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Evaluation of five longest surviving calves among a total of 54 longterm survivors, at the Brno Research Centre was performed. These calves were treated by administration of antihypertensives and partially by electrical stimulation of the right atrium to reduce central venous pressure (CVP). Treatment for CVP was effective, as compared with two groups of 5 calves each not treated in this way, and it is assumed to be an important factor for prolongation of survival. Mineralization of driving diaphragms was present in all these calves. No preventive measures of this pathologic change were performed in this group. Another complication is hemolysis, which is partly caused by immune mechanisms initiated by administration of alpha-methyl dopa and partly by mechanical destruction due to a heavily mineralized driving diaphragm. The prevention of mineralization is another important problem for future research. Other problems, concerning the survival of TAH recipients are discussed.

The degree of mineralization of TAH membranes in animals is directly related to the survival time of animals with a TAH. Large differences in mineralization of the membranes among individual animals have been observed. These differences can be explained by the simultaneous presence of infection on biomaterials (polyurethane) as well as by different levels of natural inhibitors of mineralization. No positive influence of applying sodium warfarin for the inhibition of the mineralization was observed.

The development of the adrenergic cardiac innervation was studied in premature dog fetuses, puppies and adult dogs by means of the formalin-induced fluorescence technique. A point-counting technique was used to evaluate the density of innervation. Two types of fluorescent profiles can be observed in the heart during development: (1) sprouting axons, and (2) beaded terminals. The axonal fluorescence disappears in adult neurons. A different morphology and a different time course of development enable to study separately the innervation of the myocardium (cardiomotor innervation) and that of the vessels (vasomotor innervation). The late prenatal innervation is very poor (0.1 hit). The first but very scant cardiomotor terminals appear in this period. A mature cardiomotor innervation is found in 4-month-old puppies [1.5 ± 0.3 (SD) hits]. The vasomotor innervation is shifted to the right. The development of beaded vascular terminals begins and matures 1–2 weeks later. The growing fluorescent axons reveal that the myocardium is supplied by axons of the cardiac plexus and of the perivascular nerves; the vascular wall on the other hand is supplied by the perivascular nerves only. The developmental, spatial and morphological differences in innervation suggest that two different types of neurons exist in the sympathetic ganglia: (1) neurons innervating the vessels (coronaromotor neurons), and (2) neurons innervating the myocardium (cardiomotor neurons).

The monoaminergic innervation of the coronary vessels and myocardium in the dog was studied by means of the histochemical fluorescence method. The distribution of monoaminergic terminals in the arterial wall depends on the diameter of the artery. In large arteries, the terminals are regularly distributed around the entire circumference, the fibres being situated between the elastic lamellae of the adventitia. The terminals within the media could not be detected. In small arteries, the sympathetic ground plexus aggregates in two parallel strands, the artery being between them. In arterioles, two thick fibres only accompany the vessel. The myocardium is innervated by means of a three-dimensional sympathetic ground plexus distributed between the myocardial cells independently of vessels. No difference in density of innervation was found between the right and left heart. The point-counting method, on the other hand, has revealed that the density of innervation in the left auricle is nearly twice as dense as in the left ventricle. The terminals innervating the myocardium are markedly thinner than the fibres innervating the arteries. Two-dimensional plexuses innervate the epicardium, endocardium and valves.

A special reticular basket-like system of connective tissue strips (called connective tissue skeleton – CTS) was found between the cortex and medulla in the kidney of various mammals. It enlarges the wall of the renal calyx (or pelvis) into the parenchyma. The main component of this system is collagen. A small amount of smooth muscle cells was found in one part of CTS strips situated around the papilla (the levator fornicis muscle). A dense monoaminergic and scanty cholinergic innervation was found in the whole system of the CTS. The functional importance of this system is discussed: (i) a tightly linked connection between the urine-discharging system and the kidney, (ii) ‘milking’ and similar effects in the papilla as well as perception of intrapelvic and intrarenal pressure, (iii) penetration of infection into the kidney and (iv) liberation of the monoaminergic transmitter.

The disposition of sympathetic transmitter within the wall of rat femoral and rabbit saphenous arteries has been studied during sympathetic stimulation. Using a histochemical fluorescence technique, sharply delineated bead-like fluorescent spots representing sympathetic nerve terminals were found at the adventitiomedial junction. In specimens excised during sympathetic nerve stimulation, the entire media was veiled by fluorescent material representing noradrenaline which had diffused into that layer after release. The localization of the nerve terminals in controls as well as diffusion of transmitter during stimulation in the rat femoral artery was corroborated by autoradiography of tritiated noradrenaline.

The adrenergic innervation of the juxtaglomerular complex was studied in kidneys from mice, rats, guinea-pigs, rabbits, cats, dogs, pigs, monkeys, and humans using fluorescence histochemistry of neuronal nor-adrenaline and autoradiography of 3H-noradrenaline. The localization of the nerves was established by phase contrast optics or by perfusing the vascular system with India ink. Adrenergic nerve terminals, exhibiting a formaldehyde-induced fluorescence and having the ability to take up and accumulate 3H-noradrenaline, were easily identified when they enclosed the glomerular afferent arteriole. They continued in between and close to the macula densa and lacis cells to supply the glomerular efferent arteriole. The nerves could be seen to accompany this arteriole for a considerable distance until they branched off to the vasa recta in the juxtamedullary region and to adjacent cortical veins. This innervation pattern was found to be a constant feature except in kidneys from guinea-pigs and cats, in which post-glomerular adrenergic nerves were not found in some of the superficial glomerular units. The fluorescence in all adrenergic fibres supplying the juxtaglomerular complex disappeared after removal of the aortico-renal ganglion, showing that they belong to a common system of renal sympathetic nerves.

The monoaminergic innervation of the coronary vessels and of the myocardium in dogs was examined by means of degenerative techniques: (i) local perivascular neurotomy was performed at the origin of the left coronary artery; (ii) the stellate ganglion was removed bilaterally. Evaluation of the neural degeneration pattern led the suggestion that three types of neurons at least exist within the heart: (i) neurons innervating the coronary vessels both in the ventricles and in the atria; (ii) neurons innervating the ventricular myocardium, and (iii) neurons innervating the atrial myocardium. The post-ganglionic fibres of the stellate ganglion innervate the vessels of the whole heart. Their contribution to the innervation of the ventricular myocardium represents about 30%.

Causes of the death of four animals from the total of eight were technical failures (diaphragm perforations, disconnection of the quick-connector, and a failure of the supply of electric current which could not be prevented). Causes of the death of the remaining four animals were embolisation in the brain stem, embolisation in the superior mesenteric artery leading to diffuse peritonitis, ruptures of aorta and hemocoagulation disorder causing fatal gastrointestinal bleeding.

Vasomotor disregulation, preponderantly expressed by a pathological increase of central venous pressure (CVP) in calves with total artificial heart (TAH), starts to be evident from about the 50th day of pumping. The main cause of this state is an imbalance in cardiac receptor areas. Ventricular vasodepressor mechanisms are eliminated with the ventricular tissue, which is replaced by the artificial blood pump. In the stumps of both atria, which remain in situ, all neural elements disappear immediately after TAH implantation, but within two months they are fully regenerated. Regenerated atrial receptors are the starting points of afferent neural stimuli, which in the vasomotor center of the brainstem, increase the activity of the vasoconstricting functional component. A general tendency to vasoconstriction, now not well counterbalanced, increases, and the progressive venous hypertension causes loss of liver function and morphology. Two therapeutic approaches were tried: afferent therapy by atrial electrical stimulation, and efferent therapy by the administration of antihypertensives. Both kinds of this therapy were sufficiently effective in reducing CVP, protecting the liver, and prolonging average survival.

A 150-day experiment in a calf with an implanted total artificial heart, TNS-BRNO-II, is described. The Czechoslovak TAH-BRNO-II device is a hybrid; the outer housing is made of polymethylmethacrylate, the moving parts of segmented polyurethane. The blood flow through the device after the surgery gradually increased from 7 L/min to 12 L/min by the end of the experiment. Atrial pressures were automatically maintained at the same level, about one kPa on both sides. The left systolic time was automatically derived from the pump emptying phase; the right systolic time was maintained between 40% and 50%. After the surgery, normal values of blood gases, stabilization of respiratory activity, and normalization of gastrointestinal functions were reached very soon. Anticoagulant therapy was performed during the whole experiment, with several periods of increased bleeding tendency. The clinical state of the calf was essentially physiological until the 94th day of the experiment, when acute bronchopneumonia, with a high fever, over 41°C, initiated a series of complications (pseudomembranous enterocolitis, followed by sepsis lenta). The fatal complication was sudden impairment of the coagulation mechanisms on the 149th to 150th day of the experiment. This was the basic reason for the subsequent death of the animal, caused by acute cerebral anemic anoxia, which could not be prevented by immediate therapeutic procedures. In spite of these complications, the clinical state of this calf was relatively good until the end of the experiment. Autopsy revealed minimal thromboembolization at the valves. The outer surface of the diaphragm was covered with pseudoneointimal deposits and the inlet orifices were free of pannus or other connective tissue crescents. Macroscopic signs of infection were not seen, but bacteriological cultivation from internal organs confirmed Klebsiella infection. The state of the device at the time of autopsy, with simultaneous undisturbed function of the control and driving unit, showed the possibility of still longer survival.

A modification of Pickworth's method for demonstrating open capillaries is described. The distinguishing feature of this method is freezing the organ in situ with liquid propane, cooled with liquid nitrogen, followed by lyophilization of the specimen and fixation with gaseous formaldehyde. The sections are stained strictly in accordance with Pickworth's method.

Die noradrenerge Innervation der Anastomose zwischen der V. renalis advehens und revehens des Huhnes wurde mit der histochemischen Fluoreszenzmethode nach Falck und Owman untersucht. — Ein dichtes, dreidimensionales Netz von monoaminergen Nerventerminalen kommt in der Klappe und in der benachbarten Wand der Anastomose vor. Eine ahnlich reiche monoaminerge Innervation wurde von anderen Autoren nur in der Iris und in der Membrana nictitans beobachtet. In dem freien Rande der Klappe, wo sich zahlreiche Epitheloidzellen befinden, kommt keine monoaminerge Innervation vor. Andere grose Venen des Nierengefassystems haben ein normales, zweidimensionales Geflecht von monoaminergen Endigungen auf der Mediaoberflache. Starke spezifische Fluoreszenz wird in den Zellen beobachtet, welche um kleine Gefase in der Umgebung der Anastomose angehauft sind. Diese Gefase besitzen eine dicke Muskelwand, wie sie fur arterio-venose Anastomosen, z.B. in den Fingerbeeren, typisch ist. Die spezifische Fluoreszenz verschwindet nach Reserpinverabreichung wie in den Nervenendigungen, so auch in den monoaminergen Zellen. Eine Untersuchung des durch die monoaminergen Zellen ausgesandten Lichtes mit Hilfe eines Mikroskop-Photometers liefert eine spektrale Kurve mit einem Hauptgipfel bei 500 nm und zwei Nebengipfeln bei 555 und 585 nm. Dieselbe Kurve wurde auch bei der Untersuchung der erwahnten monoaminergen Zellen beobachtet. Die spektrale Kurve der diffusen Fluoreszenz in der Wand der Klappe nach Reserpinverabreichung zeigt nur den Gipfel 555 nm, d. h. der Hauptgipfel 500 nm verschwindet. Die Empfindlichkeit nach Reserpinverabreichung und die spektrale Analyse des emittierten Lichtes zeigen, das die stark fluoreszierenden Zellen Noradrenalin enthalten.

Grosse Gaben von Diphtherietoxin ohne vorhergehende chronischeγ-Strahlung rufen vielfache kleine fuchsinophile Nekrosen in den Fasern des Myocards hervor. Eine solche chronischeγ-Strahlung sensibilisiert das Myocard gegen das Auftreten von Nekrosen vom selben Typus. Dabei erweisen sich kleine Dosen von Diphtherietoxin als pathogen vollig ausreichend, indem sie kleine Brennpunkte der Schadigung bilden und Herde infarktoider Nekrosen verschiedenen Ausmasses.

It is well-known that the ligation of nerve trunk induces an intensive accumulation of noradrenaline proximally to the ligation. A similar ligation, performed in the sympathetic ground-plexus, induces no changes in the segments of terminal fibres situated proximally.

After resection of biological ventricles, and after implantation of artificial ventricles, the mechanisms of vascular regulation are markedly changed. This situation has been observed in 42 long-term experiments on calves and 1 experiment on a goat. In our research centre we have elaborated a functional concept of the causes of changes in the vascular capacitance area in long surviving animals and we have started to influence the state of increase of central venous pressure (CVP) in these animals according to this concept. We have attained positive results, and we hope that our contribution to the explanation of the CVP increase can help to keep the CVP within acceptable limits in long-term survivors with a total artificial heart.

Der Wirkungsbereich der Querschnittsregulation der A. dorsalis pedis durch den Sympathikus bei Hunden wurde erfasst und die Relation Reizerfolg-Reizfrequenz durch eine Hyperbel charakterisiert. Mittels Fluoreszenzmethode gewonnene Befunde weisen darauf hin, dass die durch Sympathikusreizung freigesetzen Monoamine zu den nervenfreien Mediaschichten diffundieren.

Experiments on rats showed that 1 month after lowering the pressure in the blood vessels in the posterior half of the body by constricting the abdominal aorta the number of functioning capillaries in the gastrocnemius and soleus muscles was virtually the same as in the control. This suggests that a decrease in the hydraulic resistance of the resistive vessels in a region of chronic local arterial hypotension is not attributable to an increase in the number of simultaneously functioning vessels.