Ink Staining Research Articles

Douleurs osseuses fébriles chez l’enfant drépanocytaire : apport de l’IRM

Purpose The aim of this study was to report the MRI findings that can suggest a vaso-occlusive crisis in cases of febrile osseous pain in children suffering from sickle cell disease. Materials and methods MRI (T1 and T2 weighted sequences and T1 weighted sequence with fat saturation before and after gadolinium injection) was performed in 10 children with sickle cell disease, presenting with febrile osseous pain. The diagnosis of vaso-occlusive crisis was made after fast improvement due to symptomatic treatment and negative bacteriological result. Results MRI was abnormal in all cases. A multifocal localisation was found in 2 cases. Bone marrow abnormalities were constant. In 10 cases, high T1 and T2 signal and metaphyso-diaphysial lesions were noted. Heterogeneous medullar enhancement with “ink stain” feature was constant. Early periosteal abnormalities were noted in 8 cases with inflammatory or stratified features. Cortical thinning was found in 1 case. Soft tissue abnormalities were observed in 5 cases with inflammatory features in 4. Conclusion Multifocal synchronous localisation, medullar abnormalities resulting from hemoglobin degradation, heterogeneous enhancement, early periosteal abnormalities and associated soft tissues swelling are MRI findings suggesting acute vaso-occlusive disease.

Evaluation of subchondral bone mineral density associated with articular cartilage structure and integrity in healthy equine joints with different functional demands

To determine and correlate subchondral bone mineral density and overlying cartilage structure and tensile integrity in mature healthy equine stifle (low magnitude loading) and metacarpophalangeal (high magnitude loading) joints. 8 healthy horses, 2 to 3 years of age. Osteochondral samples were acquired from the medial femoral condyle (FC) and medial trochlear ridge (TR) of the stifle joint and from the dorsal (MC3D) and palmar (MC3P) aspects of the distal medial third metacarpal condyles of the metacarpophalangeal joint. Articular cartilage surface fibrillation (evaluated via India ink staining) and tensile biomechanical properties were determined. The volumetric bone mineral density (vBMD) of the underlying subchondral plate was assessed via dual-energy x-ray absorptiometry. Cartilage staining (fibrillation), tensile moduli, tensile strength, and vBMD were greater in the MC3D and MC3P locations, compared with the FC and TR locations, whereas tensile strain at failure was less in MC3D and MC3P locations than FC and TR locations. Cartilage tensile moduli correlated positively with vBMD, whereas cartilage staining and tensile strain at failure correlated negatively with vBMD. In areas of high joint loading, the subchondral bone had high vBMD and the articular cartilage surface layer had high tensile stiffness but signs of structural wear (fibrillation and low failure strain). The site-dependent variations and relationships in this study support the concept that articular cartilage and subchondral bone normally adapt to physiologic loading in a coordinated way.

Cryptococcal Meningitis Presenting as a Fever of Unknown Origin Following Immunosuppressive Therapy for Autoimmune Hepatitis

Introduction: Cryptococcus neoformans (C.N) is an opportunistic pathogen causing chronic meningitis in immunocompromised patients. The diagnosis is often missed or delayed in these patients because of the non-specific clinical features, limited usefulness of available serological tests, and failure to isolate CN in blood cultures, positive in less than one third of patients. Case Report: A 20-year-old female with congenital arthrogryposis presented with ascites, lower extremity edema, jaundice, coagulopathy and an autoantibody profile of autoimmune hepatitis. Diagnostic liver biopsy was followed by asymptomatic temp. to 103 F and the patient admitted for observation. An extensive fever workup during a 10 ten hospitalization was negative. She was readmitted one week with lethargy, confusion, bradycardia and recurrent fever. Medications included:Prednisone 7.5 mg daily and Immuran 100 mg daily for treatment of autoimmune hepatitis. P.E. revealed a febrile somnolent cushingoid female without focal neurologic or abdominal findings. Labs revealed coagulopathy secondary to liver dysfunction. On the second day of hospitalization, blood cultures were notable for a fungal species. She rapidly developed severe headache, vomiting, disconjugate gaze and meningismus. A lumbar puncture showed increased ICP and India ink staining of the CSF and spinal fluid and blood cultures confirmed the diagnosis of Cryptococcal meningitis. The patient was treated with Amphotericin B and 5-Flucytosine. Multiple lumbar drainages were required to control increased ICP. She made a complete neurological recovery. Discussion: Patients on immunosuppressive therapy are at increased risk for cryptococcal diseases. Cirrhosis may be an additional predisposing factor by impairment of host defenses including complement deficiency, defects in neutrophil chemotaxis, and lymphocyte hyporesponsiveness. To our knowledge, the diagnosis of disseminated Cryptococcosis by blood cultures in a patient with Autoimmune Hepatitis has not been described previously. This case demonstrates that prolonged fever and therapy with corticosteroids and immunomodulators should raise concern for emerging disseminated Cryptococcal disease. The acute and atypical clinical manifestations in our patient indicate the importance of early consideration of this disease as clinical deterioration and death may be precipitous.

Cryptococcal meningitis in an immunocompetent child

An immunocompetent child infected with cryptococcal meningitis was cured without any sequelae or relapse with six months of antifungal treatment. An 11-year old Japanese boy who presented with headache and vomiting without high fever was admitted to a local hospital with a diagnosis of aseptic meningitis and discharged after symptomatic relief. Cryptococcus neoformans was later detected in the cerebrospinal fluid (CSF) culture and he was referred to our hospital. His physical and neurological examination was unremarkable except for a mild stiff neck. Cryptococcus neoformans was detected in an India ink stain and culture of the CSF upon admission (Fig. 1), and the cryptococcal antigen with latex agglutination was positive. CSF revealed leukocytosis, increased protein and high opening pressure with normal glucose (53 mg/dl) (Fig. 2). Blood leukocyte count was 7600/ll. Although chest Xray was normal, CT revealed mild peribronchial inflammation in the left lower lobe (S7). An enhanced head MR imaging was negative. The initial treatment with intravenous amphotericin B (AMPH-B) and oral flucytocin (FC) was discontinued after ten days due to fever and skin rash: fluconazole (FLCZ) was administered orally for six months thereafter (Fig 2). No relapses or sequelae have been observed. A detailed developmental and medical history was unremarkable except that he had a history of repeated hospitalizations for asthma until eight years of age. It is noteworthy that he had occasionally been exposed to guano of wild pigeons. His humoral and cellular immunological parameters (CD4 900/ll, CD4/8 2.25), complement and neutrophil functions were normal. Anti-HIV antibody was negative. Although cryptococcal meningitis is uncommon among HIV-negative patients [1, 2, 6, 7, 8], severe pediatric cases have been reported [3, 4]. We infer that, in the case reported here, a pulmonary cryptococcal infection was the primary lesion preceding meningitis because of the exposure to guano of pigeons, repeated

Human Cytomegalovirus UL84 Is a Phosphoprotein That Exhibits UTPase Activity and Is a Putative Member of the DExD/H Box Family of Proteins

Human cytomegalovirus (HCMV) UL84 is required for lytic DNA replication and is proposed to be the key factor in initiation of viral DNA synthesis. We now show that UL84 has a high degree of homology to the DExD/H (where x can be any amino acid) box family of helicases, displays UTPase activity, and is phosphorylated at serine residues. Affinity column-purified UL84-FLAG fusion protein was used in an in vitro nucleoside triphosphatase (NTPase) assay to show that UL84 has NTPase activity, preferring UTP. This UTPase activity was linear with respect to enzyme concentration and slightly enhanced by the addition of nucleic acid substrates. UL84 UTPase was the highest at low salt concentrations, a pH of 7.5, and a temperature of 45 degrees C. The enzyme preferred Mg2+ as the divalent cation but was also able to catalyze the UTPase reaction in the presence of Mn2+, Ca2+, and Zn2+ albeit at lower levels. The evidence presented here suggests that the UL84 UTPase activity may be part of an energy-generating system for helicase activity associated with the initiation of HCMV DNA replication.

Community acquired pneumonia (CAP) caused byCryptococcus neoformansin a healthy individual

A 41-y-old male had been diagnosed as having community acquired pneumonia (CAP) with consolidations in the chest radiograph, fever and cough. Since clarithromycin and ss-lactam agents were not effective, bronchoscopic examination was performed. Indian ink staining of bronchial wash smears revealed yeast-like cells with a thick capsule, and Cryptococcus neoformans was isolated several d later. Serum glucuronoxylomannan antigen was > or = x 1024. The patient was treated with itraconazole for 16 weeks.

Site- and exercise-related variation in structure and function of cartilage from equine distal metacarpal condyle

Determine (1) the site-associated response of articular cartilage of the equine distal metacarpal condyle to training at a young age as assessed by changes in indentation stiffness and alterations in cartilage structure and composition, and (2) relationships between indentation stiffness and indices of cartilage structure and composition. Experimental animals (n=6) were trained on a track (increasing exercise to 1km/day by 5 months); controls (n=6) were pasture-reared. Animals were euthanized at 18 months and four osteochondral samples were harvested per metacarpal condyle from dorsal-medial, dorsal-lateral, palmar-medial, and palmar-lateral aspects. Cartilage was analyzed for India ink staining (quantified as reflectance score (RS)), short-term indentation stiffness (sphere-ended, 0.4mm diameter), thickness, and biochemical composition. Cartilage structural, biochemical and biomechanical properties varied markedly with site in the joint. Sites just medial and just lateral to the sagittal ridge showed signs of early degeneration, with relatively low RS, indentation stiffness, and collagen content, and relatively high water content. Effects of exercise and side (left vs right) were not detected for any measure. Overall, indentation stiffness correlated positively with RS and collagen content, and inversely with thickness and water content. Gentle exercise-imposed mechanical stimulation did not markedly affect articular cartilage function or structure. However, the marked site-associated variation suggests that biomechanical environment can initiate degenerative changes in immature cartilage during joint growth and maturation.

Cryptococcus neoformans CAP59 (or Cap59p) is involved in the extracellular trafficking of capsular glucuronoxylomannan.

Several genes are essential for Cryptococcus neoformans capsule synthesis, but their functions are unknown. We examined the localization of glucuronoxylomannan (GXM) in strain B-3501 and in cap59 mutants B-4131 and C536. Wild-type strain B-3501 showed a visible capsule by India ink staining and immunofluorescence with anticapsular monoclonal antibodies (MAbs) 12A1 and 18B7. B-4131, a mutant containing a missense mutation in CAP59, showed no capsule by India ink staining but revealed the presence of capsular polysaccharide on the cell surface by immunofluorescence. The cap59 gene deletion mutant (C536), however, did not show a capsule by either India ink staining or immunofluorescence. Analysis of cell lysates for GXM by enzyme-linked immunosorbent assay revealed GXM in C536 samples. Furthermore, the epitopes recognized by MAbs 12A1, 2D10, 13F1, and 18B7 were each detected in the cytoplasm of all strains by immunogold electron microscopy, although there were differences in location consistent with differences in epitope synthesis and/or transport. In addition, the cells of B-3501 and B-4131, but not those of the cap59 deletant, assimilated raffinose or urea. Hence, the missense mutation of CAP59 in B-4131 partially hampered the trafficking of GXM but allowed the secretion of enzymes involved in hydrolysis of raffinose or urea. Furthermore, the cell diameter and volume for strain C536 are higher than those for strain B-3501 or B-4131 and may suggest the accumulation of cellular material in the cytoplasm. Our results suggest that CAP59 is involved in capsule synthesis by participating in the process of GXM (polysaccharide) export.

Indentation testing of human cartilage: sensitivity to articular surface degeneration.

To determine, for clinical indentation testing of human articular cartilage, the effects of aging and degeneration on indentation stiffness and traditional indices of cartilage degeneration; the relationship between indentation stiffness and indices of degeneration; and the sensitivity and specificity of indentation stiffness to cartilage degeneration. Osteochondral cores from femoral condyles of cadaveric human donors were harvested. Samples were distributed into experimental groups based on donor age (young [20-39 years], middle [40-59 years], and old [>/=60 years]), and a macroscopic articular surface appearance that was either normal or mildly degenerate, without deep erosion. Samples were analyzed for indentation stiffness, cartilage thickness, India ink staining (quantitated as the reflected light score), and Mankin-Shapiro histopathology score. Indentation stiffness, India ink staining, and the histopathology score each varied markedly between normal-sample and degenerate-sample groups but varied relatively little between normal samples obtained from different age groups. A decrease in indentation stiffness (softening) correlated with a decrease in the reflectance score and an increase in the overall histopathology score, especially the surface irregularity component of the histopathology score. Receiver operating characteristic analysis suggested that the indentation testing could accurately detect cartilage degeneration as indicated by macroscopic appearance, India ink staining, and histopathology score. The indentation stiffness of the normal to mildly degenerate samples tested in this study was sensitive to mild degeneration at the articular surface and was insensitive to changes associated with normal aging or to slight variations in cartilage thickness. This suggests that indentation testing may be a useful clinical tool for the evaluation of early-stage degenerative changes in articular cartilage.

A yeast under cover: the capsule of Cryptococcus neoformans.

Few fungi are pathogenic to humans. Of these, Cryptococcus neoformans has emerged as an important cause of mortality in immunocompromised patients, especially those with AIDS. As a result, extensive research efforts have addressed the pathogenesis and virulence of this organism. C. neoformans is a basidiomycetous fungus that is ubiquitous in the environment, where it is found in soil, in association with certain trees, and in bird guano (16). Because of its ubiquity, it has been suggested that most people are exposed to C. neoformans early in life (41). The fungus is heterothallic, with mating types MATa and MAT. Asexual reproduction takes place either by budding or, in the case of MAT cells, by haploid fruiting in response to nutrient deprivation or exposure to the mating pheromone a factor (106). Sexual reproduction occurs when cells of opposite mating types come together to form a heterokaryon that ultimately leads to the production of basidia and basidiospores (64). Desiccated cells and the spores formed by haploid fruiting or sexual reproduction have all been suggested to serve as infective particles, which must be less than 2 m in diameter to penetrate the lung parenchyma (44, 86). Infection occurs when the fungal particles are inhaled and enter the alveolar space. In most immunocompetent individuals, this infection is either cleared or remains dormant until an immune imbalance leads to further development. In the setting of compromised immune function, however, the fungus disseminates, with particular tropism for the central nervous system. In severe cases, cryptococcal infection progresses to a meningoencephalitis that is fatal if left untreated. C. neoformans virulence is mediated predominantly by a polysaccharide capsule that surrounds its cell wall and has multiple effects on the host immune system. This structure provides a physical barrier that interferes with normal phagocytosis and clearance by the immune system. Capsule components inhibit the production of proinflammatory cytokines, deplete complement components (by efficiently binding them), and reduce leukocyte migration to sites of inflammation (11). The capsule also constitutes the major diagnostic feature of cryptococcosis, because its components can be detected in the bloodstream and it can be visualized with light microscopy by using India ink staining. The capsule excludes the ink particles and forms characteristic halos (Fig. 1A) whose diameters are often several times that of the cell. The elaborate structure of the capsule may also be appreciated by electron microscopy (Fig. 1B and C). Given the importance of the capsule in cryptococcal disease, tremendous effort has been applied in recent years to understanding its biology. This review focuses on the resulting advances in our understanding of the structure and synthesis of the capsular components, the incorporation of these components into the existing capsular network, the association between the capsule and the cell wall, and the regulation of capsule growth.

Henneguya curvata sp. n. (Myxosporea: Myxobolidae) parasitizing the gills of Serrasalmus spilopleura (Characidae: Serrasalminae), a South American freshwater fish.

The class Myxosporea of the phylum Myxozoa contains 52 genera (Kent M.L., Andree K.B., Bartholomew J.L., ElMatbouli M., Desser S.S., Devlin R.H., Feist S.W., Hedrick R.P., Hoffmann R.W., Khattra J., Hallett S.L., Lester R.J.G., Longshaw M., Palenzeula O., Siddall M.E., Xiao C.X. 2001: J. Eukaryot. Microbiol. 48: 395–413), most of which parasitize fishes. Henneguya Thelohan, 1892 is the second most common of these genera and contains more than 150 species, some of which are important pathological agents (Dykova I., Lom J. 1978: J. Fish Biol. 12: 197–202; Kalavati C., Narasimhamurti C.C. 1985: Arch. Protistenkd. 129: 199–202; Lom J., Dykova I. 1995: In P.T.K. Woo (Ed.), Fish Diseases and Disorders. Protozoan and Metazoan Infections. Vol. 1. CAB International, Wallingford pp. 97–147; Martins M.L., Souza V.N., Moraes J.R.E., Moraes F.R. 1999: Rev. Bras. Biol. 59: 527– 534). In South America, Henneguya is the most abundant genus, with 31 species. To date, two species of Henneguya have been reported in Serrasalmus spp.: Henneguya iheringi Pinto, 1928 parasitizing Serrasalmus spilopleura caught in the state of Sao Paulo, and H. striolata Casal, Matos et Azevedo, 1997 parasitizing S. striolatus collected in the Amazon River estuary near Belem, state of Para, Brazil. Fish of the genus Serrasalmus are voracious carnivorous fish popularly known as piranhas and are widely distributed throughout South American rivers. In this study, we describe a new species of Henneguya parasitizing S. spilopleura. Eighteen adult and juvenile specimens of S. spilopleura were collected locally from a lake on a farm in the municipality of Campinas, state of Sao Paulo, Brazil, and examined for the presence of myxosporeans. The fish were captured between January and June 2001, and transported alive to the laboratory where they were killed by transection of the spinal cord then measured and necropsied. The measurements of 30 fresh mature spores (Lom J., Arthur J.R. 1989: J. Fish Dis. 12: 151–156) were obtained using a micrometer incorporated into a microscope eyepiece. The dimensions were expressed as the mean ± standard deviation (SD). India ink staining was used to detect the mucus envelope. The spores were checked for the presence of an iodinophilous vacuole after adding a drop of Lugol solution. Smears containing free spores were stained with Giemsa’s solution and mounted in low viscosity medium as permanent mounts (Adriano E.A., Arana S., Ceccarelli P.S., Cordeiro N.S. 2002: Folia Parasitol. 49: 259–262). For histological analysis, portions of the gills containing plasmodia were fixed in 10% buffered formalin for 24 h, embedded in paraffin, cut into sections 4 μm thick and stained with sirius red (Adriano et al. 2002, op. cit.), haematoxylin and eosin and PAS.

Potentiation of photodynamic therapy with hypericin by mitomycin C in the radiation-induced fibrosarcoma-1 mouse tumor model.

Hypericin, a polycyclic quinone obtained from plants of the genus Hypericum, has been shown to be a promising photosensitizer. We investigated the combination of hypericin-photodynamic therapy (PDT) and a bioreductive drug mitomycin C (MMC) in the present study. The radiation-induced fibrosarcoma-1 tumors were exposed to laser light (120 J/cm2 at 595 nm) 24 h after an intravenous injection of hypericin (1 mg/kg). Hypericin-PDT alone significantly decreased tumor perfusion and oxygen tension as demonstrated by India ink staining technique and OxyLite pO2 measurement, respectively. The in vivo-in vitro cell-survival assay revealed about 60% direct tumor cell killing immediately after PDT. No significant delayed tumor cell death was observed after PDT, which suggests that vascular damage does not contribute significantly to the overall tumor cell death. Injection of a 2.5 mg/kg dose of MMC 20 min before light application significantly decreased tumor cell survival and delayed tumor growth compared with PDT or MMC alone. No greater skin reaction was observed after the combination of MMC and PDT than after PDT alone. Our study demonstrates that combining hypericin-PDT with MMC can be effective in enhancing tumor response with little side effect.

Bacteroides fragilis adherence to Caco-2 cells

The ability of ten Bacteroides fragilis strains isolated from intestinal and non-intestinal infections, normal flora and environment to adhere to human colon carcinoma cells, Caco-2, was examined. The adherence capacity varied among the strains tested from strongly adherent (76–100%) to non- or weakly adherent (0–25%). Negative staining with Indian ink showed that all the strains were capsulated, although strain 1032 (strongly adherent and originated from bacteremia) had the highest rate of capsulated cells in the culture. All strains studied presented an electron-dense layer and no fimbrial structures in their surface after PTA negative staining. The analysis of the strains with ruthenium red showed the presence of an acidic polysaccharide and also surface vesicles in all of them. The strain 1032 presented an aggregative adherence pattern toward Caco-2 cells monolayers. It could be seen trapped by elongated microvilli and surrounded by extracellular material in the scanning electron microscope. Treatment with sodium periodate (100 mM/1 h) reduced significantly its adherence capacity and also the expression of an electron-dense layer and of the capsule, detected with PTA and Indian ink staining, respectively. We suggest that the capsular polysaccharide might mediate the adherence of the B. fragilis to Caco-2 cells.

Removal of dye-based ink stains from ivory: evaluation of cleaning results based on wavelength dependency and laser type

The removal of ink stains from elephant ivory and related materials can present an intractable problem for the conservator. This research evaluates laser energy as a tool for ivory conservation and highlights the differences between removing stains that penetrate the substrate, as opposed to surface accretions, using a range of laser wavelengths. Samples of ink-stained ivory were prepared and treated with wavelengths ranging from the infrared to the far UV in order to remove ink staining. Different effects were observed at different regions of the spectrum and with different laser types (Nd:YAG, KrF excimer, ArF excimer, OPO, etc.) with the most successful removal of ink occurring in the visible range. Furthermore, there appears to be a relationship between wavelength in the visible range and the color of the ink removed, which correlates to the absorption spectra for a given ink. The results of these experiments will be discussed along with the possible mechanisms involved and some of the surface analytical techniques employed to evaluate the effectiveness of cleaning.

India ink staining after sodium dodecyl sulfate polyacrylamide gel electrophoresis and in conjunction with Western blots for peptide mapping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

We present an approach that allows matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (MALDI-TOFMS) peptide mapping of proteins separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto nitrocellulose (NC). After blocking the nitrocellulose membrane with polyvinylpyrrolidone-40 the immobilized proteins are visualized using India Ink staining which allows the detection of low nanogram amounts of protein. The utilization of a low concentration of Tween 20 (0.05%) in the India Ink staining solution does not negatively impair the quality of the mass spectra. Due to the virtual nondestructive nature of the stain proteolytic peptides could be recovered from the NC membrane. Taking into account minor precautions during the sample manipulation and concentration and by loading the sample onto a pre-crystallized matrix layer, high quality mass spectral data were obtained on <100 femtomoles of protein loaded onto the gel. Finally, the use of India Ink in conjunction with Western blot analysis is also demonstrated. A rat plasma protein, characterized by Western blot as a covalently modified protein-drug compound, was subjected to peptide mapping and post source decay (PSD) sequencing of peptides. The zomepirac-modified protein was identified as the alpha-subunit of fibrinogen.

Chondrocyte death precedes structural damage in blunt impact trauma.

Joint impact trauma has been shown to cause fissures, fibrillation, and other structural damage of the cartilage or subchondral bone. Previous studies used impact energies sufficient to fracture the underlying bone. Under these circumstances, the initial influence of impact trauma on cellular components and cartilage structure is unknown. The goal of this study was to determine whether an impact trauma first causes cellular or structural damage to a cartilage layer. Such damage might be the starting point of degenerative changes found in osteoarthrosis. Porcine patellas (n = 12) were subjected to standardized low-impact loading of three magnitudes with a spherical impactor attached to a drop tower device (0.06, 0.1, and 0.2 J). India ink staining and scanning electron microscopic analysis were used for analysis and showed no evidence of gross structural disruption. Chondrocyte viability assessed with thiazole blue staining and propidium iodide counterstaining was reduced significantly in the tangential and middle zones with increasing impact energy. These results indicate that chondrocyte death may precede excessive structural damage reported in earlier studies and might be a crucial factor in posttraumatic osteoarthrosis.

A new flap model in rats: iliac osteomusculocutaneous flap.

Although osteomusculocutaneous flaps are used frequently in clinical practice to repair defects involving soft tissue and bone, there are still many questions that remain to be answered regarding their basic physiology. To accomplish such basic science studies, simple and reliable animal osteomusculocutaneous flap models are needed. The purpose of this study was to describe a new flap model in rats--namely, the iliac osteomusculocutaneous flap. Thirty adult Wistar rats weighing 200 to 250 g were used in this experiment. In 15 rats, the vascular anatomy of the iliolumbar vessels and their relation with adjacent soft tissues and the iliac bone was determined by anatomic dissection. Based on this anatomic study, the iliac osteomusculocutaneous flap model was created in rats. The flap is comprised of a skin island (3 x 3 cm) in the flank region, a 1 x 1-cm segment of iliac bone, and an abdominal wall muscle cuff. In 10 rats, the flap was raised as an island flap based on its vascular pedicle of iliolumbar vessels, and was replaced in situ. In the remaining 5 rats, the flap was transferred to the groin region as a free flap. Direct observation on postoperative day 7 revealed that the skin island of all the flaps was completely viable. Bone scintigraphy performed on postoperative day 3 in free flaps demonstrated radionuclide uptake, indicating viability of the bony segment. The dye injection study revealed ink staining within blood vessels of the bone, confirming its viability. Microangiography of the flap demonstrated vascularity of each component of the flap by the iliolumbar vessels, including a distinct branch to the iliac bone. The authors conclude that the iliac osteomusculocutaneous flap of the rat is a simple and reliable flap model that offers the following advantages: (1) It is a true osteomusculocutaneous flap, (2) it can be used as a free flap without the need for an isogeneic rat, (3) the vascular pedicle is consistent, and (4) it is harvested from a small-animal species.

The influence of strenuous exercise on collagen characteristics of articular cartilage in Thoroughbreds age 2 years.

In order to assess the influence of strenuous exercise on collagen characteristics of articular cartilage, the response of the collagen network was studied in seven 2-year-old Thoroughbreds subjected to strenuous exercise compared to 7 nontrained individuals. After 13 weeks, the animals were subjected to euthanasia, fetlock joints of the forelimbs were scored macroscopically after Indian Ink staining, and articular cartilage from different locations of the articular surface of the proximal first phalanx was sampled and analysed for water content, collagen content, hydroxylysine content and amount of hydroxylysylpyridinoline (HP) crosslinks. Gross lesions were significantly more severe in the exercised than in the nonexercised group. In the control animals, the characteristic site-specific differences in collagen parameters were found as described earlier, but in the strenuously exercised animals this physiological biochemical heterogeneity had disappeared. In the exercised animals, an increase in water content and a sharp decrease in HP crosslinking was found that was correlated with the presence of wear lines. It is concluded that the strenuous exercise provoked significant alterations in the characteristics of the collagen network of the articular cartilage of the fetlock joint which were suggestive of microdamage and loosening of the collagen network. The collagen component of cartilage, in contrast to the proteoglycan component, is known to have a very limited capacity for repair and remodelling due to an extremely low turnover rate. Therefore, alterations within the articular collagen network might be expected to play an important role in the pathophysiology of degenerative joint disorders.

A Luminescent Ruthenium Complex for Ultrasensitive Detection of Proteins Immobilized on Membrane Supports

SYPRO Ruby protein blot stain provides a sensitive, gentle, fluorescence-based method for detecting proteins on nitrocellulose or polyvinylidene difluoride (PVDF) membranes. SYPRO Ruby dye is a permanent stain composed of ruthenium as part of an organic complex that interacts noncovalently with proteins. Stained proteins can be excited by ultraviolet light of about 302 nm or with visible light of about 470 nm. Fluorescence emission of the dye is approximately 618 nm. The stain can be visualized using a wide range of excitation sources utilized in image analysis systems including a UV-B transilluminator, 488-nm argon-ion laser, 532-nm yttrium–aluminum–garnet (YAG) laser, blue fluorescent light bulb, or blue light-emitting diode (LED). The detection sensitivity of SYPRO Ruby protein blot stain (0.25–1 ng protein/mm2) is superior to that of amido black, Coomassie blue, and india ink staining and nearly matches colloidal gold staining. SYPRO Ruby protein blot stain visualizes proteins more rapidly than colloidal gold stain and the linear dynamic range is more extensive. Unlike colloidal gold stain, SYPRO Ruby protein blot stain is fully compatible with subsequent biochemical applications including colorimetric and chemiluminescent immunoblotting, Edman-based sequencing and mass spectrometry.

Microvasculature of crotaline snake pit organs: possible function as a heat exchange mechanism.

The infrared sensory membranes of the pit organs of pit vipers have an extremely rich capillary vasculature, which has been noted passim in the literature, but never illustrated or studied in detail. We rendered the pit vasculature visible in various ways, namely, by microinjection of India ink, by a combination of ink and succinate dehydrogenase staining, and by making resin casts for scanning electron microscope study. We also used transmission electron microscopy for identifying the types (arterioles, venules, capillaries) of blood vessels. Then we compared the pit vasculature with that of the retina and the dermis. Good visualization of the vasculature was obtained with both ink and resin injection. Arterioles, venules, and capillaries could be distinguished with all methods used. The monolayer vasculature was denser in the pit membrane than in the retina or skin. Each loop of the network enclosed a small number of infrared receptors so that all receptors were in contact with a capillary on at least one side. The forward-looking areas of the pit had a denser network than side-looking areas. Since infrared rays cause nerve impulses by raising the temperature of individual receptors, the capillary network functions not only as a supplier of energy but also as a cooling mechanism to reduce afterimages. Thus the denser network in the forward-looking areas causes these areas to be more sensitive and have better image resolution than the rest of the membrane.