Protein-caloric dietary restriction inhibits mossy fiber sprouting in the pilocarpine model of TLE without significantly altering seizure phenotype.

Given the known effects of undernutrition over protein synthesis, we promoted neonatal undernutrition to evaluate its effect over the neuroplasticity induced by the pilocarpine model of epilepsy and also over spontaneous seizure expression. A well-nourished group (WN), fed ad libitum rat chow diet, and an undernourished group (UN), fed 60% of the amount of diet consumed by a WN group, were submitted to status epilepticus (SE) through pilocarpine injection at 45 days of age. Thereafter, animals were behaviorally monitored for 6h daily to quantify seizures. On the 120th day, electroencephalography (EEG) was recorded and rats were sacrificed to measure proteins and glutamate release from hippocampus. Neo-Timm staining was used to detect mossy fiber sprouting. The results indicate no statistical difference in the latency for the first spontaneous recurrent seizure (SRS), in the number of daily SRS, or in EEG epileptiform activity duration between groups. However, PILO promoted more K(+)-stimulated glutamate release in the hippocampus slices from WN animals when compared to the UN group. It was also found a lower degree of mossy fibers sprouting in UN group. Data from this work, thus, indicate that the decreased neuroplasticity as currently measured does not directly impact on the manifestation of spontaneous seizures.

The role of dorsomedial hypotalamus ionotropic glutamate receptors in the hypertensive and tachycardic responses evoked by Tityustoxin intracerebroventricular injection.

The scorpion envenoming syndrome is an important worldwide public health problem due to its high incidence and potential severity of symptoms. Some studies address the high sensitivity of the central nervous system to this toxin action. It is known that cardiorespiratory manifestations involve the activation of the autonomic nervous system. However, the origin of this modulation remains unclear. Considering the important participation of the dorsomedial hypotalamus (DMH) in the cardiovascular responses during emergencial situations, the aim of this work is to investigate the involvement of the DMH on cardiovascular responses induced by intracerebroventricular (icv) injection of Tityustoxin (TsTX, a α-type toxin extracted from the Tityus serrulatus scorpion venom). Urethane-anaesthetized male Wistar rats (n=30) were treated with PBS, muscimol or ionotropic glutamate receptor antagonists, bilaterally in DMH and later, with an icv injection of TsTX, or treated only with PBS in both regions. TsTX evoked a marked increase in mean arterial pressure and heart rate in all control rats. Interestingly, injection of muscimol, a GABAA receptor agonist, did not change the pressor and tachycardic responses evoked by TsTX. Remarkably, the injection ionotropic glutamate receptors antagonists in DMH abolished the pressor and the tachycardic response evoked by TsTX. Our data suggest that the central circuit recruited by TsTX, whose activation results in an array of physiological and behavioral alterations, depend on the activation of DMH ionotropic glutamate receptors. Moreover, our data provide new insights on the central mechanisms involved in the development of symptoms in the severe scorpion envenomation syndrome.

Effects of tityustoxin on cerebral inflammatory response in young rats.

Accidents caused by scorpion stings, mainly affecting children, are considered an important cause of morbidity and mortality in tropical countries. Clinical studies demonstrate the relevant role of systemic inflammatory events in scorpion envenoming. However, remains poorly understood whether the major lethal component in Tityus serrulatus venom, tityustoxin (TsTX), is able to induce inflammatory responses in the cerebral microcirculation. In this study, we systematically examined leukocyte recruitment into the CNS in response to TsTX injection. Accordingly, developing rats were subjected to a subcutaneous (s.c.) injection of TsTX (0.75mg/kg), and leukocyte recruitment (i.e., 4, 8 and 12h after injection) and TNF-α levels were evaluated. Rats injected with TsTX presented a significant increase in leukocyte rolling and adhesion and higher levels of TNF-α at all time points studied, compared to the control group. Altogether, this work demonstrates the triggering of neuroimmunological mechanisms induced by TsTX injection in young rats.

Murine model to study brain, behavior and immunity during hepatic encephalopathy.

AIM: To propose an alternative model of hepatic encephalopathy (HE) in mice, resembling the human features of the disease. METHODS: Mice received two consecutive intraperitoneal injections of thioacetamide (TAA) at low dosage (300 mg/kg). Liver injury was assessed by serum transaminase levels (ALT) and liver histology (hematoxylin and eosin). Neutrophil infiltration was estimated by confocal liver intravital microscopy. Coagulopathy was evaluated using prolonged prothrombin and partial thromboplastin time. Hemodynamic parameters were measured through tail cuff. Ammonia levels were quantified in serum and brain samples. Electroencephalography (EEG) and psychomotor activity score were performed to show brain function. Brain edema was evaluated using magnetic resonance imaging. RESULTS: Mice submitted to the TAA regime developed massive liver injury, as shown by elevation of serum ALT levels and a high degree of liver necrosis. An intense hepatic neutrophil accumulation occurred in response to TAA-induced liver injury. This led to mice mortality and weight loss, which was associated with severe coagulopathy. Furthermore, TAA-treated mice presented with increased serum and cerebral levels of ammonia, in parallel with alterations in EEG spectrum and discrete brain edema, as shown by magnetic resonance imaging. In agreement with this, neuropsychomotor abnormalities ensued 36 h after TAA, fulfilling several HE features observed in humans. In this context of liver injury and neurological dysfunction, we observed lung inflammation and alterations in blood pressure and heart rate that were indicative of multiple organ dysfunction syndrome. CONCLUSION: In summary, we describe a new murine model of hepatic encephalopathy comprising multiple features of the disease in humans, which may provide new insights for treatment.

Brainstem structures are primarily affected in an experimental model of severe scorpion envenomation.

Severe scorpion envenoming (SSE) is more frequent in children and is characterized by systemic dysfunctions with a mortality rate of up to 9%. Recent evidence shows that the central nervous system (CNS) plays a key role in triggering the cascade of symptoms present in SSE. The age-dependent role of the CNS in SSE lethality may be summarized in 3 hypotheses: (1) the shown increased blood brain barrier permeability of infants to the toxins would especially and primarily compromise neurovegetative control areas, (2) the neurons within these areas have high affinity to the toxins, and (3) the neurovascular interaction is such that SSE metabolically compromises proper function of toxin-targeted areas. A pharmacological magnetic resonance imaging paradigm was used to evaluate localized hemodynamic changes in relative cerebral blood volume (rCBV) for 30 min after the injection of TsTX, the most lethal toxin from the venom of the Tityus serrulatus scorpion. The brainstem showed significant rCBV reduction 1 min after TsTX administration, whereas rostral brain areas had delayed increase in rCBV (confirmed by laser Doppler measurements of cortical cerebral blood flow). Moreover, metabolic activity by 14C-2-deoxyglucose autoradiography showed the highest relative increase at the brainstem. To test whether TsTX has high affinity to brainstem neurons, the lateral ventricle was injected with Alexa Fluor 568 TsTX. Although some neurons showed intense fluorescence, the labeling pattern suggests that specific neurons were targeted. Altogether, these results suggest that brainstem areas involved in neurovegetative control are most likely within the primary structures triggering the cascade of symptoms present in SSE.

Malnutrition during central nervous system growth and development impairs permanently the subcortical auditory pathway.

The brain that grows and develops under the continued influence of malnutrition presents permanent impairment on functioning and neurotransmitter release. The aim of this study was to investigate the chronic effects of neonatal food restriction on neurochemical and neurodynamical aspects within the primary auditory sensory pathway. Our working hypothesis is that neonatal malnutrition may affect the flow of primary sensory information both at a neurochemical and neurodynamical level. To test this hypothesis, three groups of rats were assigned, from birth to 370 days of life, to the following dietary scheme: a well-nourished (WN) group fed ad libitum lab chow diet; an undernourished (UN) group fed 60% of diet consumed by WN group; and a rehabilitated group, undergoing same dietary restriction as undernourished until 42 days of age and thereafter fed ad libitum until the end of the experiment. At 370 days of age, the animals were submitted to brainstem auditory-evoked potentials (BAEPs) recordings and sacrificed for neurochemical evaluation of glutamate release. Undernutrition decreased glutamate release in the cortex, hippocampus, midbrain and brainstem, and significantly increased the latency of BAEP wave V. In addition; the re-establishment of the dietary conditions was not sufficient to reverse the neurochemical and electrophysiological alterations observed in the UN group. Taken altogether, our results suggest that malnutrition imposed at a critical development period caused an irreversible effect within the auditory primary sensory pathway.

Simultaneous quantitation of nicorandil and its denitrated metabolite in plasma by LC-MS/MS: application for a pharmacokinetic study.

A liquid chromatography-electrospray ionization tandem mass spectrometry method was developed and validated for the simultaneous quantitation of nicorandil and its denitrated metabolite, N-(2-hydroxyethyl)-nicotinamide, in rat plasma. After a liquid-liquid extraction step, chromatographic separation was performed on a ShinPack C(18) column with an isocratic mobile phase composed of methanol and 2 mM aqueous ammonium acetate containing 0.03% (v/v) formic acid (33:67 v/v). Procainamide was used as an internal standard (IS). Selected reaction monitoring was performed using the transitions m/z 212 → m/z 135, m/z 166 → m/z 106 and m/z 236 → m/z 163 to quantify nicorandil, its denitrated metabolite and IS, respectively. Calibration curves were constructed over the range of 5-15,000 ng.ml(-1) for both nicorandil and its metabolite. The mean relative standard deviation (RSD%) values for the intra-run precision were 5.4% and 7.3% and for the inter-run precision were 8.5% and 7.3% for nicorandil and its metabolite, respectively. The mean accuracy values were 100% and 95% for nicorandil and its metabolite, respectively. No matrix effect was detected in the samples. The validated method was successfully applied to a pharmacokinetic study after per os administration of nicorandil in rats.

Electroencephalographic evidence of brainstem recruitment during scorpion envenomation.

Scorpion envenomation is a public health problem in Brazil, with most severe cases occuring in children under the age of 5 years (0.6% lethality). In fact, the toxic fractions of the Tityus serrulatus scorpion venom (TSSV) have greater permeability across the BBB of weanling rats when compared to adults. Although EEG alterations have been reported in up to 75% of pediatric severe cases, the role of the CNS in envenomation morbidity is still in debate. Our working hypothesis is that the neural substrates that play a major role in morbidity generate activity undetectable from EEG scalp leads. Twenty one-day-old rats (n=18) were injected s.c. with the deadliest toxic fraction of the TSSV, tityustoxin (TsTX; 2xDL50=6 mg/kg). EEG leads were stereotaxicaly implanted in the nucleus of the solitary tract (NTS) and left parietal cortex. EEG and ECG were continuously monitored by a video EEG system until death or for a maximum period of 240 min. An experimental group pre-treated with carbamazepine (CBZ) was added in order to better access the cause-effect relationship between neural discharges and the systemic ECG alterations. High amplitude discharges in the NTS, which correlated to cardiac alterations, were recorded soon after administration of TsTX. Abnormal electrographic activity spread throughout the cortex only later in the recording. As expected, the CBZ treatment increased the latency for the first epileptiform discharge, decreased EEG/ECG alterations and increased the general survival time. In summary: peripheral scorpion toxin inoculation recruits brainstem involved in cardiovascular control and initial electrographic activity was undetectable from the cortical electrode.

Carbamazepine is effective in the treatment of 21-day-old Wistar rats injected with Tityus serrulatus crude venom.

The scorpion-envenoming syndrome has an incidence of approximately 8000 accidents/year in Brazil; with most severe cases occurring during childhood and elderly. Previous results from our laboratory suggest that the effects of scorpion toxins on the central nervous system play a major role on the lethality induced by scorpion envenoming. Our group has shown that the pre-treatment with carbamazepine (CBZ) is able to increase the latency-to-death in developing animals inoculated with tityustoxin, a toxic fraction of the Tityus serrulatus crude venom. Nevertheless, in order to perceive CBZ as potentially useful in clinical practice, the efficiency of CBZ against crude venom inoculation and the pharmacological treatment introduced after envenomation must be addressed. Thus, the objective of this work was to evaluate CBZ therapeutic efficiency against scorpion envenomation in developing rats. Animals were treated with i.p. injections of either vehicle or CBZ (50 mg/kg or 100 mg/kg) 10 min after injected with a s.c. fixed volume of either saline or crude T. serrulatus venom extract (48 mg/kg). The dose chosen for venom inoculation was 16 times its DL50 for 21-day-old Wistar rats, invariably inducing death within 2 h. Although CBZ did not significantly reduce the pulmonary edema, it was effective in increasing survival rate by approximately 75% in treated rats. In conclusion, CBZ was effective in the treatment of T. serrulatus envenomation even though not blocking the pulmonary edema.

Dose-dependent effect of carbamazepine on weanling rats submitted to subcutaneous injection of tityustoxin.

The scorpion envenoming syndrome is a serious public health matter in Brazil. The most severe cases occur during childhood and elderly. Previous results from our laboratory suggest that the effects of scorpion toxins on the central nervous system play a major role on the lethality induced by scorpion envenoming. The aim of this work is to evaluate the therapeutic potential of carbamazepine (CBZ) injected i.p. 90 min before s.c. tityustoxin (TsTX) injection in weanling rats. Rats were divided into six experimental groups according to s.c. injection (saline or TsTX) and i.p. treatment (vehicle or CBZ 12, 50 and 100 mg/kg): Sal/Veh group (n=4); Sal/CBZ100 (n=4); TsTX/CBZ12 (n=6); TsTX/CBZ50 (n=8); TsTX/CBZ100 (n=8) and, at last, TsTX/Veh (n=8). The dose of TsTX was the same for all groups: 6.0mg/kg, twice the DL50 for weanling rats. Video images were recorded until death or for a maximum period of 240 min. Lungs were excised and weighed to evaluate edema. The results showed that CBZ (12, 50 and 100mg/kg) was able to increase the survival rate and latency-to-death of the rats. Only the group treated with 100mg/kg of CBZ had a decrease in the pulmonary edema. The known effect of CBZ reducing neuronal excitability most likely protected the neural substrates targeted by TsTX. Although treatment was performed before TsTX inoculation, the results are promising regarding CBZ as a therapeutic coadjuvant in the treatment of scorpion poisoning. The pharmacokinetics of CBZ can be very much improved by either changing the form of administration or encapsulating the drug in order to enhance solubility.

Carbamazepine protects the CNS of Wistar rats against the central effects of scorpion envenomation.

The scorpion envenoming syndrome is a serious public health matter in Brazil. Previous results from our laboratory suggest that the effects of scorpion toxins on the central nervous system play a major role on the lethality induced by scorpion envenoming. The objective of this work is to evaluate carbamazepine (CBZ) as a potential therapeutic agent against tityustoxin (TsTX) envenomation. The choice of i.c.v. toxin injection assures that TsTX is readily available in the parenchyma, configuring a worst case scenario for protecting the CNS afflicted by envenomation. Adult Wistar rats were submitted to surgery for guide cannulae (lateral ventricle) and electrodes (EEG-parietal cortices and ECG-thoracic leads) implantation. Animals (n=25) were treated with i.p. injections of either vehicle or CBZ 90 min before injected i.c.v. with a fixed volume of either saline or TsTX: vehicle treated/TsTX injected (1.74 microg, i.c.v.; n=4), CBZ treated (50mg/kg, i.p.)/TsTX injected (n=12); CBZ treated/saline injected (n=5); and vehicle treated/saline injected (n=4). Video EEG/ECG was recorded until death or for a maximum period of 90 min. Lungs were excised and weighed to evaluate edema. The results showed that 10 out of 12 CBZ treated rats survived to TsTX i.c.v. microinjection. CBZ significantly decreased cardiac arrhythmias and pulmonary edema in rats injected with TsTX. Furthermore, CBZ also significantly increased the latency for the first cortical epileptiform discharge. The known effect of CBZ reducing neuronal excitability most likely protected the neural substrates targeted by TsTX. CBZ was efficient in attenuating envenoming symptoms after the i.c.v. inoculation of the TsTX in rats. Thus, CBZ can be proposed as a therapeutic coadjuvant in the treatment of scorpion poisoning.