Temporal pattern of Fos and Jun families expression after mitogenic stimulation with FGF-2 in rat neural stem cells and fibroblasts

Intense stimulation of most living cells triggers the activation of immediate early genes, such as Fos and Jun families. These genes are important in cellular and biochemical processes, such as mitosis and cell death. The present study focused on determining the temporal expression pattern of Fos and Jun families in fibroblasts and neural stem cells of cerebellum, hippocampus, and subventricular zone (SVZ) of rats of different ages at 0, 0.5, 1, 3, and 6 h after stimulation with fibroblast growth factor (FGF)-2. In neonates, a similar expression pattern was observed in all cells analyzed, with lower expression in basal condition, peak expression at 0.5 h after stimulation, returning to baseline values between 1 and 3 h after stimulation. On the other hand, cells from adult animals only showed Fra1 and JunD expression after stimulation. In fibroblasts and hippocampus, Fra1 reached peak expression at 0.5 h after stimulation, while in the SVZ, peak level was observed at 6 h after stimulation. JunD in fibroblasts presented two peak expressions, at 0.5 and 6 h after stimulation. Between these periods, the expression observed was at a basal level. Nevertheless, JunD expression in SVZ and hippocampus was low and without significant changes after stimulation. Differences in mRNA expression in neonate and adult animals characterize the significant differences in neurogenesis and cell response to stimulation at different stages of development. Characterizing these differences might be important for the development of cell cultures, replacement therapy, and the understanding of the physiological response profile of different cell types.

Modeling of post-traumatic epilepsy and experimental research aimed at its prevention

Research on the prevention of post-traumatic epilepsy (PTE) has seen remarkable advances regarding its physiopathology in recent years. From the search for biomarkers that might be used to indicate individual susceptibility to the development of new animal models and the investigation of new drugs, a great deal of knowledge has been amassed. Various groups have concentrated efforts in generating new animal models of traumatic brain injury (TBI) in an attempt to provide the means to further produce knowledge on the subject. Here we forward the hypothesis that restricting the search of biomarkers and of new drugs to prevent PTE by using only a limited set of TBI models might hamper the understanding of this relevant and yet not preventable medical condition.

Sleep pattern and learning in knockdown mice with reduced cholinergic neurotransmission

Impaired cholinergic neurotransmission can affect memory formation and influence sleep-wake cycles (SWC). In the present study, we describe the SWC in mice with a deficient vesicular acetylcholine transporter (VAChT) system, previously characterized as presenting reduced acetylcholine release and cognitive and behavioral dysfunctions. Continuous, chronic ECoG and EMG recordings were used to evaluate the SWC pattern during light and dark phases in VAChT knockdown heterozygous (VAChT-KDHET, n=7) and wild-type (WT, n=7) mice. SWC were evaluated for sleep efficiency, total amount and mean duration of slow-wave, intermediate and paradoxical sleep, as well as the number of awakenings from sleep. After recording SWC, contextual fear-conditioning tests were used as an acetylcholine-dependent learning paradigm. The results showed that sleep efficiency in VAChT-KDHET animals was similar to that of WT mice, but that the SWC was more fragmented. Fragmentation was characterized by an increase in the number of awakenings, mainly during intermediate sleep. VAChT-KDHET animals performed poorly in the contextual fear-conditioning paradigm (mean freezing time: 34.4±3.1 and 44.5±3.3 s for WT and VAChT-KDHET animals, respectively), which was followed by a 45% reduction in the number of paradoxical sleep episodes after the training session. Taken together, the results show that reduced cholinergic transmission led to sleep fragmentation and learning impairment. We discuss the results on the basis of cholinergic plasticity and its relevance to sleep homeostasis. We suggest that VAChT-KDHET mice could be a useful model to test cholinergic drugs used to treat sleep dysfunction in neurodegenerative disorders.

Protein tyrosine kinase inhibitors modify kainic acid-induced epileptiform activity and mossy fiber sprouting but do not protect against limbic cell death

Intrahippocampal administration of kainic acid (KA) induces synaptic release of neurotrophins, mainly brain-derived neurotrophic factor, which contributes to the acute neuronal excitation produced by the toxin. Two protein tyrosine kinase inhibitors, herbimycin A and K252a, were administered intracerebroventricularly, in a single dose, to attenuate neurotrophin signaling during the acute effects of KA, and their role in epileptogenesis was evaluated in adult, male Wistar rats weighing 250-300 g. The latency for the first Racine stage V seizure was 90 ± 8 min in saline controls (N = 4) which increased to 369 ± 71 and 322 ± 63 min in animals receiving herbimycin A (1.74 nmol, N = 4) and K252a (10 pmol, N = 4), respectively. Behavioral alterations were accompanied by diminished duration of EEG paroxysms in herbimycin A- and K252a-treated animals. Notwithstanding the reduction in seizure severity, cell death (60-90 percent of cell loss in KA-treated animals) in limbic regions was unchanged by herbimycin A and K252a. However, aberrant mossy fiber sprouting was significantly reduced in the ipsilateral dorsal hippocampus of K252a-treated animals. In this model of temporal lobe epilepsy, both protein kinase inhibitors diminished the acute epileptic activity triggered by KA and the ensuing morphological alterations in the dentate gyrus without diminishing cell loss. Our current data indicating that K252a, but not herbimycin, has an influence over KA-induced mossy fiber sprouting further suggest that protein tyrosine kinase receptors are not the only factors which control this plasticity. Further experiments are necessary to elucidate the exact signaling systems associated with this K252a effect.

Assessment of the progressive nature of cell damage in the pilocarpine model of epilepsy

Pilocarpine-induced (320 mg/kg, ip) status epilepticus (SE) in adult (2-3 months) male Wistar rats results in extensive neuronal damage in limbic structures. Here we investigated whether the induction of a second SE (N = 6) would generate damage and cell loss similar to that seen after a first SE (N = 9). Counts of silver-stained (indicative of cell damage) cells, using the Gallyas argyrophil III method, revealed a markedly lower neuronal injury in animals submitted to re-induction of SE compared to rats exposed to a single episode of pilocarpine-induced SE. This effect could be explained as follows: 1) the first SE removes the vulnerable cells, leaving behind resistant cells that are not affected by the second SE; 2) the first SE confers increased resistance to the remaining cells, analogous to the process of ischemic tolerance. Counting of Nissl-stained cells was performed to differentiate between these alternative mechanisms. Our data indicate that different neuronal populations react differently to SE induction. For some brain areas most, if not all, of the vulnerable cells are lost after an initial insult leaving only relatively resistant cells and little space for further damage or cell loss. For some other brain areas, in contrast, our data support the hypothesis that surviving cells might be modified by the initial insult which would confer a sort of excitotoxic tolerance. As a consequence of both mechanisms, subsequent insults after an initial insult result in very little damage regardless of their intensity.

Neo-Timm staining in the thalamus of chronically epileptic rats

The thalamus is an important modulator of seizures and is severely affected in cholinergic models of epilepsy. In the present study, chronically epileptic rats had their brains processed for neo-Timm and acetylcholinesterase two months after the induction of status epilepticus with pilocarpine. Both controls and pilocarpine-treated animals presented neo-Timm staining in the anterodorsal nucleus, laterodorsal nucleus, reticular nucleus, most intralaminar nuclei, nucleus reuniens, and rhomboid nucleus of the thalamus, as well as in the zona incerta. The intensity of neo-Timm staining was similar in control and pilocarpine-treated rats, except for the nucleus reuniens and the rhomboid nucleus, which had a lower intensity of staining in the epileptic group. In animal models of temporal lobe epilepsy, zinc seems to modulate glutamate release and to decrease seizure activity. In this context, a reduction of neo-Timm-stained terminals in the midline thalamus could ultimately result in an increased excitatory activity, not only within its related nuclei, but also in anatomical structures that receive their efferent connections. This might contribute to the pathological substrate observed in chronic pilocarpine-treated epileptic animals.

c-Fos expression induced by electroacupuncture at the Zusanli point in rats submitted to repeated immobilization

In laboratory animals, acupuncture needs to be performed on either anesthetized or, if unanesthetized, restrained subjects. Both procedures up-regulate c-Fos expression in several areas of the central nervous system, representing therefore a major pitfall for the assessment of c-Fos expression induced by electroacupuncture. Thus, in order to reduce the effect of acute restraint we used a protocol of repeated restraint for the assessment of the brain areas activated by electroacupuncture in adult male Wistar rats weighing 180-230 g. Repeated immobilization protocols (6 days, 1 h/day and 13 days, 2 h/day) were used to reduce the effect of acute immobilization stress on the c-Fos expression induced by electroacupuncture at the Zusanli point (EA36S). Animals submitted to immobilization alone or to electroacupuncture (100 Hz, 2-4 V, faradic wave) in a non-point region were compared to animals submitted to electroacupuncture at EA36S (4 animals/subgroup). c-Fos expression was measured in 41 brain areas by simple counting of cells and the results are reported as number of c-Fos-immunoreactive cells/10,000 æm². The protocols of repeated immobilization significantly reduced the immobilization-induced c-Fos expression in most of the brain areas analyzed (P < 0.05). Animals of the EA36S groups had significantly higher levels of c-Fos expression in the dorsal raphe nucleus, locus coeruleus, posterior hypothalamus and central medial nucleus of the thalamus. Furthermore, the repeated immobilization protocols intensified the differences between the effects of 36S and non-point stimulation in the dorsal raphe nucleus (P < 0.05). These data suggest that high levels of stress can interact with and mask the evaluation of specific effects of acupuncture in unanesthetized animals.

Effect of the acupoints ST-36 (Zusanli) and SP-6 (Sanyinjiao) on intestinal myoelectric activity of Wistar rats

Despite its ancient use as a therapeutic tool to treat several ailments, acupuncture still faces the challenge of scrutiny by Western science both in terms of its efficacy and in terms of the characterization of its effects and mechanisms of actions underlying these effects. We investigated under well-controlled and carefully characterized conditions the influence of electrical stimulation of acupuncture points ST-36 (Zusanli) and SP-6 (Sanyinjiao) on the myoelectric activity of the small intestine of 38 adult male Wistar rats. Electrical recordings obtained by means of four electrodes chronically implanted in the small intestine were used to assess the effects of acupuncture (electroacupuncture stimulation set at 2 Hz, intermittent stimulation, 1 V, for 30 min). Immobilization of the animals was associated with a consistent decrease (-8 ± 7 percent) in the myoelectric activity of the small intestine as measured by means of the root mean square. Conversely, acupuncture was able to significantly increase (overshoot) this activity compared to baseline (+44 ± 7 percent). In contrast, immobilized animals subjected to sham acupuncture had only modest (nonsignificant) increases in myoelectric activity (+9 ± 6 percent). Using carefully controlled conditions we confirmed previous noncontrolled studies on the ability of acupuncture to alter intestinal motility. The characterization of the topographic and temporal profiles of the effects observed here represents a basis for future dissection of the physiological and pharmacological systems underlying these effects

Effects of acupuncture on behavioral, cardiovascular and hormonal responses in restraint-stressed Wistar rats

Stress is a well-known entity and may be defined as a threat to the homeostasis of a being. In the present study, we evaluated the effects of acupuncture on the physiological responses induced by retraint stress. Acupunture is an ancient therapeutic technique which is used in the treatment and prevention of diseases. Its proposed mechanisms of action are based on the principle of homeostasis. Adult male Wistar EPM-1 rats were divided into four groups: group I (N=12), unrestrained rats with cannulas previously implanted into their femoral arteries for blood pressure and heart rate measurements; group II (N=12), rats that were also cannulated and were submitted to 60-min immobilization; group III (N=12), same as group II but with acupuncture needles implanted at points SP6, S36, REN17, P6 and DU20 during the immobilization period; group IV (N=14), same as group III but with needles implanted at points not related to acupuncture (non-acupoints). During the 60-min immobilization period animals were assessed for stress-related behaviors, heart rate, blood pressure and plasma corticosterone, noradrenaline and adrenaline levels. Group III animals showed a significant reduction (60 percent on average, P<0.02) in restraint-induced behaviors when compared to groups II and IV. Data from cardiovascular and hormonal assessments indicated no differences between group III and group II and IV animals, but tended to be lower (50 percent reduction on average) in group I animals. We hypothesize that acupuncture at points SP6, S36, REN17, P6 and DU20 has an anxiolytic effect on restraint-induced stress that is not due to a sedative action.