fractionated from kidney tissues by sieving. BMs were then isolated from the glomeruli by ultrasonic disruption, which, following low speed centrifugation, yielded "purified" but highly fragmented BM material. In an effort to obviate the mechanical damage to BMs produced by ultrasound, a sequential detergent solubilization technique was introduced that resulted in morphologically intact BMs from a variety of tissue sub-fractions. This was highly advantageous because "acellular" BMs produced by the procedure could be examined critically by light and electron microscopic methods. Subsequently, this procedure has been utilized to demonstrate the substructural heterogeneity of vascular and non-vascular BMs from a wide variety of animal species. The current review describes the results of scanning and transmission electron microscopic studies of acellular BMs prepared from renal glomeruli and from the retinal microvessels of the eye. These BMs are of particular interest to basic scientists and clinicians because they are altered in several disease states, most notably diabetes mellitus. An effort is made to point out the implications of glomerular and retinal vessel BM changes to the pathogenesis of diabetic kidney and retinal vessel BM disease.

Effect of hypothermic perfusion on corneal endothelial morphology.

The effect of moderate in-vivo hypothermic perfusion on corneal endothelial integrity was studied in the cat. Eleven cats underwent in-vivo anterior chamber perfusion for 30 minutes with either normothermic (23 degrees C) or hypothermic (5 degrees C) perfusate. Corneas were then evaluated clinically (biomicroscopy), functionally (vital staining), and morphologically (scanning electron microscopy) for changes attributable to hypothermic perfusion. All 3 modes of evaluation suggested no difference in corneal endothelial integrity under the 2 experimental perfusion conditions. At the clinical and scanning electron microscope levels hypothermic perfusion does not show any effects on the corneal endothelium. Regional hypothermia is of theoretical and potential utility in procedures involving prolonged intraocular perfusion.

An ultrastructural analysis of isolated basement membranes in the acellular renal cortex: a comparison study of human and laboratory animals.

Freshly harvested kidneys from New Zealand white rabbits, Sprague-Dawley white rats, rhesus monkeys, and transplant-quality human kidneys were used in this study. Minced renal cortical tissue blocks (less than 2 mm3) were treated with 1 mM EDTA, 3% Triton X-100, 0.025% DNAse, and 4% sodium deoxycholate in an effort to remove all cellular elements and leave the extracellular matrix (ECM) intact. These preparations showed remarkable structural preservation and all components of the ECM, including basement membranes (BMs), maintained their in vivo histoarchitectural relationships. By light microscopy, at least four major BM types were recognizable, including Bowman's capsular BM (BCBM), tubular BM (TBM), glomerular BM (GBM), and peritubular capillary BM (PTCBM). Scanning electron microscopy demonstrated that, despite the lack of supporting interstitium, GBMs in human, monkey, and rat (and rabbit to a lesser degree) exhibit intrinsic structural rigidity such that their convoluted spheroidal shapes are maintained following cell removal. Transmission electron microscopy showed that major BM types are morphologically heterogeneous and vary markedly within and between species. Randomized measurements showed that isolated BM thicknesses (lamina densa only) compared favorably with those reported in cellular preparations. Mean thicknesses of GBMs were within normal ranges in all species with or without power transformations to reduce right-sided skew of distribution curves. In all species, thickness of BCBM greater than TBM greater than GBM greater than PTCBM. The striking morphologic heterogeneity of major BM types demonstrated in the acellular renal cortex is not surprising in view of recent biochemical analyses that show that BMs derived from different sources are compositionally disparate. We conclude that BMs should be evaluated and characterized individually and that morphologic definition of isolated BMs necessary prior to further analysis.

Intrinsic fibrillar components of human glomerular basement membranes: a TEM analysis following proteolytic dissection.

At the level of ultrastructure, basement membranes (BMs) are usually described as thin layers of extracellular matrix comprised of an interwoven mat of fine 3-4 nm fibrils embedded in a granular matrix. In order to improve the resolution of the fibrillar components, we have carried out TEM studies on human glomerular BMs (GBMs) made acellular by sequential detergent solubilization. Some GBMs were pretreated with pronase, trypsin, or pepsin for 30 min to 72 h prior to preparation for microscopy. Our study shows that irrespective of which enzyme is employed, background granular matrix is first solubilized leaving a three dimensional fibrillar network comprised of 3-8 nm fibrils. Larger 7-8 nm fibrils are concentrated near subepithelial portions of the GBM and are most resistant to proteolysis. Smaller 3-4 nm fibrils are located primarily subjacent to endothelium and mesangial cells and are more protease-sensitive. An unexpected finding in pepsinized samples was a quasi-hexagonal fibrillocrystalline structure associated with mesangial matrix and subendothelial portions of the GBM. These data suggest that intrinsic fibrillar components of human GBMs are heterogeneously distributed throughout the thickness of their laminae densae. We speculate that the network consists of type IV collagen and that the hexagonal crystals may represent type VI or some previously unreported BM collagen type.

Nerve suture and grafting.

Nerve anatomy, terminology, and techniques for nerve restoration are reviewed in sufficient detail to allow the reader to quickly update his or her knowledge in this area. Ideas for future directions of study are presented.

Continued competency--a responsibility of the profession.

For the past decade the Continued Competency Committee of the American Association of Dental Examiners has explored issues in continued competency for the dental profession. The efforts have focused on creating policy and standards which must be met by any continued competency assessment mechanisms. Nine potential systems are under review. Some, such as examination for diplomate status in a recognized dental specialty are already in place. The development and pilot testing of four new mechanisms--simulations, continuing education with measurable outcomes, case presentation, and in-office audit--is being encouraged.