Stem cell factor protects against neuronal apoptosis by activating AKT/ERK in diabetic mice

Neuronal apoptosis occurs in the diabetic brain due to insulin deficiency or insulin resistance, both of which reduce the expression of stem cell factor (SCF). We investigated the possible involvement of the activation of the MAPK/ERK and/or AKT pathways in neuroprotection by SCF in diabetes. Male C57/B6 mice (20-25 g) were randomly divided into four groups of 10 animals each. The morphology of the diabetic brain in mice treated or not with insulin or SCF was evaluated by H&E staining and TUNEL. SCF, ERK1/2 and AKT were measured by Western blotting. In diabetic mice treated with insulin or SCF, there was fewer structural change and apoptosis in the cortex compared to untreated mice. The apoptosis rate of the normal group, the diabetic group receiving vehicle, the diabetic group treated with insulin, and the diabetic group treated with SCF was 0.54 ± 0.077%, 2.83 ± 0.156%, 1.86 ± 0.094%, and 1.78 ± 0.095% (mean ± SEM), respectively. SCF expression was lower in the diabetic cortex than in the normal cortex; however, insulin increased the expression of SCF in the diabetic cortex. Furthermore, expression of phosphorylated ERK1/2 and AKT was decreased in the diabetic cortex compared to the normal cortex. However, insulin or SCF could activate the phosphorylation of ERK1/2 and AKT in the diabetic cortex. The results suggest that SCF may protect the brain from apoptosis in diabetes and that the mechanism of this protection may, at least in part, involve activation of the ERK1/2 and AKT pathways. These results provide insight into the mechanisms by which SCF and insulin exert their neuroprotective effects in the diabetic brain.

Rat Dlx5 is expressed in the subventricular zone and promotes neuronal differentiation

The molecular mechanisms and potential clinical applications of neural precursor cells have recently been the subject of intensive study. Dlx5, a homeobox transcription factor related to the distal-less gene in Drosophila, was shown to play an important role during forebrain development. The subventricular zone (SVZ) in the adult brain harbors the largest abundance of neural precursors. The anterior SVZ (SVZa) contains the most representative neural precursors in the SVZ. Further research is necessary to elucidate how Dlx5-related genes regulate the differentiation of SVZa neural precursors. Here, we employed immunohistochemistry and molecular biology techniques to study the expression of Dlx5 and related homeobox genes Er81 and Islet1 in neonatal rat brain and in in vitro cultured SVZa neural precursors. Our results show that Dlx5 and Er81 are also highly expressed in the SVZa, rostral migratory stream, and olfactory bulb. Islet1 is only expressed in the striatum. In cultured SVZa neural precursors, Dlx5 mRNA expression gradually decreased with subsequent cell passages and was completely lost by passage four. We also transfected a Dlx5 recombinant plasmid and found that Dlx5 overexpression promoted neuronal differentiation of in vitro cultured SVZa neural precursors. Taken together, our data suggest that Dlx5 plays an important role during neuronal differentiation.

Participation of neuronal nitric oxide synthase in experimental neuropathic pain induced by sciatic nerve transection

Nerve injury leads to a neuropathic pain state that results from central sensitization. This phenomenom is mediated by NMDA receptors and may involve the production of nitric oxide (NO). In this study, we investigated the expression of the neuronal isoform of NO synthase (nNOS) in the spinal cord of 3-month-old male, Wistar rats after sciatic nerve transection (SNT). Our attention was focused on the dorsal part of L3-L5 segments receiving sensory inputs from the sciatic nerve. SNT resulted in the development of neuropathic pain symptoms confirmed by evaluating mechanical hyperalgesia (Randall and Selitto test) and allodynia (von Frey hair test). Control animals did not present any alteration (sham-animals). The selective inhibitor of nNOS, 7-nitroindazole (0.2 and 2 µg in 50 µL), blocked hyperalgesia and allodynia induced by SNT. Immunohistochemical analysis showed that nNOS was increased (48% by day 30) in the lumbar spinal cord after SNT. This increase was observed near the central canal (Rexed’s lamina X) and also in lamina I-IV of the dorsal horn. Real-time PCR results indicated an increase of nNOS mRNA detected from 1 to 30 days after SNT, with the highest increase observed 1 day after injury (1469%). Immunoblotting confirmed the increase of nNOS in the spinal cord between 1 and 15 days post-lesion (20%), reaching the greatest increase (60%) 30 days after surgery. The present findings demonstrate an increase of nNOS after peripheral nerve injury that may contribute to the increase of NO production observed after peripheral neuropathy.

Lack of tolerance for the anti-dyskinetic effects of 7-nitroindazole, a neuronal nitric oxide synthase inhibitor, in rats

7-Nitroindazole (7-NI) inhibits neuronal nitric oxide synthase in vivo and reduces l-DOPA-induced dyskinesias in a rat model of parkinsonism. The aim of the present study was to determine if the anti-dyskinetic effect of 7-NI was subject to tolerance after repeated treatment and if this drug could interfere with the priming effect of l-DOPA. Adult male Wistar rats (200-250 g) with unilateral depletion of dopamine in the substantia nigra compacta were treated with l-DOPA (30 mg/kg) for 34 days. On the 1st day, 6 rats received ip saline and 6 received ip 7-NI (30 mg/kg) before l-DOPA. From the 2nd to the 26th day, all rats received l-DOPA daily and, from the 27th to the 34th day, they also received 7-NI before l-DOPA. Animals were evaluated before the drug and 1 h after l-DOPA using an abnormal involuntary movement scale and a stepping test. All rats had a similar initial motor deficit. 7-NI decreased abnormal involuntary movement induced by l-DOPA and the effect was maintained during the experiment before 7-NI, median (interquartile interval), day 26: 16.75 (15.88-17.00); day 28: 0.00 (0.00-9.63); day 29: 13.75 (2.25-15.50); day 30: 0.5 (0.00-6.25); day 31: 4.00 (0.00-7.13), and day 34: 0.5 (0.00-14.63), Friedman followed by Wilcoxon test,vs day 26, P < 0.05;. The response to l-DOPA alone was not modified by the use of 7-NI before the first administration of the drug (l-DOPA vs time interaction, F1,10 = 1.5, NS). The data suggest that tolerance to the anti-dyskinetic effects of a neuronal nitric oxide synthase inhibitor does not develop over a short-term period of repeated administration. These observations open a possible new therapeutic approach to motor complications of chronic l-DOPA therapy in patients with Parkinson’s disease.

NAA and NAAG variation in neuronal activation during visual stimulation

N-acetyl-aspartyl-glutamate (NAAG) and its hydrolysis product N-acetyl-L-aspartate (NAA) are among the most important brain metabolites. NAA is a marker of neuron integrity and viability, while NAAG modulates glutamate release and may have a role in neuroprotection and synaptic plasticity. Investigating on a quantitative basis the role of these metabolites in brain metabolism in vivo by magnetic resonance spectroscopy (MRS) is a major challenge since the main signals of NAA and NAAG largely overlap. This is a preliminary study in which we evaluated NAA and NAAG changes during a visual stimulation experiment using functional MRS. The paradigm used consisted of a rest period (5 min and 20 s), followed by a stimulation period (10 min and 40 s) and another rest period (10 min and 40 s). MRS from 17 healthy subjects were acquired at 3T with TR/TE = 2000/288 ms. Spectra were averaged over subjects and quantified with LCModel. The main outcomes were that NAA concentration decreased by about 20% with the stimulus, while the concentration of NAAG concomitantly increased by about 200%. Such variations fall into models for the energy metabolism underlying neuronal activation that point to NAAG as being responsible for the hyperemic vascular response that causes the BOLD signal. They also agree with the fact that NAAG and NAA are present in the brain at a ratio of about 1:10, and with the fact that the only known metabolic pathway for NAAG synthesis is from NAA and glutamate.

Estragole blocks neuronal excitability by direct inhibition of Na+ channels

Estragole is a volatile terpenoid, which occurs naturally as a constituent of the essential oils of many plants. It has several pharmacological and biological activities. The objective of the present study was to investigate the mechanism of action of estragole on neuronal excitability. Intact and dissociated dorsal root ganglion neurons of rats were used to record action potential and Na+ currents with intracellular and patch-clamp techniques, respectively. Estragole blocked the generation of action potentials in cells with or without inflexions on their descendant (repolarization) phase (Ninf and N0 neurons, respectively) in a concentration-dependent manner. The resting potentials and input resistances of Ninf and N0 cells were not altered by estragole (2, 4, and 6 mM). Estragole also inhibited total Na+ current and tetrodotoxin-resistant Na+ current in a concentration-dependent manner (IC50 of 3.2 and 3.6 mM, respectively). Kinetic analysis of Na+ current in the presence of 4 mM estragole showed a statistically significant reduction of fast and slow inactivation time constants, indicating an acceleration of the inactivation process. These data demonstrate that estragole blocks neuronal excitability by direct inhibition of Na+ channel conductance activation. This action of estragole is likely to be relevant to the understanding of the mechanisms of several pharmacological effects of this substance.

Effect of hyperbaric oxygenation on mitochondrial function of neuronal cells in the cortex of neonatal rats after hypoxic-ischemic brain damage

The timing and mechanisms of protection by hyperbaric oxygenation (HBO) in hypoxic-ischemic brain damage (HIBD) have only been partially elucidated. We monitored the effect of HBO on the mitochondrial function of neuronal cells in the cerebral cortex of neonatal rats after HIBD. Neonatal Sprague-Dawley rats (total of 360 of both genders) were randomly divided into normal control, HIBD, and HIBD+HBO groups. The HBO treatment began immediately after hypoxia-ischemia (HI) and continued once a day for 7 consecutive days. Animals were euthanized 0, 2, 4, 6, and 12 h post-HI to monitor the changes in mitochondrial membrane potential (ΔΨm) occurring soon after a single dose of HBO treatment, as well as 2, 3, 4, 5, 6, and 7 days post-HI to study ΔΨm changes after a series of HBO treatments. Fluctuations in ΔΨm were observed in the ipsilateral cortex in both HIBD and HIBD+HBO groups. Within 2 to 12 h after HI insult, the ΔΨm of the HIBD and HIBD+HBO groups recovered to some extent. A secondary drop in ΔΨm was observed in both groups during the 1-4 days post-HI period, but was more severe in the HIBD+HBO group. There was a secondary recovery of ΔΨm observed in the HIBD+HBO group, but not in the HIBD group, during the 5-7 days period after HI insult. HBO therapy may not lead to improvement of neural cell mitochondrial function in the cerebral cortex in the early stage post-HI, but may improve it in the sub-acute stage post-HI.

Contribuição ao estudo da anatomia patológica do megaesôfago chagásico

Estudo anatomopatológico sistematizado foi feito em 56 esôfagos de chagásicos crônicos (17 com e 39 sem mega) e em 26 de não chagásicos com as seguintes finalidades: 1) avaliar as variações de calibre e espessura da parede do órgão; 2) analisar qualitativa e quantitativamente o plexo mientérico, na tentativa de avaliar a eventual relação entre suas lesões e o aparecimento de megaesôfago (ME); 3) estudar as lesões das musculares procurando verificar sua contribuição na gênese da visceromegalia; 4) pesquisar formas amastigotas do T. cruzi e sua possível relação com o processo inflamatorio; 5) identificar as principais alterações da mucosa. Confirmou-se que as lesões mais intensas localizavam-se na muscular própria e no plexo de Auerbach. Na primeira, as principais alterações foram miosite e fibrose e nos gânglios mientéricos observou-se inflamação e despopulação neuronal, maior nos esôfagos dos chagásicos com dilatação em relação aos sem dilatação e destes em relação aos controles. Entretanto, foram vistos esôfagos de calibre normal, com intensa denervação. Conclui-se que parecem ser múltiplos os fatores que desencadeiam a esofagopatia, especialmente, o ME. A pesquisa de parasitas em oito esófagos com mega e em oito sem ME foi positiva somente em quatro casos, do primeiro grupo. As lesões da mucosa e submucosa não parecem participar do processo.

Neurotoxic activity and ultrastructural changes in muscles caused by the brown widow spider Latrodectus geometricus venom

Brown widow spider (Latrodectus geometricus) venom (BrWSV) produces few local lesions and intense systemic reactions such as cramps, harsh muscle pains, nausea, vomiting and hypertension. Approximately 16 protein bands under reducing conditions and ~ 14 bands under non-reducing conditions on a 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis were observed. Neurotoxic clinical manifestations were confirmed in vivo, while proteolytic activity was demonstrated on gelatine film. Severe ultrastructural damages in mice skeletal muscles were observed at 3, 6, 12 and 24 h postinjection with at total of 45 µg of venom protein. Infiltration of eosinophils and ruptures of the cellular membranes were observed in the muscles along with swelling of the nuclear cover and interruption of the collagen periodicity. Altered mitochondrias and autophage vacuoles, nuclear indentation and mitochondria without cristae, slight increment of intermyofibrillar and subsarcolemic spaces and myelinic figures formation were also observed. In the capillary, endothelial membrane unfolding into the lumen was noticed; along with myelinic figures compatible with a toxic myopathy. Swollen sarcotubular systems with lysis of membrane, intense mitochondria autophagia and areas without pinocytic vesicles were observed. Swollen mitochondria surrounded by necrotic areas, myofibrillar disorganization and big vacuolas of the sarcotubular system, degenerated mitochondrium with formation of myelinic figure was seen. Glycogenosomes with small particulate, muscle type glycogen was noticed. Autophagic vacuole (autophagolysosomes) and necrotic areas were also noticed. These damages may be due to interactive effects of the multifactorial action of venom components. However, Latrodectus geometricus venom molecules may also be utilized as neuro therapeutic tools, as they affect neuronal activities with high affinity and selectivity. To our knowledge, the present study is the first ultrastructural report in the literature of muscle injuries and neurological and proteolytic activities caused by BrWSV.

Efects of plasma corticosteroid concentration on the osmotic threshold for vasopressin releasing in Chagas' disease

The osmotic threshold for vasopressin release was studied in normal patients (n = 7) and in patients with the chronic form of Chagas'disease (n = 11). Positive correlation between osmotic threshold and plasma cortisol concentration was obtained for the Controls (y1 = 273,30 + 0,75x i; r = 0,78;P < 0,05), suggesting a modulating effect of cortisol on vasopressin release. The lack of correlation between the two parameters for the chronic chagasic patients was interpreted, on the basis of the general denervation associated with Chagas ' disease, to be the result of neuronal destruction in hypothalamic and/or extrahypothalamic centers related to the secretory control of vasopressin.

Involvement of the autonomic nervous system in Chagas heart disease

The autonomic nervous system and especially the intracardiac autonomic nervous system is involved in Chagas' disease. Ganglionitis and periganglionitis were noted in three groups ofpatients dying with Chagas'disease: 1) Those in heart failure; 2) Those dying a sudden, non violent death and; 3) Those dying as a consequence ofaccidents or homicide. Hearts in the threegroups also revealed myocarditis and scattered involvement of intramyocardial ganglion cells as well as lesions of myelinic and unmyelinic fibers ascribable to Chagas'disease. In mice with experimentally induced Chagas' disease weobserved more intensive neuronal lesions of the cardiac ganglia in the acute phase of infection. Perhaps neuronal loss has a role in the pathogenesis of Chagas cardiomyopathy. However based on our own experience and on other data from the literature we conclude that the loss of neurones is not the main factor responsible for the manifestations exhibited by chronic chagasic patients. On the other hand the neuronal lesions may have played a role in the sudden death ofone group of patients with Chagas'disease but is difficult to explain the group of patients who did not die sudderly but instead progressed to cardiac failure.

Atividade colinesterásica no plasma e em eritrócitos de pacientes com as formas cardíaca e indeterminada da doença de Chagas

A atividade colinesterásica foi determinada no plasma e em eritrócitos de 10 indivíduos normais e em 26 pacientes chagásicos crônicos, sendo 6 com insuficiência cardíaca congestiva compensada, 10 com cardiopatia sem insuficiência cardíaca congestiva e 10 com a forma indeterminada. Os valores enzimáticos encontrados foram (média ±sd): 2196 ± 442 UI/L no plasma e 19,4 ± 3,3 Ul/g Hb em eritrócitos do grupo controla; 2291 ± 317 UI/L no plasma e 19,2 ±3,7 Ul/g Hb em eritrócitos dos chagásicos com insuficiência cardíaca congestiva compensada; 2445 ± 357 UI/L no plasma e 18,3 + 3,0 Ul/g Hb em eritrócitos dos cardiopatas chagásicos sem insuficiência cardíaca congestiva; 2006 ± 327 UI/L no plasma e 17,8 ± 3,1 Ul/g Hb em eritrócitos de chagásicos com a forma indeterminada. Nossos dados mostram que o comprometimento neuronal que ocorre nos cardiopatas chagásicos crônicos não é suficiente para levar a alterações significativas (p > 0,05) das atividades colinesterásicas no plasma e em eritrócitos.

Estudo necroscópico de um caso da forma crônica da doença de Chagas com eletrocardiograma e raio-X de tórax normais: referência especial ao sistema excito-condutor do coração

Descrevem-se as alterações anatomopatológicas observadas na necropsia de chagásico crônico, provavelmente, portador da forma indeterminada da doença. O coração mostrou lesões dos três folhetos e do sistema excito-condutor, traduzidas especialmente por pequenos focos inflamatórios. Havia discreta fibrose do miocárdio contrâtil, nódulo átrio-ventricular e origem do ramo esquerdo do feixe de His. Tais alterações, embora de leve intensidade, assemelham-se, qualitativamente, às relatadas nas demais formas crônicas da doença de Chagas e representam lesões ativas, com potencial evolutivo. No esôfago, observou-se entre outros fatos, despopulação neuronal acentuada.

Cardiopatia chagásica crônica causando insuficiência cardíaca congestiva na infância: estudo clínico e histopatológico de um caso, com ênfase para as lesões dos sistemas excito-condutor e nervoso autônomo intracardíaco

Descreve-se caso de cardiopatia chagásica em menino de nove anos, natural e procedente do sulde Goiás, que desenvolveu insuficiência cardíaca congestiva quatro meses antes do óbito. As reações sorológicaspara doença de Chagas eram reagentes, epositivo o xenodiagnóstico. Os eletrocardiogramas mostraram taquicardia sinusal, extra-sístoles ventriculares e supraventriculares, hemibloqueio anterior esquerdo, bloqueio completo do ramo direito e sinais de sobrecarga de câmaras. O exame ecocardiográfico evidenciou dilatação de câmaras com hipocontratilidade difusa. O quadro se agravou progressivamente, complicando-se por vários episódios pneumônicos, o último dos quais provocou o óbito. A necrópsia, verificou-se, no coração, inflama ção crônica dos três folhetos, com miocardite crônica fibrosante predominando no septo interventricular e no ventrículo esquerdo. As estruturas componentes do sistema excito-condutor mostraram processoflogístico crônico, essencialmente exsudativo, ora discreto, ora moderado. No sistema nervoso autônomo intracardíaco constataram-se focos esparsos de discreta periganglionite crônica, e raros fenômenos degenerativos dos neurônios sem despopulação neuronal.

Estudo dos neurônios do plexo cardíaco na infecção aguda pelo Trypanosoma cruzi em ratos albinos

O estudo histológico do plexo nervoso autonômico do coração de ratos albinos, na fase aguda da infecção pelo Trypanosoma cruzi, mostrou ganglionite, periganglionite e neurite de intensidades variáveis. Entretanto, a contagem dos neurônios dos gânglios não revelou redução (denervação) significativa dos mesmos.