Arteether-induced brain injury in Macaca mulatta. I. The precerebellar nuclei: the lateral reticular nuclei, paramedian reticular nuclei, and perihypoglossal nuclei

Malaria poses a threat across several continents: Eurasia (Asia and parts of Eastern Europe), Africa, Central and South America. Bradley (1991) estimates human exposure at 2,073,000,000 with infection rates at 270,000,000, illnesses at 110,000,000, and deaths at 1,000,000. Significant mortality rates are attributed to infection by the parasite Plasmodium falciparum, with an estimated 90% among African children. A worldwide effort is ongoing to chemically and pharmacologically characterize a class of artemisinin compounds that might be promising antimalarial drugs. The U.S. Army is studying the efficacy and toxicity of several artemisinin semi-synthetic compounds: arteether, artemether, artelinic acid, and artesunate. The World Health Organization and the U.S. Army selected arteether for drug development and possible use in the emergency therapy of acute, severe malaria. Male Rhesus monkeys (Macaca mulatta) were administered different daily doses of arteether, or the vehicle alone (sesame oil), for a period of either 14 days, or 7 days. Neuropathological lesions were found in 14-day arteether treated monkeys in the precerebellar nuclei of the medulla oblongata, namely: (1) the lateral reticular nuclei (subnuclei magnocellularis, parvicellularis, and subtrigeminalis), (2) the paramedian reticular nuclei (subnuclei accessorius, dorsalis, and ventralis), and the perihypoglossal nuclei (n. intercalatus of Staderini, n. of Roller, n. prepositus hypoglossi). The data demonstrate that the simina meduallry precerebellar nuclei have a high degree of vulnerability when arteether is given for 14 days at dose levels between 8mg/kg per day and 24 mg/kg per day. The neurological consequences of this treatment regimen could profoundly impair posture, gait, and autonomic regulation, while eye movement disorders might also be anticipated.     

On the direct and crossed components of reflex responses to unilateral stimulation of the carotid body chemoreceptors in the dog

1. In dogs under chloralose-urethane anaesthesia the chemoreceptors of the two carotid bodies were separately stimulated.2. The distribution of three primary reflex responses to carotid body stimulation was studied: parasympathetic bradycardia, sympathetic vasoconstriction, and increase in somatic phrenic nerve activity.3. The reflex bradycardia evoked by either carotid body was mediated by both vagus nerves, but when either vagus was blocked a greater response could be obtained from the contralateral than from the ipsilateral carotid body.4. The reflex vasoconstriction evoked by either carotid body was seen in both hind limbs, with no predominance in either limb.5. The reflex increase in phrenic nerve activity evoked by either carotid body was seen in both phrenic nerves, with no predominance in either nerve.     

Comparison of the reflex responses elicited by stimulation of the separately perfused carotid and aortic body chemoreceptors in the dog

1. In dogs under chloralose and urethane anaesthesia, the carotid and aortic bodies were isolated from the circulation and separately perfused with blood, the composition of which could be controlled at will. The remainder of the systemic circulation was perfused at constant blood flow, thereby enabling the reflex vascular responses to be determined. The systemic venous blood was oxygenated in the isolated perfused lungs of a second dog and the P(O2) and P(CO2) of the systemic arterial blood was maintained constant.2. Using hypoxic hypercapnic blood to stimulate the arterial chemoreceptors, carotid body excitation in spontaneously breathing animals caused an increase in respiratory minute volume approximately seven times larger than that evoked by stimulation of the aortic bodies. Whereas the hyperpnoea of carotid body origin is due to an increase in rate and depth of breathing, that from the aortic bodies is due predominantly to an increase in respiratory frequency.3. Stimulation of the carotid bodies in spontaneously breathing animals caused small variable changes in systemic vascular resistance, whereas stimulation of the aortic bodies invariably increased the vascular resistance.4. When pulmonary ventilation was maintained constant, the vascular response to stimulation of the carotid bodies was considerably modified in that constriction invariably occurred; that from the aortic bodies, however, was little affected. There was now no significant difference in the size of responses from the two groups of chemoreceptors. These constrictor responses represent the primary vascular effects.5. A similar modification of the carotid body vascular response occurred in the spontaneously breathing animal after denervation of the lungs, and is due to abolition of a lung-inflation vasodilator reflex.6. The size of the primary vasoconstrictor responses from the carotid and aortic bodies is reduced by lowering the arterial blood P(CO2).7. The results indicate that there is a fundamental difference in the functions of the carotid and aortic bodies. They exert a quantitatively similar primary control of the ;vasomotor centre' which is in striking contrast to the relatively more powerful influence on respiration by the carotid bodies. In the spontaneously breathing animal, however, the primary vasoconstrictor response from the carotid bodies is offset to a varying degree by the lung-inflation vasodilator reflex initiated by the concomitant hyperpnoea. This is not evident with the aortic bodies because of the relatively weaker respiratory response they evoke.     

The role of NADPH oxidase in carotid body arterial chemoreceptors

O(2)-sensing in the carotid body occurs in neuroectoderm-derived type I glomus cells where hypoxia elicits a complex chemotransduction cascade involving membrane depolarization, Ca(2+) entry and the release of excitatory neurotransmitters. Efforts to understand the exquisite O(2)-sensitivity of these cells currently focus on the coupling between local P(O2) and the open-closed state of K(+)-channels. Amongst multiple competing hypotheses is the notion that K(+)-channel activity is mediated by a phagocytic-like multisubunit enzyme, NADPH oxidase, which produces reactive oxygen species (ROS) in proportion to the prevailing P(O2). In O(2)-sensitive cells of lung neuroepithelial bodies (NEB), multiple studies confirm that ROS levels decrease in hypoxia, and that E(M) and K(+)-channel activity are indeed controlled by ROS produced by NADPH oxidase. However, recent studies in our laboratories suggest that ROS generated by a non-phagocyte isoform of the oxidase are important contributors to chemotransduction, but that their role in type I cells differs fundamentally from the mechanism utilized by NEB chemoreceptors. Data indicate that in response to hypoxia, NADPH oxidase activity is increased in type I cells, and further, that increased ROS levels generated in response to low-O(2) facilitate cell repolarization via specific subsets of K(+)-channels.     

The role of the carotid chemoreceptors in the CO2-hyperpnea under hyperoxia

Summary In anesthetized rabbits rebreathing oxygen or inhaling constant mixtures of CO₂ and O₂, the influence of inactivation of the carotid sinus nerves by section or temporary cold blocking on the respiratory response to increased was investigated. In some of the experiments the vagi were intact, in others they were cut. Even during respiration of pure oxygen, in part of the animals blocking of the sinus nerves caused a slight diminution of ventilation which was significant only in the vagotomized preparation (average decrease by 11.4%). However, the diminishing effect of the blockade did not increase, but rather decreased, with increasing . Similarly, section of the sinus nerves did not cause a significant shift or decrease of slope of the response curve. Evidently in the rabbit carotid chemoreceptors do not contribute essentially to the steady-state hyperpnea caused by hypercapnia under hyperoxic conditions.Supported by grant Wi 165 from the Deutsche Forschungsgemeinschaft.     

The role of the carotid body chemoreceptors and carotid sinus baroreceptors in the control of cerebral blood vessels

1. Cerebral blood flow was measured in 17 baboons, anaesthetized with pentobarbitone, paralysed with gallamine and mechanically ventilated and in which the right sinus and both aortic nerves had been cut and the left carotid sinus vascularly isolated. Later in each experiment, the head was artificially perfused with femoral arterial blood via the innominate artery.2. Stimulation of the carotid body chemoreceptors with venous blood invariably caused a rise in regional cerebral blood flow whether the head was naturally or artificially perfused. This response was almost completely abolished if the VIIth cranial nerves were cut intracranially.3. Regional cerebral blood flow varied inversely with carotid sinus pressure.4. After the remaining (left) sinus nerve had been cut, the cerebral vascular response to hypoxia was negligible and the response to hypercapnia was markedly reduced. Blood flow then varied with perfusion pressure.5. These results provide further evidence that cerebral blood vessels are reflexly controlled and that the peripheral arterial receptors are involved. Their action is most conspicuous in the vascular response to hypoxia and together with intrinsic factors in the cerebral vascular bed, they determine the size of the vascular response to changes in CO(2) and pressure.     

The analysis of reflex changes in the cardiovascular system during stimulation of the chemoreceptors of the pericardium

Summary Acute experiments were conducted on cats under urethane anesthesia. A study was made of the frequency and strength of cardiac contractions, as well as of the vascular resistance in various organs with nicotine and acetylcholine solutions acting upon the pericardial receptors. In depressor reaction occurring in response to the pericardial administration of nicotine, there always occurred a deceleration of the cardiac rhythm, whereas the strength of the cardiac contractions either diminished, increased or remained unchanged. A study of the vascular resistance of the posterior extremities, organs of the pelvis minor, kidneys, small intestine, spleen, lungs, neck and head demonstrated that reduced resistance was observed only in the vessels of the spleen, intestine and kidneys. However, the resistance changes in the mentioned organs were not always distinct and were even absent in a number of cases. Experiments with a temporary exclusion of various vascular areas from the general circulation demonstrated that the development of depressor reaction consequent upon the nicotine action, upon the pericardial receptors is connected with changes in the pulmonary vessels. There is deminished circulation rate in the venous vessels preceeding the arterial pressure reduction; as to the arteries the circulation rate remains unchanged or decreased after the arterial pressure shifts. The efferent route of this reflex passes in the vagus. With stimulation of the pericardial chemoreceptors by acetylcholine solutions depressor reaction is connected both with the reflex effects (decelerated cardiac rhythm) and the resorptive ones (reduction of peripheral resistance).(Presented by Active Member AMN SSSR P. S. Kupalov) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 54, No. 10, pp. 27–32, October, 1962     

Circulatory responses to stimulation of the carotid body chemoreceptors in the cat.

Cardiovascular responses to carotid body chemoreceptor stimulation were followed in the 'curairzed', vagotomized, artificially ventilated cat. Stimulation of the chemoreceptors by perfusion of the carotid sinus regions with venous blood induced a reflex vasconstriction in skeletal muscle, kidney, intestine and skin, and, in most cases, an increased heart rate. A comparison of the chemoreceptor reflex responses with those obtained by direct electrical stimulation of the regional vasomotor fibres indicated that in chemorecptor reflexes the vasomotor fibre activity increased to the same extent in skeletal muscle and intestinal resistance vessels and, probably, in the nutritional skin vessels but to a smaller extent in the renal vessels and the skeletal muscle capacitance vessels. The renal vessels and the muscle capacitance vessels seemed, however, to respond more during chemoreceptor stimulation than when the barorecptors were unloaded, indicating that anexcitation of the bulbar vasomotor centre is more effective than the withdroawal of an inhibitory restraint in activating the vasomotor fibres to these vascular sections.     

Axonal branching in the projections from the paramedian reticular nucleus to the cerebellar cortex

Injections of fluorescent tracers into cat cerebellar cortex gave evidence of collateral axonal branching of neurons situated in the paramedian reticular nucleus. These branched reticulocerebellar projections were distributed to opposing sides of the cerebellum, in particular the anterior lobe and the ansiform lobule. No topographical organization was observed in the PRN. Less than 30% of ipsilaterally projecting reticulocerebellar fibers had contralaterally directed collateral branches. These results are in keeping with a bilateral fastigial projection to the PRN forming a feedback loop circuit through which orthostatic reflexes may be mediated.     

The paramedian reticular nucleus: a site of inhibitory interaction between projections from fastigial nucleus and carotid sinus nerve acting on blood pressure

1. The interaction between the pressor response to electrical stimulation of the fastigial nucleus (FN), the fastigial pressor response (FPR), and the depressor response to electrical stimulation of the carotid sinus nerve (CSN) was examined in paralysed anaesthetized cats.2. Blood pressure responses evoked by electrical stimulation of the FN and the CSN were mutually inhibitory and summed algebraically.3. The FPR was augmented after denervation of buffer nerves. Lesions of the FN did not alter the depressor response to stimulation of the CSN.4. Bilateral electrolytic lesions of the paramedian reticular nucleus abolished both the FPR and the CSN depressor response without altering base line pressure.5. With micro-electrode recording neurones were discovered within the paramedian reticular nucleus which responded to electrical stimulation of the FN or the CSN. These neurones were polysynaptically excited by stimulation of either the FN or the CSN but rarely from both, and could be further subdivided into cells responding with either a single spike or a burst discharge.6. The interaction between the FN and the CSN projections to the paramedian reticular nucleus was examined by conditioning-test studies. Eleven per cent of FN- and CSN-units were inhibited by conditioning stimulation of the heteronymous input. The interaction was exclusively inhibitory and observed only in units with latencies > 4 msec and having burst responses. The latency for inhibition was > 20 msec, peaked around 100 msec and lasted up to 300 msec.7. We conclude that the FRP is buffered by baroreceptors and that there is a mutually inhibitory interaction between projections from the FN and the CSN acting on sympathetic vasomotor neurones. The paramedian reticular nucleus appears to be an important site for the interaction.8. The findings support the view that interneurones mediating pressor and depressor responses are intermixed within the medial reticular formation of the medulla.     

Cardiovascular responses in apnoeic asphyxia: role of arterial chemoreceptors and the modification of their effects by a pulmonary vagal inflation reflex

1. In the spontaneously breathing anaesthetized dog, the systemic circulation was perfused at constant blood flow; there was no pulmonary blood flow and the systemic arterial blood P(O2) and P(CO2) were controlled independently by an extracorporeal isolated pump-perfused donor lung preparation. The carotid and aortic bodies were separately perfused at constant pressure with blood of the same composition as perfused the systemic circulation.2. Apnoeic asphyxia, produced by stopping the recipient animal's lung movements and, at the same time, making the blood perfusing the systemic circulation and the arterial chemoreceptors hypoxic and hypercapnic by reducing the ventilation of the isolated perfused donor lungs, caused an increase in systemic vascular resistance.3. While the systemic arterial blood was still hypoxic and hypercapnic, withdrawal of the carotid and aortic body ;drive' resulted in a striking reduction in systemic vascular resistance. Re-establishing the chemoreceptor ;drive' immediately increased the vascular resistance again.4. Apnoeic asphyxia carried out while the carotid and aortic bodies were continuously perfused with oxygenated blood of normal P(CO2) had little or no effect on systemic vascular resistance.5. The systemic vasoconstrictor response produced by apnoeic asphyxia was reduced or abolished by re-establishing the recipient animal's lung movements, and this effect occurred in the absence of changes in the composition of the blood perfusing the systemic circulation and arterial chemoreceptors. This abolition of the vasoconstriction was due to a pulmonary reflex.6. Apnoeic asphyxia slowed the rate of the beating atria due to excitation of the carotid and aortic body chemoreceptors. This response can be over-ridden by an inflation reflex arising from the lungs.7. It is concluded that the cardiovascular responses observed in apnoeic asphyxia are due, at least in part, to primary reflexes from the carotid and aortic body chemoreceptors engendered by arterial hypoxia and hypercapnia. The appearance of these responses is, however, dependent upon there being no excitation of a pulmonary (inflation) vagal reflex.     

The function of the paramedian reticular nucleus in the control of heart rate in the cat

1. Selective electrical stimulation of the paramedian reticular nucleus (PRN) in anaesthetized and decerebrate-anaesthetized cats elicited cardiac slowing which was shown to be due to inhibition of the sympathetic input to the heart.2. In decerebrate preparations stimulation of the PRN elicited cardioacceleration which was abolished by bilateral vagotomy.3. It is suggested that these changes in heart rate in opposite directions can be accounted for by an inhibitory influence of the PRN on both neural inputs to the heart. In the absence of vagal activity, demonstrated in anaesthetized and decerebrate-anaesthetized preparations, the response to stimulation of the PRN resulted in cardiac slowing. In contrast, when both sympathetic and parasympathetic pathways were active (decerebrate preparation) stimulation of the PRN resulted in cardio-acceleration, due to simultaneous inhibition of the two neural inputs to the heart.4. It is concluded that the two inhibitory functions of the PRN are probably mediated by two different populations of neurones and that the PRN plays an integrative role in the medullary control of heart rate.     

Cerebellar afferents from the paramedian reticular nucleus studied with retrograde transport of horseradish peroxidase

Summary Details in the cerebellar projections from the paramedian reticular nucleus (PRN) were studied in cats and monkeys by means of retrograde axonal transport of horseradish peroxidase (HRP). In the cat the majority of the fibres projects to the anterior lobe and to the vermis of the posterior lobe (with the exception of lobules VIIB and VIIIA). A less conspicuous projection was found to the lobulus simplex, the crura and the flocculus. The cerebellar nuclei, the paramedian lobule and the paraflocculus appear to be weakly connected with the PRN. A similar distribution of the cerebellar afferent fibres was found in the monkey material. The three subgroups of the PRN in the cat are not equal in their projection. The dorsal group appears to be connected with the greater part of the cerebellar cortex and with all nuclei. The ventral group lacks a connection with lobulus IX, the flocculus and the paraflocculus, and the accessory group appears to have its strongest connection with lobulus I (lingula), the flocculus and the vermal lobules VII–X. The findings are discussed in relation to other studies on the efferent and afferent connections of the nucleus.On leave from the Laboratory of Neurobiology and Department of Anatomy, Faculty of Science, Mahidol University, Bankok, Thailand, under the Fellowship Program of the Norwegian Agency for International Development (NORAD)