Influence of fat pad removal on patellar tendon length during growth.

During various knee operations, the changes caused by the surgical invasion to the infrapatellar fat pad (IPF) is still unknown. If any changes exist, it will have great influence especially on growing generations. Eighty-four Japanese white rabbits (6-month-old) were divided into three groups: the resection group involving resection of the IPF, the graft group involving resection and reimplantation of the IPF, and the no-surgery group. All these surgical procedures were done in right knees. In all left knees, only arthrotomy was performed, serving as the sham side. After 3, 6, 12, and 24 weeks of the operation, the rabbits were killed. Lengths of the patellar tendon and patellar were measured in lateral X-ray. In order to eliminate individual differences in the patellar height, we defined a new index as percent patellar height (PPH) which indicated the percentage of the patellar height of surgery side compared with that of the sham side. The PPH was 90.6% (3 weeks), 83.0% (6 weeks), 73.6% (12 weeks), and 74.7% (24 weeks) in the resection group, while it was 88.4% (6 weeks), and 88.9% (24 weeks) in the graft group. Postsurgical scar tissue formation occurring where the IPF was removed prevented the normal growth of the patellar tendon. Reimplantation of the IPF lessened the adhesion of the patellar tendon to the surrounding tissue, and better growth of the tendon. These results showed that preservation of the IPF in young individuals could be crucial for the normal growth of the patellar tendon, and critical as well for the prevention of the degeneration of the articular surface.

Experimental model of knee contracture in extension: its prevention using a sheet made from hyaluronic acid and carboxymethylcellulose.

Treatment once extension contracture of the knee has completed is difficult and costly. The most effective treatment might be the prevention of contracture, especially after joint injury. In order to establish an effective method for contracture prevention we first made an extension contracture in rabbit knees, then studied the effect of a sheet made from hyaluronic acid and carboxymethyl cellulose (HA/CMC) for the prevention of knee contracture. One hundred and twenty two mature male Japanese white rabbits were divided into three groups: (1) group B (n=42), where bony holes were made at the medial and lateral epicondyles, (2) group H (n=40), where HA/CMC sheets were placed on the bony holes, and (3) group S (n=40),where only arthrotomy was performed. All surgical procedures were performed on the right knees. All right knees were fixed at 45 degrees using external fixators; this is the maximum extension angle the rabbit is able to tolerate and still walk. At 1, 3, and 6 weeks after surgery, we measured the moment necessary to flex the knee using a special device. We defined the moment as flexion moment (FM). Forty four left knees were also tested as group N, not operated on and serving as the healthy side. In all groups, FM was increased parallel to the increment of flexion angle from 45 degrees to 115 degrees . At many flexion angles, the FM in group B was higher than those of group S at 3 and 6 weeks. The FM in group H was significantly lower than those of group B at 85 degrees and 95 degrees of flexion at 6 weeks after the operation. By macroscopic observation, the area and degree of adhesion were greater in group B than those of group S. In group H, adhesions around the bony hole were less evident than in group B at 6 weeks after the operation. By histological examination, dense granulation tissue was found adjacent to the bony hole in group B at 3 and 6 weeks after the operation. In contrast, in group H the amount of granulation tissue was smaller at 3 and 6 weeks after the operation than those of group B. The usage of HA/CMC sheet should be effective for prevention of contracture occurring after trauma such as treatment for intra-articular fracture.

Synthesis and evaluation of radiolabeled piperazine derivatives of vesamicol as SPECT agents for cholinergic neurons.

To diagnose and investigate neurodegenerative diseases affecting cholinergic neuron density, piperazine derivatives of vesamicol were synthesized and evaluated. Previously, we reported that trans-5-iodo-2-hydroxy-3-[4-phenylpiperazinyl] tetralin (DRC140, 1) possessed high selectivity for vesicular acetylcholine transporter (VAChT). In present study of the effect of alkyl substituents, we observed that the introduction of a methyl group into the ortho or meta positions of the phenyl group of 1 increased affinity for VAChT. trans-5-Iodo-2-hydroxy-3-[4-[2-methylphenyl] piperazinyl]tetralin (2) displayed high affinity and specificity for VAChT. The regional distributions of radioactivity in the rat brain correlated well with known patterns of central cholinergic innervation. [(123)I]2 is a potentially useful compound for SPECT imaging.

Piperazine analog of vesamicol: in vitro and in vivo characterization for vesicular acetylcholine transporter.

The probes to detect vesicular acetylcholine transporter (VAChT) in vivo are important to evaluate the mapping and function in cholinergic system. To develop high-specific and high-affinity radiotracer for single photon emission computed tomography, we investigated piperazine analogs which replaced the piperidine ring of (-)-vesamicol with a piperazine ring. We found that the piperazine analog of iodobenzovesamicol, trans-5-iodo-2-hydroxy-3-[4-phenylpiperazinyl] tetralin (DRC140), had high affinity for VAChT in rat brain. We carried out binding assay in subcellular fraction of the rat brain. The highest B(max) for [(125)I]-DRC140 binding was observed in the synaptic vesicle fraction (1,751 fmol/mg protein), followed by the crude vesicle (821 fmol/mg protein) and the P2 fraction (187 fmol/mg protein). These K(d) values were similar to the affinity of highly purified synaptic vesicular fraction (K(d) = 0.3 nM) with a one-site model. The possibility that [(125)I]-DRC140 recognizes sigma receptor was excluded by our finding large inhibition constants (K(i) = 849 nM for haloperidol, K(i) = 3,052 nM for 1,3-di(2-tolyl)guanidine). In vivo distribution studies with the [(123)I]-DRC140 in rats showed a rapid brain uptake. The highest brain area was in striatum, followed by frontal cortex, occipital cortex, and hippocampus. The lowest brain area was cerebellum. The radioactivity of high-accumulated areas in ex vivo autoradiography was reduced by a preinjection of (-)-vesamicol and these levels were reduced to the radioactivity in cerebellum. These results show that [(125)I]-DRC140 can provide extremely high specific tracer with excellent brain permeability as a ligand for single photon emission computed tomography.

Presence of phospholipid-neutral lipid complex structures in atherosclerotic lesions as detected by a novel monoclonal antibody.

A novel monoclonal antibody (ASH1a/256C) that recognizes atherosclerotic lesions in human and Watanabe heritable hyperlipidemic (WHHL) rabbit aortae is described. When (123)I-labeled ASH1a/256C antibody is injected intravenously into WHHL rabbits, it associates specifically with fatty streaks on the aorta. The antigen recognized by the antibody is lipid, based on extraction with chloroform and methanol from WHHL rabbit tissues. The antigen, purified by high performance liquid chromatography, was shown to be phosphatidylcholine (PC), which contains unsaturated fatty acyl groups based on analyses utilizing (1)H and (13)C nuclear magnetic resonance, Fourier transfer-infrared spectrum, and mass spectrometry. The antibody did not react with other classes of phospholipids or neutral lipids when tested using an enzyme-linked immunosorbent assay. When PC was mixed with either cholesterol, cholesteryl ester, or triacylglycerol, however, the reactivity of the antibody to PC increased up to 8-fold. Homogenates of aorta tissue obtained from normal and WHHL rabbits were fractionated using sucrose density gradient ultracentrifugation in which neutral lipid droplets, cellular membranes, and proteins are separated. The phospholipid content in cellular membrane fractions from WHHL rabbits was twice as high as that of normal rabbits, and there was an enormous difference in the antigenic activity in these fractions. The content of cholesterol in the cellular membrane fraction of WHHL rabbits was approximately 50 times higher than that of normal rabbits. Addition of neutral lipids to the cellular membrane fraction of normal rabbit markedly increased the antigenic activity. Atheromatous lesions in thickened WHHL rabbit aortic intima that were rich in lipid droplets were stained positively with ASH1a/256C immunohistochemically. These results strongly suggest that PC-neutral lipid complex domains are formed in atherosclerotic lesions.

Enhancement of in vivo binding of [123I]beta-CIT by MK-801 in rat brain.

The effects of MK-801, a noncompetitive NMDA receptor antagonist, on in vivo and in vitro binding of radioactive iodine ([123I] or [125I]) labeled beta-CIT [RTI-55, 3beta-(4-iodophenyl)tropane-2beta-carboxylic acid methyl ester] were investigated in rat brain. In the in vitro binding study, 10 pM of [125I]beta-CIT was incubated with either 0.03 microM or 3 microM of MK-801 at 24 degrees C for 60 min. In vitro, no alterations in [125I]beta-CIT binding in any region of rat brain slices were detected after addition of MK-801. In the in vivo binding study, [123I]beta-CIT was intravenously injected into rats 30 min after intraperitoneal injection of 0.03-1 mg/kg of MK-801. The in vivo [123I]beta-CIT binding in the striatum, frontal cortex, occipital cortex, hypothalamus, and thalamus was significantly increased by pretreatment with 1 mg/kg of MK-801. Kinetic analysis using the cerebellum as a reference region revealed that the increases in in vivo [123I]beta-CIT binding induced by MK-801 were mainly due to increases in both input rate constant k3 and output rate constant k4. The results of this study indicate that the glutamatergic system, including NMDA receptor, plays an important role in regulating neurotransmission in the dopaminergic or serotonergic systems in intact brain.

Enhancement of [123I]beta-CIT binding in the striatum with clomipramine: is there a serotonin-dopamine interaction?

Many reports support the concept of serotonergic-dopaminergic interaction in the brain. However, at present, there are few methods to study this relationship in vivo. The purpose of this study was to investigate the effect of serotonin (5-HT) uptake inhibitor, clomipramine, on a dopamine (DA) transporter ligand, [123I]beta-CIT (RTI-55), in rat brain. Dose-dependent changes in [123I]beta-CIT specific binding induced by clomipramine were studied in the striatum (rich in DA transporter) and the hypothalamus (rich in 5-HT transporter). The changes in the time-activity curves of [123I]beta-CIT specific binding after clomipramine injection were also examined in these two regions. Using the cerebellum as the reference region, k3 and k4 values with and without clomipramine administration were estimated by a two-compartment kinetic analysis. Clomipramine inhibited [123I]beta-CIT specific binding in the hypothalamus, but enhanced its specific binding in the striatum in a dose-dependent manner. Kinetic analysis showed that k3 in the striatum was increased by 55%. In conclusion, enhancement of [123I]beta-CIT binding in the striatum after clomipramine administration indicated the possibility of 5-HT-DA interaction.

Differential kinetics of [123I]beta-CIT binding to dopamine and serotonin transporters.

Iodine-123-labelled 3beta-(4-iodophenyl)tropane-2beta-carboxylic acid ([123I]beta-CIT) labels both the dopamine transporter (DAT) and the serotonin transporter (5-HTT) and this ligand is able to clarify pathological changes in both dopaminergic and serotonergic systems. However, the differential kinetics of beta-CIT binding to DAT and 5-HTT has not been clarified fully. In this study we examined time-activity curves of [123I]beta-CIT in individual regions in the rat brain. Using cerebellum as the reference region, k3 and k4 values were estimated by a two-compartment kinetic analysis. In the striatum, the kinetics was slowest among all brain areas. In this area specific binding reached its peak 4 h after the injection. In the hypothalamus, specific binding reached its peak 1 h after the injection and its amount did not change until 4 h after the injection. In the occipital cortex, the binding and washout of the ligand were fastest among all brain regions. Estimated k3 values were 0.040+/-0.003 in the striatum, 0.019+/-0.002 in the hypothalamus and 0.082+/-0.011 in the occipital cortex (min-1, mean +/-SD). Estimated k4 values were 0.0034+/-0.0005 in the striatum, 0.0071+/-0.0009 in the hypothalamus and 0.083+/-0.013 in the occipital cortex (min-1, mean +/-SD). Therefore binding kinetics of [123i]beta-cit in the region rich in dat is apparently different from that in the region rich in 5-HTT. These results will provide fundamental data to image both DAT and 5-HTT in one series of examinations with [123I]beta-CIT.