Ventrolateral periaqueductal gray matter integrative system of defense and antinociception.

Defensive responses are neurophysiological processes crucial for survival during threatening situations. Defensive immobility is a common adaptive response, in rodents, elaborated by ventrolateral periaqueductal gray matter (vlPAG) when threat is unavoidable. It is associated with somatosensory and autonomic reactions such as alteration in the sensation of pain and rate of respiration. In this study, defensive immobility was assessed by chemical stimulation of vlPAG with different doses of NMDA (0.1, 0.3, and 0.6 nmol). After elicitation of defensive immobility, antinociceptive and respiratory response tests were also performed. Results revealed that defensive immobility was followed by a decrease in the nociceptive perception. Furthermore, the lowest dose of NMDA induced antinociceptive response without eliciting defensive immobility. During defensive immobility, respiratory responses were also disturbed. Interestingly, respiratory rate was increased and interspersed with prolonged expiratory phase of breathing. These findings suggest that vlPAG integrates three different defensive behavioral responses, contributing to the most effective defensive strategies during threatening situations.

The quantitative analysis of the mechanism involved in pertussis toxin-mediated cell clustering and its implications in the in vitro quality control of diphtheria tetanus and whole cell pertussis vaccines.

Some of the adverse side-effects such as leukocytosis, hyperinsulinemia, hypoglycemia and sensitization to histamine, caused by diphtheria, tetanus and whole cell pertussis (DTwP) vaccines are related to the presence of non-inactivated pertussis toxin (PTx) residues (NiPTxR). The CHO cell clustering assay is an in vitro assay to measure NiPTxR in DTwP vaccines based on the ability of active PTx to cause cellular clustering. To study the biochemical mechanism involved in the clustering effect in CHO cells induced by PTx and by two DTwP vaccines, the levels of total cyclic cAMP were measured and compared to those obtained after treatment with cholera toxin (CTx) able to induce CHO cells elongation instead of cell clustering. Our results showed an increment of cAMP levels by CTx and total cell elongation in CHO cells. However, changes in cAMP levels were not associated with the total clustering induced by PTx or by DTwP vaccines. The high correlation seen between the levels of NiPTxR in the DTwP vaccines determined by the in vivo lethal histamine sensitization (HIST) assay and the in vitro CHO cell clustering assay indicated that the latter could be a suitable alternative test to HIST assay for the toxicological approval and release of batches of DTwP vaccines in their final formulation for human use in accordance with the application of the 3R's principle.

Chemical and Pharmacological Screening of Rhinella icterica (Spix 1824) Toad Parotoid Secretion in Avian Preparations.

The biological activity of Rhinella icterica parotoid secretion (RIPS) and some of its chromatographic fractions (RI18, RI19, RI23, and RI24) was evaluated in the current study. Mass spectrometry of these fractions indicated the presence of sarmentogenin, argentinogenin, (5ß,12ß)-12,14-dihydroxy-11-oxobufa-3,20,22-trienolide, marinobufagin, bufogenin B, 11α,19-dihydroxy-telocinobufagin, bufotalin, monohydroxylbufotalin, 19-oxo-cinobufagin, 3α,12ß,25,26-tetrahydroxy-7-oxo-5ß-cholestane-26-O-sulfate, and cinobufagin-3-hemisuberate that were identified as alkaloid and steroid compounds, in addition to marinoic acid and N-methyl-5-hydroxy-tryptamine. In chick brain slices, all fractions caused a slight decrease in cell viability, as also seen with the highest concentration of RIPS tested. In chick biventer cervicis neuromuscular preparations, RIPS and all four fractions significantly inhibited junctional acetylcholinesterase (AChE) activity. In this preparation, only fraction RI23 completely mimicked the pharmacological profile of RIPS, which included a transient facilitation in the amplitude of muscle twitches followed by progressive and complete neuromuscular blockade. Mass spectrometric analysis showed that RI23 consisted predominantly of bufogenins, a class of steroidal compounds known for their cardiotonic activity mediated by a digoxin- or ouabain-like action and the blockade of voltage-dependent L-type calcium channels. These findings indicate that the pharmacological activities of RI23 (and RIPS) are probably mediated by: (1) inhibition of AChE activity that increases the junctional content of Ach; (2) inhibition of neuronal Na+/K+-ATPase, leading to facilitation followed by neuromuscular blockade; and (3) blockade of voltage-dependent Ca2+ channels, leading to stabilization of the motor endplate membrane.

Low-intensity laser therapy improves tetanic contractions in mouse anterior tibialis muscle injected with Bothrops jararaca snake venom

Abstract Introduction Envenomation by Bothrops snakes can produce local pain, edema, hemorrhage and myonecrosis. However, standard antivenom therapy is generally ineffective in neutralizing these effects so that alternative methods of treatment have been investigated. In experimental animals, low-level laser therapy (LLLT) attenuates the local effects of Bothrops venoms, but the benefits of LLLT on muscle function after envenomation are unclear. In this study, we examined the influence of LLLT on the contractile activity of mouse skeletal muscle injected with venom from Bothrops jararaca, the principal cause of snakebite in southeastern Brazil. Methods Twenty-seven male mice were used. Mice were injected with venom (40 μg in 50 μl) in the right anterior tibialis muscle, after which the muscle tendon was exposed, connected to an isometric transducer and subjected to a resting tension of 1 g. A bipolar electrode was attached to the tibial nerve for electrical stimulation. The mice were randomly allocated to five groups: A – Control (n = 3), B – Venom 3 h (n = 6), C – Venom 9 h (n = 6), D – Venom + Laser 3 h (n = 6), E – Venom + Laser 9 h (n = 6). Results The two groups that received LLLT post-venom showed improved muscle contraction and contracture in relation to muscle treated with venom alone. Conclusion These results indicate that LLLT can improve muscle function after damage induced by B. jararaca venom.

5-Hydroxytryptamine 1A receptors in the dorsomedial hypothalamus connected to dorsal raphe nucleus inputs modulate defensive behaviours and mediate innate fear-induced antinociception.

The dorsal raphe nucleus (DRN) is an important brainstem source of 5-hydroxytryptamine (5-HT), and 5-HT plays a key role in the regulation of panic attacks. The aim of the present study was to determine whether 5-HT1A receptor-containing neurons in the medial hypothalamus (MH) receive neural projections from DRN and to then determine the role of this neural substrate in defensive responses. The neurotracer biotinylated dextran amine (BDA) was iontophoretically microinjected into the DRN, and immunohistochemical approaches were then used to identify 5HT1A receptor-labelled neurons in the MH. Moreover, the effects of pre-treatment of the dorsomedial hypothalamus (DMH) with 8-OH-DPAT and WAY-100635, a 5-HT1A receptor agonist and antagonist, respectively, followed by local microinjections of bicuculline, a GABAA receptor antagonist, were investigated. We found that there are many projections from the DRN to the perifornical lateral hypothalamus (PeFLH) but also to DMH and ventromedial (VMH) nuclei, reaching 5HT1A receptor-labelled perikarya. DMH GABAA receptor blockade elicited defensive responses that were followed by antinociception. DMH treatment with 8-OH-DPAT decreased escape responses, which strongly suggests that the 5-HT1A receptor modulates the defensive responses. However, DMH treatment with WAY-100635 failed to alter bicuculline-induced defensive responses, suggesting that 5-HT exerts a phasic influence on 5-HT1A DMH neurons. The activation of the inhibitory 5-HT1A receptor had no effect on antinociception. However, blockade of the 5-HT1A receptor decreased fear-induced antinociception. The present data suggest that the ascending pathways from the DRN to the DMH modulate panic-like defensive behaviours and mediate antinociceptive phenomenon by recruiting 5-HT1A receptor in the MH.

Effect of low-level laser therapy (GaAlAs - λ660 nm) on muscle function

Introduction:Low-level laser therapy (LLLT) is effective in preventing fatigue and in stimulating the microcirculation and cellular activity. In this study, we examined the effect of LLLT on injured tibial muscle in vivo by assessing muscle function during fatigue.MethodsTwenty-four male mice were used. Each mouse received an injection of sterile 0.9% saline solution (50 µL) in the right tibialis anterior muscle, after which the tendon of the muscle was exposed, connected to an isometric transducer and subjected to a resting tension of 1 g. A bipolar electrode was attached to the tibial nerve for electrical stimulation. The mice were randomly allocated to one of two groups: G1 (control: 3 h – n=8 and 9 h – n=5) and G2 (treated with GaAlAs laser, λ660 nm, 35 mW, 0.6 J, 17 s: 3 h – n=6 and 9 h – n=5).ResultsIn G1 mice, the amplitude of the tetanic contracture in response to induced fatigue remained unchanged during six consecutive tetani. The amplitude of the tetanic contractions in response to electrical stimulation (4-8 mV) was also unchanged. These results indicated muscle intactness in response to the load imposed by tetanus. In G2 mice, there was an increase in the amplitude of contraction after 3 h and 9 h when compared to G1 at 83% tetanus.ConclusionThese results indicate that exposure of muscle to LLLT enhanced the contractile force and increased the resistance to muscle fatigue without causing morphological damage to cellular structures.

Dialysis headache.

We describe the various side effects occurring in dialysis sessions and indicate measures for their prevention and/or treatment. Next, we analyze dialysis headache in terms of incidence, prevalence, criteria for its inclusion in the classification of the International Headache Society, and factors related to its triggering, ie, bradykinin and nitric oxide (NO), which have increased plasma levels during dialysis. This permits a critical analysis in comparison with NO donor headache in terms of the pathophysiologic mechanisms that mediate the triggering of both conditions. A similarity is particularly detected in terms of the role of NO as the last link in the chain of events that precedes the onset of headache, which is preceded by a latency period of 3 to 4 hours, much longer than the few seconds needed to inactivate NO. A hypothesis is raised to explain this phenomenon, opening new perspectives for the study of the pathophysiology of headaches, including primary headaches.

Pharmacological and structural characterization of a novel phospholipase A2 from Micrurus dumerilii carinicauda venom.

We have isolated a new phospholipase A2 (MiDCA1) from the venom of the coral snake Micrurus dumerilii carinicauda. This toxin, which had a molecular mass of 15,552Da, shared high sequence homology with the PLA2 toxins MICNI A and B from Micrurus nigrocinctus venom (77.7% and 73.1%, respectively). In chick biventer cervicis preparations, MiDCA1 produced concentration- and time-dependent neuromuscular blockade that reached 100% after 120 min (2.4 microM, n = 6); contractures to exogenously applied carbachol (8 microM) and KCl (13 mM) were still seen after complete blockade. In mouse phrenic-nerve diaphragm preparations, MiDCA1 (2.4 microM; n = 6) caused triphasic changes followed by partial neuromuscular blockade. Intracellular recordings of end-plate potentials (EPPs) and miniature end-plate potentials (MEPPs) from mouse diaphragm preparations showed that MiDCA1 increased the quantal content by 386+/-12% after 10 min (n = 14; p<0.05) and caused a triphasic change in the frequency of MEPPs. MiDCA1 also decreased the resting membrane potential, an effect that was prevented by tetrodotoxin and/or low extracellular calcium, but not by d-tubocurarine. The toxin increased the amplitude of mouse sciatic-nerve compound action potentials by 30+/-9% (0.6 microM; p<0.05). Potassium currents elicited in freshly dissociated dorsal root ganglia neurones were blocked by 31+/-1% (n = 4; p<0.05) in the presence of 2.4 microM MiDCA1. These results show that MiDCA1 is a new presynaptic phospholipase A2 that produces neuromuscular blockade in vertebrate nerve-muscle preparations. The triphasic effects seen in mammalian preparations and the facilitatory response were probably caused mainly by the activation of sodium channels, complemented by the blockade of nerve terminal potassium channels. The inability of d-turocurarine to prevent the depolarization by MiDCA1 indicated that cholinergic nicotinic receptors were not involved in this phenomenon.

New evidence for a presynaptic action of prednisolone at neuromuscular junctions.

The action of prednisolone at the neuromuscular junction was studied in mouse isolated phrenic nerve-diaphragm and rat external popliteal/sciatic nerve-tibialis anterior muscle preparations. Prednisolone (0.03 mM and 0.3 mM) did not alter the twitch-tension in phrenic nerve-diaphragm preparations after 120 min, but increased the frequency (170 +/- 4%) and amplitude (200 +/- 13%) of miniature end-plate potentials. Quantal content was not influenced by the glucocorticoid treatment. Prednisolone (400 microg/kg) did not change the twitch-tension in rat external popliteal/sciatic nerve-tibialis anterior muscle preparations. However, this steroid (0.3 mM) prevented the neuromuscular blockade by d-tubocurarine (1.45 microM) in mouse preparations by 70 +/- 10% (P < 0.05). A similar effect (82 +/- 6% protection, P < 0.05) occurred in rats treated with prednisolone (400 microg/kg) before d-tubocurarine (225 microg/kg). In phrenic nerve-diaphragm preparations, prednisolone (0.3 mM) increased (13 +/- 4%, p < 0.05) the twitch-tension in the presence of beta-bungarotoxin (1 microM), and prevented the blockade produced by this toxin (0.15 microM) in its third phase of action. This presynaptic facilitatory effect may contribute to the usefulness of prednisolone in myasthenia gravis.

Natural probes for cholinergic sites: L-bebeerine actions on the neuromuscular transmission, the nicotinic receptor/ionic channel complex, and contraction of skeletal muscles

The mechanisms underlying the muscle relaxant of 1-bebeerine (BB), a tertiary alkaloid isolated from the roots of Chondrodendron platyphyllum, were examined in mammalian and amphibian skeletal muscles. Injections of BB (0.05 - 1 g/kg,i.p.) in rats caused a dose-related flaccid paralysis and respiratory arrest at high doses. In isolated rat diaphragmand toad sartorius muscles, BB depressed the indirectly elicited muscles twitches (IC50:228 muM and 5.4 muM, respectively, at 22 degree) and blocked the nerve-elicited muscle action potential. The neuromuscular blockade was not reserved by neostigmine (10 muM). High concentrations of BB (170 and 340 muM) caused muscle contracture unrelated to the junctional blockade, and intensified by increasing the bath temperature. Analysis of the contraction properties showed that BB (40 and 80 muM)increaded the twitch/tetanus ratio (46 percent and 125 percent) and prolonged the relaxation time; the falling phase of the directly elicited action potential in toad sartorius muscle fibers was slower probably by a decreased potasium conductance. BB (0.1 - 340 muM) reduced the binding of [1251]alpha- -bungarotoxin to the junctional AACh receptor of the rat diaphragm (IC50:47.7 muM, at 37 degree. At low concentrations BB (1.5 - 15 muM) induced either opening or blockade of the Ach receptor-ionic channel. The results showed that BB blocked noncompetitively the neuromuscular transmission through a mechanism that affects the Ach recognition site and the ionic channel properties. The alkaloid also produced muscle contracture and changed the contractile properties through its extra-junctional action at the calcium handling by the sarcoplasmic reticulum or the contractile machinery.

Natural probes for cholinergic sites: L-bebeerine actions on the neuromuscular transmission, the nicotinic receptor/ionic channel complex, and contraction of skeletal muscles

The mechanisms underlying the muscle relaxant of 1-bebeerine (BB), a tertiary alkaloid isolated from the roots of Chondrodendron platyphyllum, were examined in mammalian and amphibian skeletal muscles. Injections of BB (0.05 - 1 g/kg,i.p.) in rats caused a dose-related flaccid paralysis and respiratory arrest at high doses. In isolated rat diaphragmand toad sartorius muscles, BB depressed the indirectly elicited muscles twitches (IC50:228 muM and 5.4 muM, respectively, at 22 degree) and blocked the nerve-elicited muscle action potential. The neuromuscular blockade was not reserved by neostigmine (10 muM). High concentrations of BB (170 and 340 muM) caused muscle contracture unrelated to the junctional blockade, and intensified by increasing the bath temperature. Analysis of the contraction properties showed that BB (40 and 80 muM)increaded the twitch/tetanus ratio (46 percent and 125 percent) and prolonged the relaxation time; the falling phase of the directly elicited action potential in toad sartorius muscle fibers was slower probably by a decreased potasium conductance. BB (0.1 - 340 muM) reduced the binding of [1251]alpha- -bungarotoxin to the junctional AACh receptor of the rat diaphragm (IC50:47.7 muM, at 37 degree. At low concentrations BB (1.5 - 15 muM) induced either opening or blockade of the Ach receptor-ionic channel. The results showed that BB blocked noncompetitively the neuromuscular transmission through a mechanism that affects the Ach recognition site and the ionic channel properties. The alkaloid also produced muscle contracture and changed the contractile properties through its extra-junctional action at the calcium handling by the sarcoplasmic reticulum or the contractile machinery. (AU)