The inhibiting effect of a plasma globulin on arterial thrombus formation, (as compared to dipyridamole).

The inhibiting effect on arterial white thrombus formation of a globulin prepared from beef plasma has been compared to dipyridamole in white Wistar rats. It was demonstrated that the globulin fraction had a greater effect in inhibiting thrombus formation as judged by the lag time [t(l)], the maximal thrombus value [m(T)], the maximal thickness of the thrombus [m(D)] and the maximal thrombus surface [m(O)].

The tachyphylactic effect of arachidonic acid on in-vivo ADP induced arterial platelet thrombosis.

The continuous superfusion of arachidonic acid over a branch of the mesenteric artery in an animal in-vivo model results in an initial increase followed by a decrease to control values of ADP induced platelet thrombogenesis. This phenomenon is observed in normal rats as well as in rats submitted to the intravenous administration of ASA three hours prior to the investigation. In the latter group the cyclooxygenase of the platelets is permanently affected while the cyclooxygenase of the endothelial cells is regenerated at the time the investigation is started. The tachyphylactic-like phenomenon observed with arachidonic acid is probably related to the dynamics of the enzymatic reactions of the arachidonate cascade involving the platelets and the endothelial cells surrounding the local de-endothelialized area.

Exchange of cyclooxygenase dependent metabolites between vessel wall and platelets in arterial thrombosis.

In this investigation, transport of endoperoxides from the vessel wall to the platelets in an in vivo model for the induction and quantification of platelet thrombosis is described. Thrombi are induced in a branch of the mesenteric artery of the male white Wistar rat through topical superfusion of adenosine diphosphate solutions following focal de-endothelialization by means of a small electrical current in two groups, comparing untreated rats to acetylsalicylic acid (ASA) treated ones. In the latter group the cyclooxygenase activity of the platelets is completely inhibited but not so that of the endothelial cells. In both groups arachidonic acid enhances adenosine diphosphate induced arterial platelet thrombosis most likely through the conveyance of endoperoxides generated in the vessel wall to the platelets as thromboxane synthetase antagonists as well as thromboxane receptor blocking agents completely inhibit this enhancement in both groups. Furthermore it is demonstrated that ASA treated animals show an increased propensity to thrombosis induced by adenosine diphosphate.

The effect of acute anoxia on the cortical somatosensory evoked potential in the rabbit.

In the course of this study it was found that the functional state of the cortex during anoxic anoxia could be more accurately monitored by the cortical somatosensory evoked potentials (EPs) than by the electrocorticogram (ECoG). Compared with the ECoG, the EPs yield more detailed information. Five different steps may be distinguished in the evolution of EPs; the ECoG, however, exhibits only two (almost trivial) abnormal patterns (epileptic pattern and flat response). Of particular practical interest are the two steps preceding the disappearance of the EPs: (1) a rounding of the P wave accompanied by an increased voltage, followed later by (2) a decrease of the latter. If, at this stage, anoxia is maintained for 20 seconds, then permanent dysfunction is observed, at least during the time interval of the experimentation.

Properties of cortical somatosensory evoked potentials in the awake rabbit.

By appropriate experimental procedures it was possible to distinguish the components of the contra- and ipsilateral cerebral somatosensory potentials evoked by electrical stimulation of an extremity in the unrestrained rabbit. The components of the contralateral response occur in the following order; an early positive primary wave (P1), a positive associative wave (P2), and ultimately a late negative N wave. The components of the ipsilateral response occur in the following order; a small positive wave P, the latency of which is intermediate between those of the contralateral P1 and P2 waves, and a large negative wave N, similar to the contralateral N wave. The different topographical distributions of these waves were elucidated by the use of insulated, chronically implanted electrodes glued onto the cortical surface. The properties of the waveform components were studied by various methods such as varying the stimulation parameters, simultaneous application of somesthetic and acoustic stimuli, and administration of narcotic drugs. The properties of P1 were similar to those of P28 in humans; the properties of P2 can be compared to those of P45; and, finally, the N wave resembles the late negative components observed in humans. Inconstant small positive waves of shorter latencies, which will be discussed in a following paper, may also be seen. Interestingly enough, no early negative wave such as that observed in humans (N20) was ever found. If, as is presently thought, this wave is, in fact, due to the folding of the cortical surface (Broughton, 1969), its absence is to be expected in the rabbit because the cortical surface of this species is lissencephalic and thus devoid of gyri.

Standardization of a device for continuous observation of local flow in tissue.

As our experimental set-up for continuous recording of local blood flow in the cerebral cortex of a laboratory animal with chronically implanted miniature thermistors (based on the heat clearance principle) gave satisfactory results for routine tests of pharmacological agents during anoxia, hypoxia, hypercapnia, etc., experiments, we intended to standardize the apparatus, to increase accuracy, to facilitate calibration and to enhance flexibility with respect to the operator. The exponential aspect in the thermistor resistance/temperature characteristic is linearized by applying a logarithmic converter in the thermistor amplifier. Calibration to the centigrade temperature scale is performed by a three digit numerical adaptation of two thermistor constants determined in a thermostatic-cryostatic bath (zero and slope). A heating power measuring circuit is provided so that the dissipation constant of the thermistor implanted in tissue can be obtained and the thermal conductivity of the tissue can be estimated. Linearity of the relation between cooling of the heated thermistor and local flow, for small cooling values as they are registered in vivo, is still being investigated.

PAF-acether induced arterial thrombosis and the effect of specific antagonists.

Platelet-vessel wall interactions and local thrombosis are investigated in vivo in a branch of the mesenteric artery of the guinea pig, using optoelectronic registration and ultrastructural control. Following an electrical challenge resulting in changes of cell membrane polarization, subsequent superfusion by PAF-acether or a stable analogue, (1-O-alkyl-2-N-methylcarbamyl-sn-glycero-3-phosphocholine, 10(-8) M focal concentration (f.c.)) for a restricted period results in endothelial cell retraction and bleb formation followed by platelet adhesion and the development of a thrombus which over time becomes invaded by leukocytes and eventually occludes the vascular lumen. It was demonstrated in a previous investigation that these phenomena are triggered by the generation of endogenous PAF-acether by the endothelial cells. Specific PAF-acether-antagonists, such as BN 52021 a ginkgolide, but also synthetic molecules, derivatives of the triazolo-pyridino-diazepine group (BN 50727, BN 50755 and BN 50789), significantly inhibit platelet-vessel wall interactions and thrombosis, but not the formation of blebs in the endothelial cells. Hydrogen peroxide (10(-5)M f.c.) not only primes the effect of PAF-acether, but is by itself capable of inducing thrombosis through a PAF-acether-mediated mechanism. Inhibition of acetyl hydrolase by PMSF (phenyl-methyl-sulfonyl-fluoride, 10(-5)M f.c.) invariably results in a significant enhancement of thrombosis, while conversely, inhibition of acetyl transferase by 27584 RP (4-(naphtylvinyl)pyridine hydrochloride, 10(-6)M f.c.) inhibits thromboformation indicating that the remodeling pathway is involved.

Assessment of the in vivo recording of local cerebral blood flow using a thermistor device.

In order to obtain a continuous measurement of local blood flow in the cerebral cortex of a laboratory animal using chronically implanted sensors, we have developed a device based on the heat clearance principle. Flow information is obtained from temperature measurement by means of two thermistors one of them being heated at a defined level above ambient tissue temperature; as such, cooling of the heated thermistor caused by convection phenomena in its vicinity, can be related to local perfusion rate. In a first step "in vitro" measurements were performed in order to study the behaviour, sensitivity and reliability of the device; a physical model was established explaining the results. In this paper we describe "in vivo" tests in the rabbit's brain cortex with the miniature thermistors (0.5 mm diameter) chronically implanted (at the cortical surface). Results are correlated with oxygen tension measurements using (smaller) pO2 electrodes inserted into the cortical tissue. We have observed that all sensors are well tolerated by the animals who remain symptom free. Test experiments, inducing a well known physiological effect on local blood flow, such as arterial clamping, inhalation of CO2 gas mixtures, etc., are performed. The phenomena during induced anoxic anoxia are also shown. These preliminary investigations are essential in order to attempt by future experiments the establishment of a correlation between "in vivo" recorded flow signals and the "in vitro" measured characteristics.

In vivo arterial platelet-vessel wall interaction and thrombosis: induction, on-line registration and ultrastructural control.

A technique for induction and on-line quantification of local platelet thrombi in mesenteric arteries of small laboratory animals was developed and standardized in our laboratory. In the past, this model was used to study the nature of platelet-vessel wall interaction in the living animal. The ultrastructure of the experimental intimal lesion and the vessel wall regeneration were assessed by transmission electron microscopy (TEM), both in normal and pathologic conditions. Scanning electron microscopy (SEM) now shows the ultramorphology of platelet thrombi on the experimentally injured arterial segment following topical superfusion with ADP, mepacrine or platelet-activating factor (PAF). The application of these substances, each with proper bio-activity, leads to distinct types of platelet thrombi. Mepacrine or PAF superfusion causes large thrombotic masses, as compared to control, ADP induced thrombi, and seems toxic for the endothelial cells. Mepacrine thrombi differ significantly from PAF thrombi in their platelet density, degree of platelet activation and in their relation to the endothelium that surrounds the experimental lesion. Furthermore, PAF superfusion induces a phenomenon of spontaneous regeneration of the thrombus after its forced embolization. This is probably due to some unknown bio-action of PAF in the vessel wall.

Discriminant parameters representing cerebral cortical function during anoxic anoxia investigations.

In order to quantify the effect of specific drugs on the cerebral cortex an "in vivo" model has been developed for the induction and the observation of anoxic anoxia. Rabbits are used as test animals. Sensors for the assessment of local parameters are chronically implanted: ECoG electrodes are applied; pO2 electrodes are inserted into the cortical tissue. The derived somatosensory evoked potentials are used for evaluating the cerebral cortical function. Animals are cannulated with a tracheal tube, curarized and artificially ventilated. Anoxic anoxia, controlled by a special purpose microprocessor system, may then be induced and repeated in a reproducible way. During the experiments local (pO2, ECoG, SEP) as well as general parameters (ECG, heart rate, systemic blood pressure, rectal temperature) are recorded and stored on analog magnetic tape as well as digitized with the microprocessor system. The cortical pO2 is measured with a polarographic method, the SEP's are obtained on-line by time coherent averaging and the ECoG states (e.g. epilepsy during anoxia) are derived by using band-pass filters and rms detectors. Off-line the signals (pO2, heart rate, mean systemic blood pressure, temperature) are standardized and represented together with parameters derived from ECoG and SEP. SEP-waveform parameters indicating intensity (norm) and similarity with a reference SEP signal (correlation value) are used. The measuring and processing method is still being optimized; special attention is being paid towards the quality of the calculated SEP's which are to be used for the quantification of the cortical function during reference, anoxia and recovery period. As such, in order to improve the signal-to-noise ratio of the SEP's and, consequently, of the derived parameters, ECoG signals are digitized off-line and subjected to a preprocessing, implying filtering and spectral analysis procedures.

A standardized 'in vivo' model for the study of experimental arterial thrombosis: description of a method.

A method for "in vivo" induction and registration of arterial platelet thrombosis has been developed and standardized in a branch of the mesenteric artery of the white Wistar rat. It consists in local deendothelialization by electrical current. Thrombus induction is performed by topical superfusion with ADP; when the superfusion is discontinued the thrombus disappears but can again be induced reproducibly by renewal of the ADP superfusion after a time interval not exceeding 15 minutes. Registration of the thrombotic phenomenon is made possible by microprojection of the investigated arterial segment. An appropriate optoelectronic device allows the on-line derivation of discriminating parameters. Off-line storage and processing of the experimental data by computer is provided. The method is automatized and can easily be applied to other species of laboratory animals.

Enhancement of prostacyclin generation by cicletanine (an in vivo investigation).

The effect of cicletanine, an antihypertensive drug, was studied in two in vivo models. It was demonstrated that the drug markedly affected the response of intravenously administered arachidonic acid in the rabbit. In this model a marked increase of 6-oxo-PGF1 alpha was observed when challenged by the intravenous injection of 50 micrograms/kg b.w. of arachidonic acid. In the rat model used for the study of platelet-vessel wall interaction, it was demonstrated that the inhibition of prostacyclin synthetase could be offset by cicletanine. These results indicate that the drug modulates the generation of prostacyclin and as such is capable of affecting the peripheral resistances which determine the level of the blood pressure.

Modulation of in vivo generation of prostanoids by drugs affecting membrane ion transport.

An in vivo animal model was developed in the rabbit for the study of the mechanisms involved in the generation and release of prostanoids. Following heparinization and, if required, further sensitization of the animals by intravenous administration of haemolysed blood, injection of doses of arachidonic acid not exceeding 180 micrograms/kg induced a marked fall in arterial blood pressure on condition that the plasma anti-inflammatory protein levels were within the normal range. Cicletanine, certain diuretics (furosemide and bumetanide), as well as calcium-entry blockers such as verapamil and the association of insulin and potassium ions, all markedly decreased the AA50 value and were accompanied by a significant increase in the plasma levels of 6-oxo-PGF1 alpha, enhancing as such the ratio of 6-oxo-PGF1 alpha versus TXB2 in plasma. The infusion of insulin in association with potassium ions induced a similar but less sustained effect. Drugs which affect membrane ion transport were investigated in relation to an enhancing effect on the generation and release of prostanoids following the administration of arachidonic acid.

[Arterial thrombosis--an experimental approach]. / Arteriële trombose--een experimentele benadering.

In this study description is given of a method developed in our laboratory for the investigation of platelet-vessel wall interaction and in vivo thrombus formation. The development of this method is based on the recording of the optical density of an arteriole. Following a standardized challenge by an electrical current onto the arterial wall and subsequent topical superfusion by adenosine diphosphate (ADP), endothelial cells demonstrate specific lesions and platelets start to adhere onto the site of lesion, they aggregate and a platelet thrombus develops. Using an optoelectronic device, which functions as an analogue computer, the generation and the growth of the thrombus in the arteriole can be followed and recorded over time. This continuous on-line recording of the thrombotic phenomenon made it possible to develop discriminating parameters. As such it was possible to study thromboformation in relation to the effect of the metabolites of the arachidonate cascade. Because it is possible to register the thrombotic phenomenon over time, ultrastructural analysis using transmission and scanning electronmicroscopy was performed and allowed us to investigate the interaction of the platelets, the involvement of leukocytes and the onset of fibrin deposition in the thrombus. In further investigations it was demonstrated that as a result of an appropriate challenge of the arterial wall, Paf-acether was generated within the arterial endothelial cells, inducing local thromboformation which perpetuated itself. As such, it could be demonstrated that as a result of the local generation of endogenous Paf-acether thromboformation by an inflammatory process was maintained, leading eventually to complete obstruction of the artery.

[Effect of cicletanine on prostacyclin generation in vivo]. / Effet du ciclétanine sur la génération de prostacycline in vivo.

The administration of arachidonic acid to live rabbits if followed by the generation of prostacyclin and/or thromboxane. Cicletanine increased the production of prostacyclin in a first group of rabbits and amplified the prostacyclin/thromboxane ratio in a second group of the thromboxane type. The most probable mechanism for this action is activation of prostacyclin synthase by cicletanine. This was confirmed in the in vivo model by a study of the platelet-vascular wall interaction: tranylcyprominE, a prostacyclin synthase inhibitor, increased the interaction. Under these experimental conditions, cicletanine inhibited the effect of tranylcypromine and completely restored the enzymatic activity of prostacyclin synthase.