Intraoperative evaluation of blood flow for soft tissues in orthopaedic surgery using indocyanine green fluorescence angiography: A pilot study.

OBJECTIVES: Indocyanine green (ICG) fluorescence angiography is an emerging technique that can provide detailed anatomical information during surgery. The purpose of this study is to determine whether ICG fluorescence angiography can be used to evaluate the blood flow of the rotator cuff tendon in the clinical setting. METHODS: Twenty-six patients were evaluated from October 2016 to December 2017. The participants were categorized into three groups based on their diagnoses: the rotator cuff tear group; normal rotator cuff group; and adhesive capsulitis group. After establishing a posterior standard viewing portal, intravenous administration of ICG at 0.2 mg/kg body weight was performed, and fluorescence images were recorded. The time from injection of the drug to the beginning of enhancement of the observed area was measured. The hypovascular area in the rotator cuff was evaluated, and the ratio of the hypovascular area to the anterolateral area of the rotator cuff tendon was calculated (hypovascular area ratio). RESULTS: ICG fluorescence angiography allowed for visualization of blood flow in the rotator cuff in all groups. The adhesive capsulitis group showed significantly earlier enhancement than the other groups. Furthermore, the adhesive capsulitis group had a significantly smaller hypovascular area ratio than the other groups. CONCLUSION: ICG fluorescence angiography allowed for evaluation of real-time blood flow of the rotator cuff in arthroscopic shoulder surgery. The techniques of ICG fluorescence angiography are simple and easy to observe, observer reliability is high, and it has utility for evaluating blood flow during surgery.Cite this article: N. Doi, T. Izaki, S. Miyake, T. Shibata, T. Ishimatsu, Y. Shibata, T. Yamamoto. Intraoperative evaluation of blood flow for soft tissues in orthopaedic surgery using indocyanine green fluorescence angiography: A pilot study. Bone Joint Res 2019;8:118-125. DOI: 10.1302/2046-3758.83.BJR-2018-0151.R1.

Determination of urinary Tamm-Horsfall protein by ELISA using a maleimide method for enzyme-antibody conjugation.

A method for enzyme-antibody conjugation using a new maleimide derivative as coupling reagent has been developed. Since a monomeric conjugate of horseradish peroxidase and Fab' antibody could be readily prepared with high efficiency and reproducibility, the enzyme activity and antigen-binding activity were well preserved and nonspecific staining was greatly reduced. The conjugate is suitable for use in both ELISA procedures and immunohistochemistry. Using both methods we examined the pathophysiological significance of Tamm-Horsfall protein (THP) and the present study describes the ELISA method to quantify urinary THP using the new method with rabbit anti-THP antibody. A low concentration (0.04 M) of urea added to the urine samples increased the linearity of the standard curve and the sensitivity of the assay, permitting the detection of as little as 20 ng/ml THP. Freezing and thawing the urine resulted in variable or lower values of THP concentration. THP concentrations in urine as determined by ELISA were stable for at least one month after -70 degrees C storage, but not after -30 degrees C storage. There was no correlation between THP concentrations in 24 h urine samples and the morning urine of the same patient. These results suggest that it is essential to use fresh or -70 degrees C stored 24 h urine samples with added urea (0.04 M) for the determination of THP concentrations in urine by the present enzyme-antibody conjugation method. The THP concentration in normal 24 h urine of young children was found to be less than 51.8 mg/g Cr.

Development of a magnetic sensing device for tooth displacement under orthodontic forces.

We have developed a system for measuring tooth displacement from orthodontic force. Eight small magnetic sensors and a magnet are combined to measure three-dimensional displacement. Sensors, arranged cubically in the three planes of space, are placed in the mouth and fixed to the posterior teeth by a splint. A magnet is placed in the center of the eight sensors and attached to a front tooth that is subjected to orthodontic force. Sensors detect the magnet's movement as target tooth displacement. The system was designed to achieve displacement resolution of 1 microm. The mean percentage of measurement errors was determined to be less than 1% in a 600-cubic-microm volume from calibration. The system was tested clinically on human teeth. Although the oral environment, with high temperature and humidity, was not agreeable with the sensors, this system was stable and accurate enough for quantitative measurement of tooth displacement. The advantage of this system is the ability to detect tooth trajectories by decomposing displacement into translation and rotation and to determine the position of the center of rotation from these parameters.

Hageman factor dependent kinin generation system in guinea pig skin: extravascular localization of the components, and prolonged vascular reaction in inhibitor-depleted animal of this system.

1. Monospecific antibodies against components of the guinea pig Hageman factor-dependent system, such as Hageman factor, prekallikrein and high-molecular-weight (HMW) kininogen were prepared. These antibodies demonstrated the presence of all of these components in the normal guinea pig skin extract with the properties identical to them in plasma on western immunoblotting analysis. 2. Guinea pig macroalbumin which is a major circulating inhibitor of activated Hageman factor was immunologically depleted from three animals. Duration of a vascular permeability reaction caused by an intradermal injection of activated Hageman factor was prolonged in all of these guinea pigs approximately twice as long as the control, indicating a presence of negative feedback regulation of the Hageman factor-dependent kinin generation cascade in dermal tissue via the circulating protease inhibitor.

[Pulsatile administration of LH-RH for hypogonadotropic hypogonadism].

A case of hypothalamic-pituitary failure is reported. A 28-year-old man who had been treated with testosterone for hypogonadism for two years was admitted to our hospital with the chief complaint of sexual problems. The serum level of gonadotropins was very low and hypophysial responses to luteinizing hormone-releasing hormone (LH-RH) were good. Treatment was started with subcutaneous pulses of 10 micrograms LH-RH every 120 minutes using a portable infusion pump. Gradually, his potency improved and ejaculation returned. The serum concentration of FSH, LH and testosterone increased to the normal range of adult males and the testicular volume increased rapidly from 5 ml to 14 ml after 13 weeks of treatment. Sperms appeared in the ejaculated fluid after 25 weeks and, after following 16 weeks, the concentration of sperms increased up to 20 x 10(6)/ml. Prolonged pulsatile subcutaneous administration of a low-dose of LH-RH at a physiologic frequency was an effective therapy for pubertal induction and maturation in hypogonadotropic hypogonadism due to hypothalamic-pituitary failure.

Possible binding sites for inositol 1,4,5-trisphosphate in canine tracheal smooth muscle cells and rat liver cells.

To search for binding site of inositol 1,4,5-trisphosphate (InsP3) in relation to the release of Ca2+ from smooth muscle cells of the canine trachea, InsP3 coupled with p-azidobenzoyl beta-alanine (AB beta A) was synthesized for photoaffinity labelling, using N,N'-carbonyldiimidazole. With subsequent photolysis of the smooth muscle cells following incubation with this derivative (InsP3-AB beta A), the InsP3-induced Ca2+ release from saponin-permeabilized dispersed smooth muscle cells no longer occurred. The irreversible release of Ca2+ induced by InsP3-AB beta A was prevented by adding 10-fold excess amounts of InsP3. These observations strongly suggest that there is a covalent binding of InsP3-AB beta A to a specific "receptor" on the sarcoplasmic reticulum in tracheal smooth muscle cells. To determine the specific binding sites on the endoplasmic reticulum, I synthesized an isotope-labelled arylazide derivative of InsP3; InsP3 was coupled with p-[125I]azidosalicyl beta-alanine, using carbonyldiimidazole as a catalyst. I obtained three proteins with molecular weights of 44K-Da, 27K-Da and 22K-Da, which were preferentially labelled with the [125I]arylazide derivative of InsP3 (InsP3-[125I]AS beta A) in photoirradiated microsomal fractions of canine tracheal smooth muscle cells. The labelling of these proteins was partially blocked by an excess amount of unlabelled InsP3 but not by inositol 1,4-bisphosphate (Ins(1,4)P2). Similar binding proteins with molecular weights of 48K-Da, 32K-Da and 18K-Da were also labelled in the endoplasmic reticulum fraction of the rat liver, but such proteins were not evidenced in the plasma membrane fraction.

Localization of ferritin in human liver diseases studied by immuno-histochemical and immuno-electron microscopic procedures.

Localization of ferritin using a pre-embedding diffusion technique and an indirect localization sequence has been made in 34 cases of human liver under normal and several pathological conditions. With light microscopic observation, positive immuno-staining for ferritin was demonstrated as diffuse deposits in the hepatocytes and Kupffer cells. Intensity of the positive immuno-staining for ferritin in these cells appeared to roughly coincide with serum ferritin levels of each patient, but showed no disease specificity, although hepatoma cells contained weak deposits or were negative from immuno-staining for ferritin. With electron microscopic studies, intracellular antigen was well preserved in the hepatocytes and Kupffer cells in most cases with the positive immuno-staining for ferritin being observed in cytosol and a few cisternae of rough endoplasmic reticulum. Content of the positive immuno-staining for ferritin differed considerably from one case to another and one cell to another even in the same case. There was no immuno-staining for ferritin in hemosiderin pigment, lysosome, most of rough endoplasmic reticulum, Golgi complexes, and nucleus in both cells.

Mechanism of vasodilation induced by alpha-human atrial natriuretic polypeptide in rabbit and guinea-pig renal arteries.

Effects of alpha-human atrial natriuretic polypeptide (alpha-HANP) on electrical and mechanical properties of smooth muscle cells of the guinea-pig and rabbit renal arteries and of the guinea-pig mesenteric artery were investigated. alpha-HANP (up to 10 nM) modified neither the membrane potential nor resistance of smooth muscle cells of the guinea-pig and rabbit renal arteries. In the guinea-pig mesenteric and renal arteries, alpha-HANP (up to 10 nM) had no effect on the amplitude and facilitation (mesenteric artery) or depression (renal artery) of excitatory junction potentials nor on action potentials. In the guinea-pig renal artery, alpha-HANP (up to 10 nM) had no effect on the depolarization induced by noradrenaline (NA) (up to 10 microM) but markedly inhibited NA-induced contraction. alpha-HANP (10 nM) slightly inhibited the K-induced contraction. In the rabbit renal artery, alpha-HANP (10 nM) inhibited the NA-induced contraction and to a lesser extent the K-induced contraction. In the rabbit renal artery, the effects of alpha-HANP on the release of Ca from the cellular storage by two applications of NA, and its re-storage, were investigated in Ca-free solution containing 2 mM-EGTA. When 5 nM-alpha-HANP was applied before and during the first application of 0.5 microM-NA, the contraction was markedly inhibited but the contraction to a second application of 10 microM-NA was potentiated. If the first dose of NA was 10 microM the effect was very small. Under the same experimental procedures, nitroglycerine (10 microM) showed almost the same effects as alpha-HANP on the NA-induced contractions. When both the first (3 mM) and second (10 mM) contractions were evoked by caffeine in Ca-free solution, alpha-HANP (5 nM) and nitroglycerine (10 microM) inhibited both contractions to the same extent. In the rabbit renal artery, applications of alpha-HANP or nitroglycerine increased the amount of guanosine 3',5'-phosphate (cyclic GMP) in a dose-dependent manner. However, a much higher concentration of nitroglycerine was required (2 X 10(3) times). In the rabbit renal artery, hydrolysis of phosphatidyl inositol 4,5-bisphosphate (PI-P2) activated by 0.5 microM-NA was inhibited by alpha-HANP, in a dose-dependent manner, but activation by 10 microM-NA was not inhibited by alpha-HANP (up to 100 nM).(ABSTRACT TRUNCATED AT 400 WORDS)

Ca2+ dependence of inositol 1,4,5-trisphosphate 3-kinase activity in the cytosol fraction of pig coronary artery.

1. The activity of inositol 1,4,5-trisphosphate 3-kinase in subcellular fractions of smooth muscles of the pig coronary artery was examined. 2. Incubation of [3H]inositol 1,4,5-trisphosphate (IP3) with muscle homogenates produced more polar 3H-radioactivity (probably as inositol 1,3,4,5-tetrakisphosphate, IP4) than IP3, in the Mg2+- and ATP-dependent manner, thereby indicating the presence of IP3 3-kinase activity in homogenates of the muscle. 3. Most of the kinase activity was present in the cytosol fraction. The enzyme activity was reversibly activated by Ca2+ with a half-maximal effective concentration of 2.5 x 10(-7) M. 4. The calmodulin antagonists, W-7 and chlorpromazine inhibited the Ca2+-activated enzyme activity.

Inflammatory activity of a trypsin-like protease in the tuberculin reaction sites in guinea pig skin.

A trypsin-like protease(s) was found in the extracts from tuberculin reaction sites in the guinea pig skin. Extractable enzyme activities were chronologically paralleled with the gross appearance of the inflammation, and the maximum activity from 24-hour old inflamed sites was about 12 times stronger than that from control skin, suggesting a potential role in the pathogenesis of the delayed hypersensitivity reaction (DHR). The enzyme suitably hydrolyzed t-butyloxycarbonyl-phenylalanyl-seryl-arginine 4-methylcoumaryl-7-amide, and was partially purified by gel filtration. It showed an apparent molecular weight of 700,000 with optimal pH of 9.0-9.5. The activity was inhibited by various serine protease inhibitors but not by thiol or metal protease inhibitors. It was relatively heat-stable. These findings suggest that the protease is similar to a trypsin-like protease from DHR to bovine gamma-globulin. Intradermal injection of the enzyme into normal guinea pigs induced a mixed neutrophil-mononuclear infiltrate. The reaction was reduced in intensity with the diisopropylfluorophosphate-inhibited enzyme preparation. The enzyme did not induce any increased vascular permeability. These findings suggest that the protease may be present generally in the DHR sites and probably participates in the cellular response of DHR.

[An autopsied case of double carcinomata consisting of hepatocellular and renal cell carcinoma associated with adrenocortical adenoma].

Reported is an autopsied case of a double carcinomata associated with an adrenocortical adenoma in a 68-year-old Japanese female. The hepatocellular carcinoma was classified as being type II, according to Edmondson's classification, and showed massive necrosis caused by TAE and metastases to both lungs the diaphragm, the portal veins, the hepatic veins, and the inferior vena cava. The renal cell carcinoma was latent and diagnosed as being a mixed-cell type, with clear and granular cells also present. Double carcinomata of hepatocellular and a renal cell carcinoma are extremely rate and such a combination occupies merely 0.27-1.04% of the total double carcinomata reported in the Japanese literature. The adrenocortical adenoma in the present case was considered to be a non-functioning adenoma, based on no specific clinical symptoms during the woman's hospitalization.

Guinea pig macroalbumin. A major inhibitor of activated Hageman factor in plasma with an alpha 2-macroglobulin-like nature.

A major inhibitor of the beta form of activated Hageman factor (beta-HFa) with an apparent molecular weight of 28,000, which was reported as a strong permeability factor (Yamamoto and Cochrane, Am J Pathol 1981, 105: 164-175), was purified from guinea pig plasma. When it was depleted in vitro, the plasma lost 71% of the total inhibitory activity toward beta-HFa. The inhibitor, termed "macroalbumin," with an apparent molecular weight of 720,000 and an apparent pI of 4.6, seemed to be an inhibitor similar to alpha 1- or alpha 2-macroglobulin of man and other mammalian species in physicochemical characteristics and in enzymologic properties. Though the inhibitory activity to beta-HFa was negligible in normal skin extract, a significant inhibitory activity appeared in extracts of permeability-enhanced skin sites which were induced by intradermal beta-HFa injection. The inhibitory activity that appeared was macroalbumin-dependent, with more than a 10-fold increase in the concentration. These results indicate the roles of macroalbumin as a negative feedback regulator in situ to the Hageman-factor-dependent pathway in a permeability enhancement system.

A high-molecular-weight trypsinlike protease in the skin sites of delayed hypersensitivity in guinea pigs.

A trypsinlike protease was extracted from the delayed hypersensitivity skin sites in guinea pigs. Extractable amounts of the enzyme were chronologically paralleled with the gross appearance of the inflammation, and the maximum activity from the inflamed sites at 24-36 hours was about 20 times stronger than that from normal skin, suggesting a potential role in the pathogenesis of the delayed hypersensitivity reaction. The enzyme, which suitably hydrolyzed t-butyloxycarbonyl-phenylalanyl-seryl-arginine 4-methylcoumaryl-7-amide, was partially purified by isoelectric focusing or by gel filtration. The enzyme demonstrated a single peak of activity on the former column with an apparent isoelectric point of 4.2, and in the latter it showed an apparent molecular weight of 600,000 (600K-protease). When incubated with 3H-diisopropylfluorophosphate, the enzyme lost all amidolytic activity and yielded a single band of radioactivity in polyacrylamide disk gel electrophoresis in the presence of sodium dodecyl sulfate, and a single peak of radioactivity in gel filtration, both having an apparent molecular weight of 31,000-33,000 (31K-protease). That the 600K-protease might be a complex with alpha 2-macroglobulin was ruled out. The 31K-protease was separated from the 600K-protease by gel filtration in the presence of 6 M guanidine hydrochloride, and was renatured to an active form. An apparent isoelectric point of the 31K-protease observed was 9.4, suggesting that the 600K-protease may be a complex of 31K-protease with an acidic carrier molecule(s). Both proteases, 31K- and 600K-protease, had identical substrate specificity, a pH profile of amidolytic activity, and susceptibility to exogenous protease inhibitors. However, when sensitivities to intrinsic protease inhibitors in guinea pig plasma, two kinds of trypsin inhibitor, and alpha 2-macroglobulin were compared, the 600K-protease was at least 100 times more resistant than the 31K-protease. It was supposed that one of the pathophysiologically significant functions of the complex formation might be to maintain the enzyme activity longer in vivo.

Possible binding sites for inositol 1,4,5-trisphosphate in macrophages.

We reported that an arylazide derivative of inositol 1,4,5-trisphosphate (InsP3) caused irreversible InsP3-induced Ca2+ release from saponin-permeabilized macrophages after photoirradiation. To determine the specific receptors for InsP3, presumably present on the endoplastic reticulum, we synthesized isotope-labeled arylazide derivatives of InsP3; InsP3 was coupled with p-azidobenzoyl [3H]beta-alanine (InsP3-[3H]AB beta A) or p-[125I]azidosalicyl beta-alanine (InsP3-[125I]AS beta A). We report here that three proteins may be associated with the Ca2+ releasing mechanism, in photoirradiated saponin-permeabilized macrophages and in the microsomal fraction.

A case of amyloidosis associated with diphenylhydantoin therapy.

Prolonged administration of diphenylhydantoin (DPH) has been implicated as a possible etiologic factor in immunological aberrations and lymphoproliferative disorders. Diphenylhydantoin may account for the increase in susceptibility to lymphoproliferative diseases, as a result of its immunosuppressive effect. We report a case of amyloidosis with monoclonal gammopathy which developed during DPH treatment, without multiple myeloma or lymphoproliferative disorders. The association between DPH and monoclonal gammopathy is very rare, and such a case of amyloidosis associated with DPH has not been reported previously. DPH, however, may have played a role in the development of monoclonal gammopathy, which was the precursor of amyloid protein.

Mechanisms of flunarizine-induced vasodilation in the rabbit mesenteric artery.

The vasodilating effects of flunarizine on smooth muscle strips of rabbit mesenteric artery have been investigated and compared with those of nifedipine. Flunarizine (30-300 nM) dose-dependently inhibited Ca2+-induced contractions in Ca2+-free solution containing 100 mM K+. Double reciprocal analysis showed that this inhibition was either competitive at low concentrations (30-100 nM; nifedipine-like) or noncompetitive at high concentrations (0.3-1 microM). The latter seemed to be partly related to an inhibition of contractile proteins as estimated from Ca2+-induced contractions in saponin-treated chemically skinned muscle strips. In contrast to the actions of nifedipine, flunarizine inhibited norepinephrine (NE)-induced contractions more than those induced by high K+, and at 0.3 microM, this agent totally blocked NE-induced contraction. Flunarizine also inhibited NE-induced contraction in Ca2+-free solution containing 2 mM EGTA. In Ca2+-free solution, NE rapidly hydrolyzed phosphatidylinositol 4,5-bisphosphate (PI-P2) and produced phosphatidic acid (PA). Flunarizine (30 and 300 nM), but not nifedipine (100 nM), inhibited NE-induced hydrolysis of PI-P2 and production of PA. However, flunarizine (100 nM) did not modify the contraction induced by 10 microM inositol 1,4,5-trisphosphate in chemically skinned muscle strips. It is concluded that flunarizine inhibits both voltage-dependent (nifedipine-like) and receptor-operated Ca2+ influx induced by NE and also inhibits NE-induced Ca2+ release from intracellular stores due to inhibition of the hydrolysis of PI-P2.

Urinary Tamm-Horsfall protein excretion in patients with primary vesicoureteral reflux.

The excretion of urinary Tamm-Horsefall protein (THP) was determined by enzyme-linked immunosorbent assay and glomerular filtration rate (GFR) was calculated with technetium-99m diethylenetriamine pentacetic acid (99mTc-DTPA) renal scintigraphy in 26 consecutive patients with primary vesicoureteral reflux (VUR) before and after antireflux surgery. Wide variations of urinary THP excretion and GFR were seen in all grades of VUR. On the basis of the relationship between urinary THP excretion and GFR before the surgery, patients were divided into three groups. The first group (group A, n = 8) had normal urinary THP values and normal values of GFR. The second group (group B, n = 11) had high THP excretion and moderately decreased GFR, the third group (group C, n = 7) had normal urinary THP excretion and severely decreased GFR. In group A, urinary THP values remained normal and GFR improved in all patients after surgery. In group B, GFR improved when urinary THP dropped immediately, but GFR did not improve when urinary THP remained high after surgery. In group C, GFR did not improve and urinary THP continued to be low or tended to drop again after the surgery. The results suggested that serial measurements of urinary THP excretion and GFR by 99mTc-DTPA renal scintigraphy before and after antireflux surgery are useful for the evaluation of renal function in patients with primary VUR.

Inositol 1,4,5-trisphosphate affinity chromatography.

Inositol 1,4,5-trisphosphate (IP3) affinity columns were made by coupling IP3 analogs to a supporting matrix. Sepharose 4B. IP3 5-phosphatase activity. IP3 3-kinase activity and IP3 binding activity from rat brain were absorbed on the IP3 columns. and were eluted by increasing KC1 concentration. This purification procedure increased the specific activities of these parameters 5-200-fold. Thus Sepharose 4B immobilized IP3 analogs can specifically interact with IP3-binding proteins, demonstrating that IP3 affinity columns are a good method for purifying such proteins. Furthermore, our results suggest that IP3 analogs can be linked to other molecules to make useful derivatives without loss of their biological activities.

Assessment of the role of surgery for stage III bronchogenic carcinoma.

In order to assess the role of surgery for stage III bronchogenic carcinoma, we reviewed our results of 282 patients who were treated with surgical resection or various other therapy modalities. The survival in patients who underwent surgical resection was significantly better than those with nonresectable carcinoma. However, superior survival results were demonstrated only in patients with complete resection. In contrast, the survival in patients with palliative resection was similar to that in patients with exploratory thoracotomy and inoperable carcinoma. On the other hand, all patients with T3N2 lesions died of their disease within two years after surgery even when undergoing complete resection. Thus, we conclude that radical resection should be indicated in patients with T3N0-1 and T1-2N2 lesions if complete resection is expected.