Ultracytochemical demonstration of soluble guanylate cyclase activation in rat aorta by NCX4016, a NO-releasing aspirin derivative.

Biochemical studies demonstrate that the NO-releasing-aspirin derivative (NCX4016) stimulates soluble guanylate cyclase (sGC) activity and increases cyclic GMP (cGMP) in human platelet and monocytes by releasing NO. In the present study, an ultracytochemical technique for electron microscopy was used to investigate the effects of NCX4016 (2 mM) on sGC activity in rat thoracic aorta, using sodium nitroprusside (0.01 mM) as reference NO-donor. Guanylyl-imidodiphosphate sodium salt [Gpp(NH)p], a synthetic non-hydrolyzable analogue of GTP, was used as sGC substrate. NO-activated sGC released imidodiphosphate ions which were precipitated with lead ions, giving rise to deposits of electron-dense granules (reaction product). Ultracytochemistry allowed us to demonstrate that NCX4016 stimulated sGC activity in smooth muscle cells, and particularly in vascular endothelial cells, as sodium nitroprusside did. This result could explain the protective effects of chronic treatment with NCX4016 on aortic endothelium of diabetic rats demonstrated by scanning and transmission electron microscopy.

Ultrastructural investigations on protective effects of NCX 4016 (nitroaspirin) on macrovascular endothelium in diabetic Wistar rats.

The effect of a nitric oxide-donating aspirin derivative, 2-acetoxy-benzoate 3-(nitroxy-methyl)phenyl ester (NCX 4016), and aspirin on the aortic endothelium of diabetic rats was investigated by using scanning and transmission electron microscopy. Control and streptozotocin-treated rats were used. Metabolic control was assessed by measuring blood and urine metabolites, and 24-h urine volume. The ultrastructural study was performed after 7 weeks of diabetes and 6 weeks of therapy. Streptozotocin treatment induced a persistent hyperglycemia which was not influenced by the pharmacological treatments. Values of blood metabolites were in line with the diabetic status. Both scanning and transmission electron microscopy revealed that aortic endothelium was severely damaged in all diabetic rats except for the NCX 4016 treated ones. Our data document the protective effects of NCX 4016 on the vascular endothelium of diabetic rats. Since aspirin had no protective action, NCX 4016 may have exerted its beneficial action by releasing nitric oxide.

Induction of cerebellar hsp72 in rats learning a two-way active avoidance task.

The involvement of brain heat shock proteins in learning was examined by Western analyses in rats trained for an active avoidance task, and in passive and active controls. Expression of the constitutive hsp73 was intense in brain, liver, and kidney of all rats. Conversely, expression of the inducible hsp72 occurred in the cerebellum of most trained rats, but not in passive or active controls. Significant correlations were present between avoidances and cerebellar scores determined 8 h after training. Induction of hsp72 may therefore be attributed to learning in the cerebellum, while in other brain regions, liver and kidney stress-related stimuli may play a prevalent role.

The sequential hypothesis of the function of sleep.

In addition to modulatory roles concerning bodily functions, sleep is assumed to play a main processing role with regard to newly acquired neural information. Elaboration of memory traces acquired during the waking period is assumed to require two sequential steps taking place during slow wave sleep (SWS) and eventually during paradoxical sleep (PS). This view is suggested by several considerations, not the least of which concerns the natural sequence of appearance of SWS and PS in the adult animal. While the involvement of PS in memory processing is well documented, the involvement of SWS is supported by the results of baseline and post-trial EEG analyses carried out in rats trained for a two-way active avoidance task or a spatial habituation task. Together with control analyses, these data indicate that the marked increase in the average duration of post-trial SWS episodes does not reflect the outcome of non-specific contingent factors, such as sleep loss or stress, but is related to memory processing events. Several considerations have furthermore led to the proposal that, during SWS, after a preliminary selection step, the first processing operation consists in the weakening of non-adaptative memory traces. The remaining memory traces would then be stored again under a better configuration during the ensuing PS episode. This view is in agreement with several relevant features of sleep, including the EEG waveforms prevailing during SWS and PS, as well as the ontogenetic sequence of appearance of SWS and PS. Some theoretical considerations on the role of sleep are also in agreement with the sequential hypothesis. More recent data indicate that the learning capacity of rats is correlated with several baseline EEG features of sleep and wakefulness. They include the average duration of PS episodes and of SWS episodes followed by wakefulness (longer in fast learning rats), and the waking EEG power spectrum of fast learning rats whose output is more balanced in the frequency range below 10 Hz than in slow learning and in non-learning rats. Additional EEG data suggest that fast learning rats may accomplish 'on line' processing of newly acquired information according to a sequence of events not dissimilar from the one proposed by the sequential hypothesis.

Nitro-aspirin (NCX4016) reduces brain damage induced by focal cerebral ischemia in the rat.

The potential neuroprotective effects of the novel nitro-derivate of aspirin (NCX4016) on permanent focal cerebral ischemia in spontaneously hypertensive rats (SHRs) was investigated. Reference compounds were acetylsalicilic acid (ASA) and FK506 (tacrolimus). Ten minutes after surgery, SHRs were randomly divided into four groups of ten, pharmacologically treated and sacrificed 24 h after treatment. Brains were removed and processed to measure infarct volume, 70 kDa heat shock protein (hsp70), glial fibrillary acidic protein (GFAP) and vimentin (Vim) immunoreactivity (IR), and apoptosis using terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end-labeling (TUNEL) assay. NCX-4016 significantly reduced total infarct volume compared to ASA (-20%, P < 0.05), FK506 (-18%, P < 0.05) and vehicle treatment (-20%, P < 0.05). Experimental groups did not differ in hsp70-IR and GFAP-IR. Conversely, hyperplastic astrocytes, measured by Vim-IR, were significantly lower in NCX-4016 than in the vehicle group (-36%, P<0.01). TUNEL assay indicated a significantly lower degree of apoptosis in NCX-4016 group than vehicle in both the homolateral (-27%, P < 0.01) and contralateral hemisphere (-29%, P < 0.05). These findings indicate that NO release associated with aspirin confers neuroprotective effects against ischemic injury.

Sleep fragmentation, and changes in locomotor activity and body temperature in trypanosome-infected rats.

The rest-activity and body temperature 24 h cycles, as well as the structure of spontaneous sleep, were studied in rats 3 weeks after infection with monomorphic Trypanosoma brucei brucei. This parasite belongs to the species of trypanosomes that causes in humans African sleeping sickness, a neuropsychiatric syndrome that involves alterations of endogenous biological rhythms. In the infected rats, entrained to a 12 h:12 h photoperiod, a considerable hypokinesia was detected during the hours of darkness. A significant oscillation of the body temperature during 24 h was lost in some infected animals. In the other infected animals, the body temperature cycle displayed a lower amplitude and a phase advance. The mean temperature was slightly higher in the infected than in control rats during the period of light. A detailed analysis of the structure of spontaneous sleep, based on daytime electroencephalographic recordings, revealed during trypanosome infection an increased relative proportion of wake, and a decreased percent value of synchronized sleep. A marked reduction of the mean REM latency and a fragmented pattern of synchronized sleep, resulting in a considerable alteration of the REM-non-REM sleep sequences, were also observed in the infected animals. These findings indicate that trypanosomiasis in the rat results in a striking sleep fragmentation, as well as in changes of locomotor activity and body temperature rhythm. Thus, trypanosome infection in the rat provides an experimental model of sleep dysregulation in a structurally intact brain, and may provide an animal model of endogenous rhythm changes documented in African sleeping sickness.

Effect of sleep on cerebral DNA synthesized during shuttle-box avoidance training.

Female Wistar rats weighing 200 g were implanted with cortical electrodes and two intraventricular cannulae. Five days later they were given 3H-thymidine and exposed to shuttle-box training for four hours. They were then left free to sleep in the following three hours during which their EEG activity was recorded. In comparison with control animals (C), learning (L) and non-learning (NL) rats exhibited an increase in SS. In comparison to the EEG recording made the previous day, all animal groups displayed an increase in SS, but only NL rats suffered a decrease in PS(%). The specific radioactivity of DNA measured in several brain regions was tendentially lower in NL rats, but significance was achieved only in the cerebellum in the comparison between NL rats and C rats. No change occurred in liver. More marked and significant decrements in the DNA specific radioactivity of all brain regions were observed in the subgroup of NL rats displaying relatively high values of PS time in comparison to the analogous subgroups of C and L animals. Comparable decrements were present with regard to the subgroup of NL rats endowed with relatively low PS time. Less widespread and more limited changes were observed in the concentration of acid-soluble radioactivity. In addition, several significant correlations were detected by Spearman's analysis among behavioral, biochemical and sleep parameters. The results are consistent with the interpretation that the selective decrease in brain radioactive DNA observed in NL rats reflects a loss of DNA synthesized during the training period. The loss is related to the amount of post-training PS and is associated to a lengthening of the mean duration of PS episodes. It may be concluded that the loss of newly-synthesized brain DNA reflects the elimination of molecules associated with neural information devoid of adaptive value.

The sequential hypothesis of sleep function. III. The structure of postacquisition sleep in learning and nonlearning rats.

EEG methods were used to examine the structure of postacquisition sleep in learning (L) and nonlearning (NL) rats previously exposed to a session of two-way active avoidance training, and in control rats (C) left in their home cages. In agreement with literature data, the number and total amount of paradoxical sleep (PS) episodes were higher in L rats than in NL rats. In addition, significant differences between L and NL rats concerned the episodes of synchronized sleep followed by wakefulness or by PS (SS-W and SS-PS, respectively). The average duration and related parameters of SS-W episodes, and the average duration, number, amount and related parameters of SS-PS episodes increased in NL and L rats in comparison with C rats. Longer SS-W episodes occurred early in NL and L rats, but the effect lasted longer in NL rats. On the other hand, the increments concerning SS-PS episodes occurred earlier, were more pronounced and laster longer in L rats. The results support a role of SS in brain information processing, as envisaged by the sequential hypothesis on the role of sleep. They suggest, furthermore, that memory traces lacking adaptive value may be destabilized and cleared away during SS-W and SS-PS episodes, while the remaining memory traces may be retained and eventually stored again in more integrated form during SS-PS and PS episodes, respectively.

The sequential hypothesis of sleep function. IV. A correlative analysis of sleep variables in learning and nonlearning rats.

Female adult rats were trained for a two-way active avoidance task (4 h), and allowed free sleep (3 h). Control rats (C) were left in their home cages during the acquisition period. Dural electrodes and an intraventricular cannula, implanted one week in advance, were used for EEG recording during the period of sleep and for the injection of [3H]thymidine at the beginning of the training session, respectively. Rats were killed at the end of the sleep period, and the DNA-specific activity was determined in the main brain regions and in liver. Correlations among sleep, behavioral and biochemical variables were assessed using Spearman's nonparametric method. In learning rats (L), the number of avoidances was negatively correlated with SS-W variables, and positively correlated with SS-PS variables (episodes of synchronized sleep followed by wakefulness or paradoxical sleep, respectively) and with PS variables. An inverse pattern of correlations was shown by the number of escapes or freezings. No correlations occurred in rats unable to achieve the learning criterion (NL). In L rats, the specific activity of brain DNA was negatively correlated with SS-W variables and positively correlated with SS-PS variables, while essentially no correlation concerned PS variables. On the other hand, in NL rats, comparable correlations were positive with SS-W variables and negative with SS-PS and PS variables. Few and weak correlations occurred in C rats. The data support a role of SS in brain information processing, as postulated by the sequential hypothesis on the function of sleep. In addition, they suggest that the elimination of nonadaptive memory traces may require several SS-W episodes and a terminal SS-PS episode. During PS episodes, adaptive memory traces cleared of nonadaptive components may be copied in more suitable brain sites.

Post-trial sleep in old rats trained for a two-way active avoidance task.

Nine male Wistar rats aged 27 months were trained for a two-way active avoidance task and tested for retention the following day. At variance with young adult rats, most of which succeed in mastering the task, all old rats displayed a large majority of freezing responses throughout the training and the retention sessions, thereby confirming the condition of learning impairment of aged rats. Comparison of baseline and post-trial sleep indicated the presence of a transient, but marked, increment in the average duration and total amount of post-trial slow-wave sleep followed by waking, and of a decrease in total amount of quiet waking. On the other hand, variables of paradoxical sleep and of slow-wave sleep followed by paradoxical sleep or by transition sleep did not show significant variations. Because these sleep variables are known to undergo significant variations in learning in young adult rats, the present data confirm that the latter effects are related to memory-processing events rather than to nonspecific effects of training. An additional outcome of training consisted in a marked post-trial decrement in the number of spike-wave discharges, which are known to occur in old rats during periods of quiet waking.

Sequential hypothesis of sleep function. V. Lengthening of post-trial SS episodes in reminiscent rats.

Rats failing to learn a two-way active avoidance task during the training session were tested for performance the following day. One group of rats maintained its low level of avoidances (non improving or NI rats), while the remaining rats dramatically improved their avoidance score (improving or I rats). EEG recording during the posttrial period demonstrated significant variations in the sleep structure of I rats, in comparison with NI rats. The main change consisted in an increase in the average duration of the episodes of slow wave sleep followed by wakefulness or by paradoxical sleep. These variations occurred in the third hour of the posttrial period, while an increment in the amount of PS was observed in the sixth hour. In I rats, but not in NI rats, comparable variations emerged from the comparison of baseline sleep (determined the day before training) with posttrial sleep. The data are in agreement with the main postulate of the sequential hypothesis of sleep function which attributes a primary role to slow wave sleep in the processing of newly acquired memories.

The sequential hypothesis on sleep function. I. Evidence that the structure of sleep depends on the nature of the previous waking experience.

The sequential hypothesis on sleep function assumes that the information gathered by brain during the waking period is processed during sleep in two main steps occurring during synchronized sleep (SS) and, eventually, during paradoxical sleep (PS). To verify the main consequences of the hypothesis, i.e., (1) that SS is involved in brain information processing; and (2) that the structure of sleep is dependent on the nature of the previous waking experience, an experiment was designed involving rats exposed to a training session (two-way active avoidance) but failing to learn (NL), and rats left in their home cages in the same training room (C). The structure of sleep, determined by EEG techniques in the postacquisition period (3 hr), was different in NL rats in comparison to C rats, chiefly because SS episodes were markedly longer in the former group. A more detailed analysis indicated that, in NL rats, SS episodes not followed by PS increased their duration first, while those followed by PS became longer in the second half of the sleep period. Comparable results were obtained in the comparison of NL and C subgroups deprived of PS at the end of the acquisition period by chlomipramine treatment. The data support the sequential hypothesis and provide evidence for a primary role of SS in brain information processing.

The sequential hypothesis on sleep function. II. A correlative study between sleep variables and newly synthesized brain DNA.

The information acquired by brain during wakefulness (W) may be processed in two sequential steps occurring during synchronized sleep (SS) and paradoxical sleep (PS), respectively. On the assumption that brain molecules synthesized during the acquisition step undergo a comparable sleep processing, we have designed an experiment aimed at the verification of the sequential hypothesis. Groups of adult female Wistar rats received [3H-methyl] thymidine by intraventricular injection 30 min before being exposed to a 4 hr session of a two-way active avoidance training. Animals failing to achieve the learning criterion were further allowed a period of 3 hr during which they were left free to sleep, or were deprived of PS or of total sleep. Control rats were similarly treated, but were left in their home cages in the same training room during the period of acquisition. The results of correlative study among behavioral, sleep and biochemical variables demonstrate that the specific radioactivity of DNA in the cerebral cortex, cerebellum and brainstem is correlated with several variables of postacquisition sleep, mostly SS parameters. The correlations depend on the previous waking experience of the rats. The data substantiate the two main consequences of the hypothesis, i.e., (1) the involvement of SS in brain information processing; and (2) the dependence of the operations performed by the sleeping brain on the nature of the previous waking experience. The results also provide some insight into the kind of processing which occurs in the sleeping brain.

Synthesis of brain DNA during acquisition of an active avoidance task.

The possible involvement of cerebral DNA synthesis in the learning process was examined in rats injected intracerebrally with 3H thymidine. During the period of incorporation (4.5 hr) one rat was trained to an active avoidance task while a second animal was kept in the same experimental room. In comparison with control rats paired to learning animals, the concentration of PCA-soluble radioactivity and of radioactive DNA of the cerebral cortex increased in all animal groups, i.e., control rats paired to non-learning animals, learning rats and non-learning rats. No change occurred in liver. In the cerebral cortex the slope of the regression line obtained by plotting the concentration of radioactive DNA versus the concentration of PCA-soluble radioactivity was lower in learning rats than in the group of pooled control animals. A comparable effect was noted in the hippocampus. In non-learning animals a similar decrease was present in the cerebral cortex and in cerebellum. In addition, it was found that in learning animals the percent incorporation was inversely related to the total number of avoidances only in the cerebral cortex. In non-learning rats a similar inverse relationship was present in the cerebral cortex and in cerebellum. In the former region the regression line of learning rats was shifted upwards in comparison with the regression line of non-learning animals. These results are interpreted to indicate that the incorporation of 3H-thymidine into cerebral DNA is directly related to the level of stress and is increased by learning.

Sleep-wake variables and EEG power spectra in Mongolian gerbils and Wistar rats.

Using electroencephalographic methods (EEG), we have analyzed the basal sleep structure and the EEG power spectra of gerbils and rats during periods of wakefulness (W), synchronized sleep (SS) and paradoxical sleep (PS). During the 6 hr light period examined, duration of sleep was similar for rats and gerbils, but gerbils showed fewer PS episodes and a longer amount of SS episodes followed by wakefulness. In addition, SS episodes preceding PS were of longer duration in gerbils than in rats. EEG power spectral analysis indicated a higher relative output in the 1-4 Hz range in gerbils in comparison with rats. On the whole, the data indicate the existence of significant differences in the basal sleep structure and EEG power spectra of gerbils and rats. This background information might be useful in the comparison of the effects of a given experimental treatment, such as cerebral ischemia, on the EEG activity of these two animal species.

Effects of light physical exercise on sleep regulation in rats.

PURPOSE: Acute physical exercise is known to enhance slow-wave sleep (SWS) and reduce paradoxical sleep (PS) in humans. In this study, we examined the effects of moderate physical exercise on sleep in rats. METHOD: Young adult Wistar rats underwent a 4-h baseline electroencephalographic (EEG) recording session. The following day, they were induced to walk (0.8 m x min(-1)) or run (4 m x min(-1)) for 45 min in a rota-rod treadmill. Active control rats (ACR) were placed on the locked rota-rod for 45 min, whereas passive control rats (PCR) remained in their home cages. They were then left free to sleep for 4 h during which EEG activity was recorded. Rectal temperature (Tre) was monitored before and after exercise in ACR, walking and running rats (WR and RR, respectively) and at 45 min intervals in PCR. RESULTS: WR were able to walk for 45 min consecutively whereas in RR performances differed. Posttraining Tre was unchanged in ACR, PCR, and WR and resulted about 1.8 degrees C above baseline in RR. In both WR and RR after exercise i) length of SWS and PS, ii) intensity of SWS (spectral power density in 1-4 Hz range), and iii) propensity for falling asleep were enhanced. Interestingly, there was a more conspicuous increment in PS than SWS. In ACR and PCR there were no changes in sleep. CONCLUSIONS: Due to the complexity of sleep regulation, the interaction of several factors might underlie the observed increment in SWS and PS. Nevertheless, it is interesting that light physical exercise favors sleep and above all a harmonic enhancement of both sleep phases.

Induction of heat shock protein 70 reduces the alteration of striatal electrical activity caused by mitochondrial impairment.

Since mild thermal stress seems to exert neuroprotection via induction of heat-shock protein 70 kDa (hsp70), we tested whether hsp70 would preserve striatal bioelectrical activity under conditions of mitochondrial impairment. Corticostriatal slices from rats that had undergone mild thermal stress were exposed to either rotenone or 3-nitropropionic acid (3-NP), that selectively inhibits mitochondrial complex I and complex II, respectively. Rotenone is utilized to obtain an experimental model of Parkinson's disease while 3-NP replicates Huntington's disease phenotype in experimental animals. The cerebral hsp70 increase did not alter field potential amplitude of the slices but partially protected them against rotenone-induced neurotoxicity. Similarly, induction of hsp70 had also a partial neuroprotective effect on the neurotoxicity caused by 3-NP on striatal field potential. Since rotenone and 3-NP treatments mimic the mitochondrial impairment and oxidative stress that contribute to development of Parkinson's disease and Huntington's disease, these data suggest that induction of hsp70 might represent a possible neuroprotective mechanism against the pathophysiological chain of events implicated in these neurodegenerative disorders.