Adrenergic control of functional characteristics of the cardiovascular system in the South American rattlesnake, Crotalus durissus.

In squamate reptiles, extensive innervation of the heart and vascular beds allows for continuous modulation of the cardiovascular system by the autonomic nervous system. The systemic vasculature is the main target of excitatory sympathetic adrenergic fibers, while the pulmonary circulation has been described as less responsive to both nervous and humoral modulators. However, histochemical evidence has demonstrated the presence of adrenergic fibers in pulmonary circulation. Besides, reduced responsiveness is intriguing since the balance of regulation between systemic and pulmonary vascular circuits has critical hemodynamic implications in animals with an undivided ventricle and consequent cardiovascular shunts. The present study investigated the role and functional relevance of α and ß-adrenergic stimulation in regulating systemic and mainly the pulmonary circulations in a decerebrate, autonomically responsive rattlesnake preparation. The use of the decerebrate preparation allowed us to observe a new diverse functional modulation of vascular beds and the heart. In resting snakes, the pulmonary vasculature is less reactive to adrenergic agonists at 25 °C. However, the ß-adrenergic tone is relevant for modulating resting peripheral pulmonary conductance, while both α- and ß-adrenergic tones are relevant for the systemic circuit. Active dynamic modulation of both pulmonary compliance and conductance effectively counterbalances alterations in the systemic circulation to maintain the R-L shunt pattern. Furthermore, we suggest that despite the great attention given to cardiac adjustments, vascular modulation is sufficient to support the hemodynamic adjustments needed to control blood pressure.

Liver dysfunction and energy storage mobilization in traíra, Hoplias malabaricus (Teleostei, Erythrinidae) induced by subchronic exposure to toxic cyanobacterial crude extract.

Microcystins (MC) are hepatotoxic for organisms. Liver MC accumulation and structural change are intensely studied, but the functional hepatic enzymes and energy metabolism have received little attention. This study investigated the liver and hepatocyte structures and the activity of key hepatic functional enzymes with emphasis on energetic metabolism changes after subchronic fish exposure to cyanobacterial crude extract (CE) containing MC. The Neotropical erythrinid fish, Hoplias malabaricus, were exposed intraperitoneally to CE containing 100 µg MC-LR eq kg-1 for 30 days and, thereafter, the plasma, liver, and white muscle was sampled for analyses. Liver tissue lost cellular structure organization showing round hepatocytes, hyperemia, and biliary duct obstruction. At the ultrastructural level, the mitochondria and the endoplasmic reticulum exhibited disorganization. Direct and total bilirubin increased in plasma. In the liver, the activity of acid phosphatase (ACP) increased, and the aspartate aminotransferase (AST) decreased; AST increased in plasma. Alkaline phosphatase (ALP) and alanine aminotransferase (ALT) were unchanged in the liver, muscle, and plasma. Glycogen stores and the energetic metabolites as glucose, lactate, and pyruvate decrease in the liver; pyruvate decreased in plasma and lactate decreased in muscle. Ammonia levels increased and protein concentration decreased in plasma. CE alters liver morphology by causing hepatocyte intracellular disorder, obstructive cholestasis, and dysfunction in the activity of key liver enzymes. The increasing energy demand implies glucose mobilization and metabolic adjustments maintaining protein preservation and lipid recruitment to supply the needs for detoxification allowing fish survival.

Baroreflex responses of decerebrate rattlesnakes (Crotalus durissus) are comparable to awake animals.

A decerebrate rattlesnake, Crotalus durissus, has previously been used as a model Squamate for cardiovascular studies. It enabled instrumentation for concomitant recordings of diverse variables that showed autonomic responses. However, to validate the preparation and its scope for use, it is necessary to assess how close its cardiovascular variables are to non-decerebrate snakes and the effectiveness of its autonomic responses. Similarly, it is important to analyze its recovery profile after instrumentation and observe if it maintains stability throughout the duration of experimental protocol. Here we have objectively assessed these points by comparing decerebrate preparations and non-decerebrate snakes, after the occlusive cannulation of the vertebral artery. We have assessed cardiovascular variables and the baroreflex to analyze the presence, magnitude and stability of complex autonomic-controlled parameters as indicators of autonomic nervous system (ANS) functionality. After instrumentation, mean heart rates were high but recovered to stable values within 24 h. Mean arterial pressure stabilized within 24 h in control snakes and 48 h in decerebrate preparations. After that, both parameters remained stable. The operational gain and effectiveness index of the baroreflex recovered within the first 6 h after instrumentation in both experimental groups. In addition, the baroreflex capacities and its limits were also equivalent between the groups. These experiments demonstrated that decerebrate preparations and inactive, non-decerebrate snakes showed comparable recovery profiles following anesthesia and cannulation, maintained similar values of cardiovascular variables during experimental manipulation and exhibited functional, ANS modulated reflexes. Accordingly, the present results attest the relevance of this decerebrate preparation for studies on cardiovascular modulation.

Role of Nitric Oxide in the Cardiovascular System of South American Rattlesnakes (Crotalus durissus).

AbstractUnderstanding the basis of vascular tonus regulation is fundamental to comprehending cardiovascular physiology. In the present study, we used the recently developed decerebrate rattlesnake preparation to investigate the role of nitric oxide (NO) in the control of vascular tonus in a squamate reptile. This preparation allowed multiple concomitant cardiovascular parameters to be monitored, while avoiding the deleterious effect of anesthetic drugs on autonomic modulation. We observed that both systemic and pulmonary circuits were clearly responsive to NO signaling. NO increased vascular conductance in the systemic and pulmonary systems. Vasodilation by NO of the systemic circulation was compensated by cardiovascular alterations involving venous return, cardiac output, and cardiac shunt adjustments. The cardiac shunt seemed to be actively used for hemodynamic adjustments via modulation of the pulmonary artery constriction. N(ω)-nitro-L-arginine methyl ester injection demonstrated that NO contributes to modulating resting vasodilation in the systemic circuit. In contrast, NO-mediated vasodilation did not have an important role in the pulmonary circulation in inactive decerebrated snakes at 25°C. These responses vary importantly from those described for anesthetized snakes.

A Decerebrate Preparation of the Rattlesnake, Crotalus durissus, Provides an Experimental Model for Study of Autonomic Modulation of the Cardiovascular System in Reptiles.

AbstractThe South American rattlesnake, Crotalus durissus, has been successfully used as an experimental model to study control of the cardiovascular system in squamate reptiles. Recent technical advances, including equipment miniaturization, have lessened the impact of instrumentation on in vivo recordings, and an increased range of anesthetic drugs has improved recording conditions for in situ preparations. Nevertheless, any animal-based experimental approach has to manage limitations regarding the avoidance of pain and stress the stability of the preparation and duration of experiments and the potentially overriding effects of anesthesia. To address such aspects, we tested a new experimental preparation, the decerebrate rattlesnake, in a study of the autonomic control of cardiovascular responses following the removal of general anesthesia. The preparation exhibited complex cardiovascular adjustments to deal with acute increases in venous return (caused by tail lifting), to compensate for blood flow reduction in the cephalic region (caused by head lifting), for body temperature control (triggered by an external heating source), and in response to stimulation of chemoreceptors (triggered by intravenous injection of NaCN). The decerebrate preparation retained extensive functional integrity of autonomic centers, and it was suitable for monitoring diverse cardiac and vascular variables. Furthermore, reanesthetizing the preparation markedly blunted cardiovascular performance. Isoflurane limited the maintenance of recovered cardiovascular variables in the prepared animal and reduced or abolished the observed cardiovascular reflexes. This preparation enables the recording of multiple concomitant cardiovascular variables for the study of mechanistic questions regarding the central integration of autonomic reflex responses in the absence of anesthesia.

Cholinergic regulation along the pulmonary arterial tree of the South American rattlesnake: vascular reactivity, muscarinic receptors, and vagal innervation.

Vascular tone in the reptilian pulmonary vasculature is primarily under cholinergic, muscarinic control exerted via the vagus nerve. This control has been ascribed to a sphincter located at the arterial outflow, but we speculated whether the vascular control in the pulmonary artery is more widespread, such that responses to acetylcholine and electrical stimulation, as well as the expression of muscarinic receptors, are prevalent along its length. Working on the South American rattlesnake (Crotalus durissus), we studied four different portions of the pulmonary artery (truncus, proximal, distal, and branches). Acetylcholine elicited robust vasoconstriction in the proximal, distal, and branch portions, but the truncus vasodilated. Electrical field stimulation (EFS) caused contractions in all segments, an effect partially blocked by atropine. We identified all five subtypes of muscarinic receptors (M1-M5). The expression of the M1 receptor was largest in the distal end and branches of the pulmonary artery, whereas expression of the muscarinic M3 receptor was markedly larger in the truncus of the pulmonary artery. Application of the neural tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindo-carbocyanine perchlorate (DiI) revealed widespread innervation along the whole pulmonary artery, and retrograde transport of the same tracer indicated two separate locations in the brainstem providing vagal innervation of the pulmonary artery, the medial dorsal motor nucleus of the vagus and a ventro-lateral location, possibly constituting a nucleus ambiguus. These results revealed parasympathetic innervation of a large portion of the pulmonary artery, which is responsible for regulation of vascular conductance in C. durissus, and implied its integration with cardiorespiratory control.

Biotransformations, Antioxidant System Responses, and Histopathological Indexes in the Liver of Fish Exposed to Cyanobacterial Extract.

Radiocystis fernandoi, a microcystin (MC) producer, has been common in cyanobacterial blooms in tropical regions. Microcystin is a hepatotoxin that causes tissue damage and even death in animals, including humans; its detoxification process may involve biotransformation and activation of the antioxidant defense system. We evaluated the detoxification pathway, examined the antioxidant defense system responses, and determined the alterations and the organ histopathological indexes in the liver of the tropical fish Hoplias malabaricus after acute and subchronic intraperitoneal exposure to microcystin. The crude microcystin extract of R. fernandoi had predominantly MC-RR and MC-YR. The detoxification process was activated by increasing ethoxyresorufin-O-deethylase activity, whereas glutathione S-transferase was inhibited. The activity of the antioxidant defense enzymes superoxide dismutase (SOD) and glutathione peroxidase decreased after acute exposure; the SOD-catalase system and the glutathione level increased after subchronic exposure. The carbonyl protein level, lipid peroxidation (LPO), and DNA damage were unchanged after acute exposure, whereas protein carbonyl was unchanged, LPO decreased, and DNA damage increased after subchronic exposure. Histopathological alteration indexes differed between acute and subchronic exposure, but the histopathological organ indexes indicate liver dysfunction in both exposure periods. We conclude that MC-RR and MC-YR induce different liver responses depending on the time of exposure, and the antioxidant defense responses after subchronic exposure may help to partially restore the liver function. Environ Toxicol Chem 2020;39:1041-1051. © 2020 SETAC.

Heart rate variability in the tegu lizard, Salvator merianae, its neuroanatomical basis and role in the assessment of recovery from experimental manipulation.

Using long-term, remote recordings of heart rate (fH) on fully recovered, undisturbed lizards, we identified several components of heart rate variability (HRV) associated with respiratory sinus arrhythmia (RSA): 1.) A peak in the spectral representation of HRV at the frequency range of ventilation. 2.) These cardiorespiratory interactions were shown to be dependent on the parasympathetic arm of the autonomic nervous system. 3.) Vagal preganglionic neurons are located in discrete groups located in the dorsal motor nucleus of the vagus and also, in a ventro-lateral group, homologous to the nucleus ambiguus of mammals. 4.) Myelinated nerve fibers in the cardiac vagus enabling rapid communication between the central nervous system and the heart. Furthermore, the study of the progressive recovery of fH in tegu following anesthesia and instrumentation revealed that 'resting' levels of mean fH and reestablishment of HRV occurred over different time courses. Accordingly, we suggest that, when an experiment is designed to study a physiological variable reliant on autonomic modulation at its normal, resting level, then postsurgical reestablishment of HRV should be considered as the index of full recovery, rather than mean fH.

Osmoregulatory disturbance in Neotropical fish exposed to the crude extracts of the cyanobacterium, Radiocystis fernandoi.

Blooms of cyanobacteria, a common event in eutrophic environments, result in the release of potentially toxic substances into the water. The cyanobacterium Radiocystis fernandoi produces microcystin (MC) and other peptides that may disturb homeostasis. This study evaluated the effect of intraperitoneal injections containing the crude extract (CE) of R. fernandoi strain R28 on the gills and kidneys of neotropical fish, Piaractus mesopotamicus, 3, 6 and 24 h post-injection. CE contained MC-RR, MC-YR and minor other oligopeptides. Plasma ions and the activities of the enzymes PP1 and PP2A, Na+/K+-ATPase (NKA), H+-ATPase (HA) and carbonic anhydrase (CA) were determined and morphological changes in both the gills and kidneys were characterized. Compared to controls, the concentration of Na+ within the plasma of P. mesopotamicus decreased after treatment with CE 3 h post treatment and increased after 24 h; the concentration of K+ decreased after 6 h. The activity of the endogenous PP1 and PP2A was unchanged in the gills and was inhibited in the kidneys 6 h after i.p. injection. In the gills, NKA activity increased after 3 h and decreased 6 h post i.p. exposure. Further, NKA activity did not differ from the controls 24-h post injection. In the kidneys, NKA, HA and CA activities were unaffected by treatment. The mitochondria-rich cell (MRC) density in the gills decreased after 3 h in the filament and 3 and 6 h in the lamellae and was restored to the control levels 24 h post-exposure. Filament epithelial hyperplasia and hypertrophy, lamellar atrophy and rupture of the lamellar epithelium were the most common effects of treatment in the gills. No histopathological changes occurred in the kidneys. This study demonstrates that a single dose of toxic CE from R. fernandoi can cause a transitory ion imbalance in P. mesopotamicus which is related to the changes in MRC levels and NKA activity. Ionic balance was recovered 24 h post i.p. injection, however, morphological changes that occurred in the gills took a longer amount of time to return to normal. To conclude, the effects of components contained within the CE of R. fernandoi may be harmful to P. mesopotamicus. In particular, the recovery of ionic regulation depends on MRC responses and histopathological changes produced by CE may affect gas exchange and other gill functions.

Crude extract of cyanobacterium Radiocystis fernandoi strain R28 induces anemia and oxidative stress in fish erythrocytes.

The cyanobacterium Radiocystis fernandoi has been frequently identified in cyanobacterial blooms in Brazil. Recently, R. fernandoi strain R28, which produces microcystin (MC)-RR and MC-YR, was isolated from the Furnas reservoir, Minas Gerais, Brazil. The present study evaluated the hematological variables and erythrocyte antioxidant responses, lipid peroxidation (LPO), and genotoxicity in a neotropical fish (Hoplias malabaricus) after acute and subchronic exposure to a crude extract (CE) of R. fernandoi strain R28. Acute exposure (12 or 96 h) consisted of a single intraperitoneal (i.p.) CE injection, and subchronic exposure consisted of one i.p. CE injection every 72 h for 30 days. After acute exposure, fish exhibited macrocytic anemia (12 h post-injection) followed by normocytic anemia (96 h post-injection). The increased activity of superoxide dismutase, glutathione peroxidase, glutathione-S-transferase, and the glutathione level in the erythrocytes did not prevent oxidative stress, manifested as lipid peroxidation and elevated DNA damage after acute exposure. After subchronic exposure, the hematological variables recovered, and the absence of erythrocyte oxidative stress suggests possible modulation by other biological factors, including a possible decrease in MC uptake by the cells and/or increasing detoxification efficiency that precludes erythrocyte damage.

Evaluation of the sequence method as a tool to assess spontaneous baroreflex in reptiles.

The sequence method is an alternative to the traditional pharmacological approach (i.e., the Oxford technique) used to calculate baroreflex gain (G) in mammals. Although the sequence method assesses baroreflex by measuring spontaneous events of blood pressure regulation, the pharmacological method relies on the injection of vasoactive drugs that impact the baroreflex mechanism itself. The sequence method might be relevant for dynamic measurement of baroreflex modulation but it was never validated for vertebrates with low heart rate. Hence, we tested the sequence method in three species of reptiles and compared the results with those provided by the classic pharmacological method. G was similar between both methods and values correlated when parameters for the sequence method were set at delay 0 or 1 (i.e., the baroreflex system responds immediately to blood pressure changes or after 1 heartbeat). Calculation of the baroreflex effectiveness index was adequate at a minimum of 300 cycles and a delay of 1 for the three species. Therefore, the sequence method has been validated to investigate baroreflex regulation in reptiles, enabling studies during dynamic alterations in homeostasis.

Overview of the toxic effects of titanium dioxide nanoparticles in blood, liver, muscles, and brain of a Neotropical detritivorous fish.

The toxicity of titanium dioxide nanoparticles (TiO2 -NP) in the blood, liver, muscle, and brain of a Neotropical detritivorous fish, Prochilodus lineatus, was tested. Juvenile fish were exposed to 0, 1, 5, 10, and 50 mg L-1 of TiO2 -NP for 48 hours (acute exposure) or 14 days (subchronic exposure) to evaluate changes in hematology, red blood cell (RBC) genotoxicity/mutagenicity, liver function (reactive oxygen species (ROS) production, antioxidant responses, detoxification, and histopathology), acetylcholinesterase (AChE) activity in muscles and brain, and Ti bioaccumulation. TiO2 -NP did not cause genetic damage to RBC, but acutely decreased white blood cells (WBC) and increased monocytes. Subchronically, RBC decreased, mean cell volume and hemoglobin increased, and WBC and lymphocytes decreased. Therefore, NP has the potential to affect immune system and increase energy expenditure, reducing the fish's ability to avoid predator and to resist pathogens. In the liver, acute exposure decreased ROS and increased glutathione (GSH) content, while subchronic exposure decreased superoxide dismutase activity and increased glutathione-S-transferase (GST) activity and GSH content. GSH and GST seem to play an essential role in metabolizing NP and ROS, likely increasing hepatocytes' metabolic rate, which may be the cause of observed cell hypertrophy, disarrangement of hepatic cords and degenerative morphological alterations. Although most studies indicate that the kidney is responsible for metabolizing and/or eliminating TiO2 -NP, this study shows that the liver also has a main role in these processes. Nevertheless, Ti still accumulated in the liver, muscle, and brain and decreased muscular AChE activity after acute exposure, showing neurotoxic potential. More studies are needed to better understand the biochemical pathways TiO2 -NP are metabolized and how its bioaccumulation may affect fish homeostasis and survival in the environment.

Biochemical and morphological biomarkers of the liver damage in the Neotropical fish, Piaractus mesopotamicus, injected with crude extract of cyanobacterium Radiocystis fernandoi.

Cyanobacterial proliferation in river and lakes is the result of eutrophication. The cyanobacterium Radiocystis fernandoi strain R28 produces mostly two MC variants MC-RR and MC-YR and small amounts of other oligopeptides, but does not produce MC-LR. The present study investigated the hepatotoxic potential of the crude extract of the R. fernandoi strain R28 on the Neotropical fish, Piaractus mesopotamicus, at 3, 6, and 24 h after intraperitoneal injection (100 µg MC-LR equivalent per kg-1 body mass) using biochemical and morphological biomarkers of liver damage. Although the protein phosphatases PP1 and PP2A were not inhibited during the 24-h treatment, liver parenchyma and hepatocyte structure were disrupted. Alkaline phosphatase increased at 3 h post-injection and decreased after 24 h; alanine aminotransferase and aspartate aminotransferase increased in a time-dependent manner up to 24 h indicating impaired liver function. Progressive histopathological changes were consistent with biochemical results demonstrating alterations in liver structure and function. In conclusion, the crude extract of R. fernandoi strain R28 has high hepatotoxic potential and can severely compromise fish health.

Alternagin-C (ALT-C), a Disintegrin-Like Cys-Rich Protein Isolated from the Venom of the Snake Rhinocerophis alternatus, Stimulates Angiogenesis and Antioxidant Defenses in the Liver of Freshwater Fish, Hoplias malabaricus.

Alternagin-C (ALT-C) is a disintegrin-like protein isolated from Rhinocerophis alternatus snake venom, which induces endothelial cell proliferation and angiogenesis. The aim of this study was to evaluate the systemic effects of a single dose of alternagin-C (0.5 mg·kg-1, via intra-arterial) on oxidative stress biomarkers, histological alterations, vascular endothelial growth factor (VEGF) production, and the degree of vascularization in the liver of the freshwater fish traíra, Hoplias malabaricus, seven days after the initiation of therapy. ALT-C treatment increased VEGF levels and hepatic angiogenesis. ALT-C also enhanced hepatic antioxidant enzymes activities such as superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase, decreasing the basal oxidative damage to lipids and proteins in the fish liver. These results indicate that ALT-C improved hepatic tissue and may play a crucial role in tissue regeneration mechanisms.

Hepatotoxicity and metabolic effects of cellular extract of cyanobacterium Radiocystis fernandoi containing microcystins RR and YR on neotropical fish (Hoplias malabaricus).

The toxicological effect of cellular extract of cyanobacterium Radiocystis fernandoi strain R28 containing RR and YR microcystins was analyzed in the fish Hoplias malabaricus with emphasis on the liver structure and energetic metabolism, after short-term exposure. Fish were intraperitoneally (i.p.) injected with 100 µg of equivalent MC-LR kg-1 body mass containing in the cellular extract of R. fernandoi strain R28. Twelve and 96 h post-injection, the plasma, liver and white muscle were sampled for biochemical analyses and liver was also sampled for morphological analyses. After i.p. injection, the activity of acid phosphatase (ACP), alanine aminotransferase (ALT) and direct bilirubin increased in the plasma, while ALT and aspartate aminotransferase (AST) decreased in the liver. Glucose, lactate and pyruvate increased while protein decreased in the plasma; glycogen, pyruvate and lactate decreased in the liver; and glycogen and glucose increased in the muscle. Ammonia increased in the plasma, liver and muscle. The hepatocyte cell shape changed from polyhedral to round after cellular extract injection; there was loss of biliary canaliculus organization, but the biliary duct morphology was conserved in the liver parenchyma. In conclusion, microcystins present in the cellular extract of R. fernandoi strain R28 affect the liver structure of H. malabaricus, but the liver was able to continuously produce energy by adjusting its intermediate metabolism; glycogenolysis and gluconeogenesis maintained glucose homeostasis and energy supply.