Comparison of the effects of intra-articular injections of Hyaluronan and its chemically cross-linked derivative (Hylan G-F20) in normal rabbit knee joints.

OBJECTIVE: Intraarticular injection of native hyaluronan (HA) or a cross-linked derivative are commonly utilized in the treatment of osteoarthritis. Unlike from native hyaluronan, the crosslinked HA derivative is a gel containing also other chemical entities. This study compares the local tolerability of these different preparations in normal rabbit knees, in order to provide further information on their biological effects. METHODS: Synovial fluids were aspirated after single or repeated weekly injections (up to three) of the therapeutic agents and cell count was determined in a Burker chamber and in an automatic cell counter. The percentage of the different cell types was determined by light microscopy in semithin sections of fixed synovial fluid cytocentrifugate. Fragments of synovial membrane were also morphologically analyzed. RESULTS: In the synovial membrane no signs of inflammation were evident either after a single or repeated injections of native Hyaluronan (Hyalgan or Artz). In addition, the cell recruitment and the percentage of cell types in the synovial fluid was not statistically different from saline treated joints. After 3 weekly injections of the crosslinked HA derivative (Hylan G-F20, Synvisc) about 50% of the treated joints appeared slightly inflamed and in these joints a statistically significantly higher cell content was determined in the synovial fluid compared to placebo and native Hyaluronan treatment. In addition an unexpectedly high percentage of eosinophils was found in the synovial fluid and in the synovial membrane of slightly inflamed joints treated with crosslinked HA. CONCLUSION: The data obtained after repeated intra-articular injections in normal rabbit knee joints confirm the safety profile of native Hyaluronan.

Cisplatin ototoxicity in the Sprague Dawley rat evaluated by distortion product otoacoustic emissions.

The present study has evaluated the use of distortion product otoacoustic emission (DPOAE) responses in the detection of cisplatin-induced ototoxicity in a Sprague Dawley rat animal model. The cisplatin was administered as a 16 mg/kg, dose introduced by a slow 30-min intraperitoneal infusion. Data from three DP-gram protocols, DPOAE input-output responses at 8 kHz, and auditory brainstem responses (ABRs) at 8, 12 and 16 kHz were collected before and 72 h after treatment. The post-treatment ABRs at 16 kHz showed the greatest mean threshold shift of 33.6 dB. The post-treatment DP-gram data showed significant reduction of the signal to noise ratios in the majority of the frequencies tested, across all tested protocols. The data suggest that the most sensitive DPOAE procedure for the early detection of the cisplatin-induced ototoxic damage is the DPOAE I/O protocol. Morphological analyses indicated that the inner hair cells remained intact, while several types of alterations were observed in the arrangement of the stereocilia in the outer hair cells.

Transvalvular impedance (TVI) recording under electrical and pharmocological cardiac stimulation.

The cardiac electric impedance was recorded between right atrium and ventricle, throughout the cardiac cycle, by means of a tripolar single pass lead for VDD pacing. The transvalvular impedance signal (TVI) is a sharp periodic wave, with high signal-to-noise ratio, that is detected exclusively in the presence of cardiac mechanical activity. The minimum TVI value is attained during the atrial systole, the maximum at the end of ventricular systole. Different parameters of TVI waveform are affected by changes in the inotropic state, and could therefore be proposed as potential signals for new rate responsive algorithms based on the correlation between inotropic and chronotropic regulation. The signal might be used, moreover, for pacing and sensing validation in autoregulating pacemakers and for fibrillation recognition in ICDs.

Acute carotid artery occlusive thrombosis and its pharmacological prevention in the rabbit.

A simple and reproducible method to induce an occlusive thrombus in rabbit carotid artery is reported. Rabbits were anesthetized and prepared to record arterial pressure, heart rate, and carotid blood flow. A critical stenosis of a damaged carotid artery was obtained using an external plastic cylinder. Complete occlusion occurred within 6 to 12 minutes, as measured by the decrease in blood flow. Both stenosis of the vessel and deliberate damage (clamping by surgical forceps) were found essential to occlusion. Occlusion was prevented by administration of heparin (200 IU/kg), tissue plasminogen activator (300 micrograms/kg), iloprost (10 micrograms/kg) or the synthetic thrombin inhibitor, FPRCH2Cl (0.5 mg/kg), while ASA (100 mg/kg) was uneffective. The procedure permits an easy and rapid evaluation of thrombus formation and of anti-thrombotic drugs affecting the hemostatic process.

Molecular aspects of cloricromene (AD6) distribution in human platelets and its pharmacological effects.

Cloricromene (AD6) is an investigational drug which inhibits platelet aggregation and release reaction. We studied the relationship between its action and its distribution and metabolism in platelets. Incubation of anticoagulated whole blood or platelet-rich plasma (PRP) without exogenous aggregating agents resulted in a progressive decrease of platelet count with a concomitant increase of beta-thromboglobulin (BTG) release. AD6 (20-50 mumols/l), but not acetylsalicylic acid (ASA), incubated with whole blood or PRP, prevented the fall in platelet count and the release of BTG for at least 150 min. Moreover, incubation of PRP with AD6 (50 mumols/l) and subsequent stimulation by ADP at threshold concentrations resulted in a significant reduction (about 30%) in aggregation for at least 90 min. AD6 (20 mumols/l) added to PRP was rapidly metabolized by hydrolysis of an ester bond to AD6 acid, a stable catabolite pharmacologically inactive in platelets. Significant amounts of AD6 acid (up to 13.26 +/- 2.80 pmol/10(6) platelets) were associated with the platelets after incubation either at 37 degrees C or 4 degrees C. The amount of AD6 acid in the platelet pellet was proportional to AD6 concentration (2 to 100 mumols/l). PRP incubation with AD6 acid (20 mumols/l) resulted in very low levels (less than 1 pmol/10(6) platelets) of the same compound in the platelet pellet after 1, 5 or 30 min. These data suggest that AD6 is taken up as an ester and converted to its acid catabolite with a consequent long-lasting inhibition of platelet function.

Stenosis and vascular damage as a cause of thrombosis in the dog femoral artery.

We describe here an experimental model of peripheral arterial thrombosis and the effect of several drugs which are known to affect vessel and platelet biological functions. A similar method has been previously applied by us and others on dog coronary arteries. Male Beagle dogs, under pentobarbital anesthesia, were instrumented to measure arterial pressure, heart rate, ECG, femoral blood flow and expired CO2. A segment of the femoral artery was squeezed with forceps to damage the endothelium, and a plastic cylinder was placed around the vessel in the area of the damage. The cylinders had a length of 2 mm and an internal diameter of 1.6-1.8 mm. Under these circumstances blood flow in the stenosed artery was reduced by about 60-70% from control value and showed cyclic blood flow variations (CBFV). CBFV eventually led either to a total occlusion of the vessel (documented by blood flow measurement and by angiographic analysis), or to a spontaneous partial restoration of flow, followed by another decrease, in a repetitive fashion. Drug effect was monitored by observing the changes in frequency and amplitude of CBFV. Ketanserin (0.25 mg/kg), dazmegrel (0.5 mg/kg), and chlorpromazine (0.5 mg/kg), abolished or greatly reduced CBFV in all the experiments, while acetylsalycilic acid (ASA, 10 mg/kg) reduced or abolished CBFV in 60% of the treated dogs. Heparin (50 I.U./kg), dipyridamole (1.0 mg/kg) and prazosin (0.1 mg/kg) did not change CBFV. These results emphasize the importance of serotonin and thromboxane as mediators of vascular occlusion in this particular experimental model. This approach provides a reproducible in vivo preparation to study the pharmacological control of peripheral arterial thrombosis.

General toxicity and peripheral nerve alterations induced by chronic vincristine treatment in the rabbit.

The effects of five 0.3 mg/kg intravenous administrations of vincristine (VCR) at weekly intervals were studied in the rabbit. Body weight gain was impaired starting from the first injection, while gross signs of motor paralysis and hair loss initiated from the third week. At the end of the observation period blood analysis revealed normocytic normochromic anemia, elevated serum creatine kinase, and low serum alkaline phosphatase, whereas all the tested parameters related to liver and kidney functions where within normal limits. The decreased number of red blood cells was the consequence of a complete, although reversible, blockade of staminal hematopoietic activity. Two important indexes of peripheral nerve function were clearly altered at the end of the treatment: (i) the sciatic nerve conduction velocity in vitro was 27% reduced and (ii) the latency between sciatic nerve stimulation and extensor digitorum longus (EDL) twitch in vivo was 34% prolonged. The usefulness of the rabbit as an animal model to study side-effects of VCR treatment is discussed.

Prostacyclin release in the coronary circulation during sustained stimulation in in vitro and in in vivo experimental systems.

Prostacyclin release from rat isolated perfused hearts and from dog coronary circulation was studied by measuring immunoreactive 6-keto-PGF1 alpha (6-keto-PGF1a) in heart perfusate and in plasma obtained from the great cardiac vein respectively. Continuous infusion of arachidonic acid at constant concentration in isolated perfused hearts induced an increased prostacyclin release. This release showed a rapid peak within 10 min and a subsequent decrease. Low-flow ischemia induced an increased perfusate concentration of 6-keto-PGF1a but, considering the decreased flow, prostacyclin release was actually reduced. During the whole period of ischemia (60 min) prostacyclin release was constant. In open-chest anesthetized dogs 6-keto-PGF1a concentration in the great cardiac vein was increased after ligation of the left anterior descending coronary artery. A prolonged period of coronary occlusion (4.5 hours) resulted in a progressive rise of prostacyclin release. 6-keto-PGF1a determinations in the femoral vein and in the aorta did not show relevant variations during the observation period.

Enkephalin modulation of neural transmission in the cat stellate ganglion: pharmacological actions of exogenous opiates.

Neural ganglionic transmission was studied in vivo in the cat, using closed chest anesthetized preparations. The right stellate ganglion and its branches were exposed retropleurally and prepared for electrical stimulation of pre- and postganglionic nerve fibers. The axillary artery was cannulated allowing direct administration of drugs in the arterial blood supplying the ganglion. Stimulation of postjunctional receptors could thus be obtained by local administration of selective agents. Local administration of nicotinic, muscarinic or histaminergic agents increased heart rate and blood pressure. Opiates were given either i.v. or locally through the axillary artery: we tested the effects of morphine, Leu-enkephalin (Leu-enk), Met-enkephalin (Met-enk), [D-ala2]-Met-enkephalinamide (DAME) and etorphine. When given locally, Leu-enk (from 10 micrograms), Met-enk (from 20 micrograms), DAME (from 5 micrograms) and etorphine (from 0.2 micrograms) inhibited tachycardia induced by preganglionic stimulation and reduced the amplitude of the compound action potential recorded from the postganglionic nerve. Morphine (10-200 micrograms) had no effect. On the other hand, tachycardia induced by postganglionic nerve stimulation was unaffected by opiates in the same experimental conditions. Intravenous administration of similar doses of opiates had no effect on ganglionic transmission. When tachycardia was induced by chemical stimulation of nicotinic (DMPP), muscarinic (McN-A-343-11) or histamine receptors in the stellate ganglia, opiates were still active in reducing the effect of these chemicals. These data provide evidence that exogenous opiates exert a depressing action on postsynaptic responses of sympathetic ganglia tested in vivo, although an additional action on presynaptic terminals is not excluded. As endogenous opiates are normally present in various sympathetic ganglia, including the stellate ganglion of the cat, it is possible that they play some modulatory role on ganglionic transmission in physiological conditions.

The action of AD6 on experimental arrhythmias and on action potentials of cardiac fibers.

AD6 is a coumarinic derivative which increases both coronary blood flow and prostacyclin production, while it decreases platelet responsiveness. We tested its action on experimental cardiac arrhythmias. AD6 (2.5-10 mg/kg) was able to antagonize the arrhythmogenic action of aconitine in rats and of adrenaline in cats. AD6 action was also tested in vitro. The drug (20-50 microM) prolonged the functional refractory period of guinea-pig atrial and ventricular muscle and lengthened the refractory period shortened by hypoxia. Intracellular electrophysiological experiments showed that AD6 prolongs action potential duration (APD) of guinea-pig atrial myocardium, sino-atrial node and cat Purkinje fibers. The results obtained in vitro may explain the effect on experimental arrhythmias, therefore suggesting a protective action on cardiac rhythm disturbances.

Inhibition by AD6 (8-monochloro-3-beta-diethylaminoethyl-4-methyl-7-ethoxycarbonyl methoxy coumarin) of platelet aggregation in dog stenosed coronary artery.

The action of AD6 as an anti-thrombotic agent was studied in a model of coronary artery thrombosis and on platelet aggregation in the dog. AD6 (10-100 microM) in vitro inhibited aggregation induced by ADP, epinephrine, collagen and PAF (platelet aggregating factor) used at their threshold concentration for maximal aggregation. Arterial thrombosis was induced in a coronary vessel by critically reducing (about 70%) the vessel lumen. Thrombus formation was estimated by measuring coronary flow in the stenosed vessel. Using this procedure on the left descending coronary artery (LAD), we obtained reproducible blood flow changes in 18 dogs. AD6 was given i.v. at three different doses. At 0.25 mg/kg two out of four dogs showed decreased thrombus formation at the stenosis site. Seven out of eleven dogs treated with 0.5 mg/kg and two out of three treated with 1.5 mg/kg showed decreased thrombus formation. Major decreases in coronary resistance, evaluated by measuring blood flow in the unstenosed left circumflex artery (LCX), were evident only after the highest dose. We conclude that AD6 has an inhibitory action on dog platelet aggregation and reduces thrombus formation in a stenosed coronary vessel.

Coronary and systemic 6-ketoprostaglandin F1 alpha and thromboxane B2 during myocardial ischemia in dog.

The left anterior descending coronary artery (LAD) of five dogs was ligated, and blood was withdrawn from the great cardiac vein, left marginal cardiac vein, femoral vein, and aorta. After ligation, immunoreactive 6-ketoprostaglandin (PG) F1 alpha rose from less than 0.1 to a mean value of 1.2 pmol/ml plasma in the great cardiac vein (GCV) and 0.88 pmol/ml in the left marginal vein, with no change in peripheral circulation. Immunoreactive thromboxane (TX) B2 remained below 0.075 pmol/ml throughout the experiments. LAD of 11 dogs was stenosed 60-80% with consequent cyclical reductions in blood flow. 6-Keto-PGF1 alpha in GCV rose in seven dogs (range 0.5-2.2 pmol/ml) and remained unchanged in four. No change was observed in peripheral plasma levels of 6-keto-PGF1 alpha. In these experimental conditions TXB2 remained below 0.075 pmol/ml. Lactate concentrations rose in both experimental conditions in GCV but not in peripheral circulation or in the left marginal vein. This study confirms a link between cardiac ischemia and increased coronary prostacyclin release, but we were unable to detect a similar correlation with TXB2 in plasma.

Muscle reinnervation--I. Restoration of transmitter release mechanisms.

Following sciatic nerve crush the restoration of neuromuscular transmission in the extensor digitorum longus muscle of rat proceeds in a well defined manner: (a) as soon as the nerve-muscle contact is reformed, a subthreshold end-plate potential is recorded; no 'non-transmitting stage' is observed; (b) 24 hours later muscle action potentials are induced by nerve stimulation; (c) miniature end-plate potentials are absent or very rare at the newly reinnervated end-plates; their frequency returns to normal in about 4 weeks; (d) the frequency is also very much reduced in 30 mM K+ and hypertonic solutions and recovers slowly, in 4 and 5 weeks, respectively, while black widow spider venom is from the beginning as powerful as in normal neuromuscular junctions; (e) at the early stages of reinnervation the Ca2+-dependent release mechanisms are much stronger than control cases, while the Ca2+-independent mechanisms are weaker and recover in 5 weeks. The gradual reassembly and restoration of neurotransmitter release mechanisms of the extensor digitorum longus nerve terminal indicate the complexity of pre-synaptic ending organization.

Muscle reinnervation--II. Sprouting, synapse formation and repression.

Extensor digitorum longus muscle is reinnervated by the regenerating neurites at the end-plate region; as soon as the contact is made, the rate of neurite elongation inside the cleft decreases about 1000-fold while interfibre growth and sprout formation proceed unchanged. Polyinnervation reaches the maximum level 7-10 days after reinnervation, then synaptic repression begins. The elimination of redundant innervation takes place when the biophysical properties of the muscle are again normal. There is no sign of either phagocytosis or degeneration, therefore the process of synaptic repression is probably due to retraction, as neurites do when in culture. The role of Schwann cells and nerve sheath in the process of maintenance is suggested.

PGF2 alpha, thromboxane B2 and HETE levels in gerbil brain cortex after ligation of common carotid arteries and decapitation.

The effects of ligation of both common carotid arteries in the gerbil on the levels of PGF2 alpha, TXB2, HETE and of energy metabolites in brain cortex, have been investigated. Also, in the same experimental conditions the changes of cyclic AMP in brain cortex, cerebellum, striatum and hippocampus have been monitored. ATP, glycogen, glucose and phosphocreatine decrease whereas, lactate and cyclic AMP are enhanced in the ischemic brain, as previously reported. In contrast, levels of arachidonic acid metabolites are not modified. During ischemia following decapitation, instead, PGF2 alpha, and TXB2, show considerable increase.