The effect of sulforaphane on perinatal hypoxic-ischemic brain injury in rats.

Perinatal hypoxic-ischemic insult (HII) is one of the main devastating causes of morbidity and mortality in newborns. HII induces brain injury which evolves to neurological sequelae later in life. Hypothermia is the only therapeutic approach available capable of diminishing brain impairment after HII. Finding a novel therapeutic method to reduce the severity of brain injury and its consequences is critical in neonatology. The present paper aimed to evaluate the effect of sulforaphane (SFN) pre-treatment on glucose metabolism, neurodegeneration, and functional outcome at the acute, sub-acute, and sub-chronic time intervals in the experimental model of perinatal hypoxic-ischemic insult in rats. To estimate the effect of SFN on brain glucose uptake we have performed 18F-deoxyglucose (FDG) microCT/PET. The activity of FDG was determined in the hippocampus and sensorimotor cortex. Neurodegeneration was assessed by histological analysis of Nissl-stained brain sections. To investigate functional outcomes a battery of behavioral tests was employed. We have shown that although SFN possesses a protective effect on glucose uptake in the ischemic hippocampus 24 h and 1 week after HII, no effect has been observed in the motor cortex. We have further shown that the ischemic hippocampal formation tends to be thinner in HIE and SFN treatment tends to reverse this pattern. We have observed subtle chronic movement deficit after HII detected by ladder rung walking test with no protective effect of SFN. SFN should be thus considered as a potent neuroprotective drug with the capability to interfere with pathophysiological processes triggered by perinatal hypoxic-ischemic insult.

Microbiota affects the expression of genes involved in HPA axis regulation and local metabolism of glucocorticoids in chronic psychosocial stress.

The commensal microbiota affects brain functioning, emotional behavior and ACTH and corticosterone responses to acute stress. However, little is known about the role of the microbiota in shaping the chronic stress response in the peripheral components of the hypothalamus-pituitary-adrenocortical (HPA) axis and in the colon. Here, we studied the effects of the chronic stress-microbiota interaction on HPA axis activity and on the expression of colonic corticotropin-releasing hormone (CRH) system, cytokines and 11ß-hydroxysteroid dehydrogenase type 1 (11HSD1), an enzyme that determines locally produced glucocorticoids. Using specific pathogen-free (SPF) and germ-free (GF) BALB/c mice, we showed that the microbiota modulates emotional behavior in social conflicts and the response of the HPA axis, colon and mesenteric lymph nodes (MLN) to chronic psychosocial stress. In the pituitary gland, microbiota attenuated the expression of Fkbp5, a gene regulating glucocorticoid receptor sensitivity, while in the adrenal gland, it attenuated the expression of genes encoding steroidogenesis (MC2R, StaR, Cyp11a1) and catecholamine synthesis (TH, PNMT). The pituitary expression of CRH receptor type 1 (CRHR1) and of proopiomelanocortin was not influenced by microbiota. In the colon, the microbiota attenuated the expression of 11HSD1, CRH, urocortin UCN2 and its receptor, CRHR2, but potentiated the expression of cytokines TNFα, IFNγ, IL-4, IL-5, IL-6, IL-10, IL-13 and IL-17, with the exception of IL-1ß. Compared to GF mice, chronic stress upregulated in SPF animals the expression of pituitary Fkbp5 and colonic CRH and UCN2 and downregulated the expression of colonic cytokines. Differences in the stress responses of both GF and SPF animals were also observed when immunophenotype of MLN cells and their secretion of cytokines were analyzed. The data suggest that the presence of microbiota/intestinal commensals plays an important role in shaping the response of peripheral tissues to stress and indicates possible pathways by which the environment can interact with glucocorticoid signaling.

Morphine decreases social interaction of adult male rats, while THC does not affect it.

The aim of the present study was to compare effect of three low doses of morphine (MOR) and delta9-tetrahydrocannabinol (THC) on social behavior tested in Social interaction test (SIT). 45 min prior to testing adult male rats received one of the drugs or solvents: MOR (1; 2.5; 5 mg/kg); saline as a solvent for MOR; THC (0.5; 1; 2 mg/kg); ethanol as a solvent for THC. Occurrence and time spent in specific patterns of social interactions (SI) and non-social activities (locomotion and rearing) was video-recorded for 5 min and then analyzed. MOR in doses of 1 and 2.5 mg/kg displayed decreased SI in total. Detailed analysis of specific patterns of SI revealed decrease in mutual sniffing and allo-grooming after all doses of MOR. The highest dose (5 mg/kg) of MOR decreased following and increased genital investigation. Rearing activity was increased by lower doses of MOR (1 and 2.5 mg/kg). THC, in each of the tested doses, did not induce any specific changes when compared to matching control group (ethanol). However, an additional statistical analysis showed differences between all THC groups and their ethanol control group when compared to saline controls. There was lower SI in total, lower mutual sniffing and allo-grooming, but higher rearing in THC and ethanol groups than in saline control group. Thus, changes seen in THC and ethanol groups are seemed to be attributed mainly to the effect of the ethanol. Based on the present results we can assume that opioids affect SI more than cannabinoid.

Effect of 7-nitroindazole, a neuronal nitric oxide synthase inhibitor, on behavioral and physiological parameters.

The role of brain derived nitric oxide in the physiology and behavior remains disputable. One of the reasons of the controversies might be systemic side effects of nitric oxide synthase inhibitors. Therefore, under nNOS inhibition by 7-nitroindazole (7-NI) we carried out recordings of blood gasses, blood pressure and spontaneous EEG in conscious adult rats. Locomotion and spontaneous behavior were assessed in an open field. In addition skilled walking and limb coordination were evaluated using a ladder rung walking test. The blood gas analysis revealed a significant increase in pCO(2) 180 min and 240 min after the application of 7-NI. The power and entropy decreased simultaneously with a shift of the mean frequency of the spontaneous EEG toward slow oscillations after 7-NI treatment. The thresholds of evoked potentials underwent a significant drop and a trend towards a slight increase in the I-O curve slope was observed. 7-NI significantly suppressed open field behavior expressed as distance moved, exploratory rearing and grooming. As for the ladder rung walking test the 7-NI treated animals had more errors in foot placement indicating impairment in limb coordination. Therefore our findings suggest that 7-NI increased cortical excitability and altered some physiological and behavioral parameters.

Impacts of perinatal induced photothrombotic stroke on sensorimotor performance in adult rats.

Perinatal ischemic stroke is a leading cerebrovascular disorder occurring in infants around the time of birth associated with long term comorbidities including motor, cognitive and behavioral deficits. We sought to determine the impact of perinatal induced stroke on locomotion, behavior and motor function in rats. A photothrombotic model of ischemic stroke was used in rat at postnatal day 7. Presently, we induced two lesions of different extents, to assess the consequences of stroke on motor function, locomotion and possible correlations to morphological changes. Behavioral tests sensitive to sensorimotor changes were used; locomotion expressed as distance moved in the open field was monitored and histological changes were also assessed. Outcomes depicted two kinds of lesions of different shapes and sizes, relative to laser illumination. Motor performance of rats submitted to stroke was poor when compared to controls; a difference in motor performance was also noted between rats with small and large lesions. Correlations were observed between: motor performance and exposition time; volume ratio and exposition time; and in the rotarod between motor performance and volume ratio. Outcomes demonstrate that photothrombotic cerebral ischemic stroke induced in early postnatal period and tested in adulthood, indeed influenced functional performance governed by the affected brain regions.

Rebound increase in seizure susceptibility but not isolation-induced calls after single administration of clonazepam and Ro 19-8022 in infant rats.

The purpose of our study was to determine whether a single administration of anticonvulsant doses of two ligands of benzodiazepine receptors, clonazepam and Ro 19-8022, leads to development of rebound phenomena in immature 12-day-old rats. Three tests were used: pentylenetetrazole (PTZ)-induced seizures, isolation-induced ultrasonic vocalizations, and motor performance. Susceptibility to the convulsant effects of PTZ decreased 24 hours, but increased 48 hours, after clonazepam administration. Ultrasonic vocalizations were completely suppressed 30 minutes and 3 hours after clonazepam; a moderate inhibitory effect persisted even at 48 hours. Motor abilities were slightly compromised up to 3 hours. Similar effects of Ro 19-8022 on PTZ-induced seizures and ultrasonic vocalizations were observed 24 and 48 hours after administration; motor performance was not affected. Rebound proconvulsant effects followed different time courses after administration of the two benzodiazepine receptor ligands in developing animals. Anxiolytic-like effects of these drugs were still present at the time when animals exhibited rebound proconvulsant effects.

Postictal behavior after two types of cortical epileptic afterdischarges in rats.

OBJECTIVE: The aim of this study was to determine if behavioral patterns during the postictal state depend on the type of seizure. METHODS: Rhythmic electrical stimulation of sensorimotor cortex can elicit two types of epileptic afterdischarges (ADs) in adult rats: the spike-and-wave type accompanied by clonic convulsions, and a transition to a nonconvulsive type characterized by behavioral automatisms (mixed type). Rats in which stimulation did not induce ADs constituted the control group. The behavior of the animals was recorded for 20 minutes after the end of ADs or stimulation. RESULTS: Stimulation elicited only shortlasting minimal changes in control rats. Spike-and-wave ADs led to alternation of normal and unsteady walking, a decrease in normal sitting and lying (rigid lying appeared instead), and wet dog shakes (WDS). Mixed-type ADs abolished normal walking and sitting and induced unsteady walking, rigid lying, and a substantial number of WDS. CONCLUSIONS: Abnormal phenomena induced by the two types of ADs differ mostly quantitatively, but also qualitatively.

Anticonvulsant action of allopregnanolone in immature rats.

Anticonvulsant activity of allopregnanolone, a neurosteroid allosterically modulating GABA(A) receptor was tested in a model of motor seizures elicited by pentetrazol in immature rats. Rats 7, 12, 18, 25 or 90 days old were pretreated with allopregnanolone in doses from 5 to 40 mg/kg i.p. and 15 min later pentetrazol was injected subcutaneously in a dose of 100 mg/kg. Rats were observed in isolation for 30 min. Allopregnanolone dose-dependently suppressed both generalized tonic-clonic and minimal clonic seizures with the highest efficacy in 12-day-old rats. Anticonvulsant action was least expressed in adult animals. Duration of anticonvulsant action tested after a dose of 20 mg/kg in 12- and 90-day-old rats demonstrated markedly longer effects in young rats. Allopregnanolone compromised motor performance of rats but duration of this unwanted effect in 12-day-old rats was shorter than duration of anticonvulsant action. This difference can be important for possible clinical use of neurosteroids.

NMDA receptor antagonists impair motor performance in immature rats.

RATIONALE: Antagonists of NMDA receptors are excellent anticonvulsants in adult animals but serious side effects prevent their clinical use. The effects of two antagonists on motor performance were studied to find out if they develop in parallel with previously described anticonvulsant action. METHODS: Motor performance of 12-, 18- and 25-day-old rats was studied using a battery of tests (surface righting, negative geotaxis, bar holding and wire mesh ascending and three age-specific tests). A competitive NMDA antagonist CGP 40116 (0.1, 0.5 and/or 1 mg/kg IP) and a noncompetitive one dizocilpine (0.1, 0.5 and/or 1 mg/kg IP) were tested. RESULTS: Ten minutes after CGP 40116, the performance was compromised in all tests but there was negative geotaxis in all age groups. A decrease in efficacy with age was clearly demonstrated. Righting ability remained untouched in 25-day-old animals. Dizocilpine also influenced the performance in all tests but righting (compromised only in the youngest group) when studied 10 min after the injection. The relation to age was not so marked as with CGP 40116. When the tests were applied 4 h after dizocilpine administration the results were similar to those at 10-min interval. Twenty-four hours after dizocilpine only cliff avoidance exhibited prolonged latencies in 12-day-old rats but significant effects were observed in 18-day-old (negative geotaxis, bar holding and wire mesh ascending) as well as 25-day-old animals (bar holding, jumping down with choice). CONCLUSIONS: The acute effects of both NMDA antagonists studied decreased with age; this age-related change was more marked with CGP 40116 than with dizocilpine. In contrast, duration of dizocilpine effects did not exhibit a clear developmental tendency.

An animal model of nonconvulsive status epilepticus: a contribution to clinical controversies.

PURPOSE: To characterize electroencephalographic and behavioral effects as well as electrophysiologic and morphologic consequences of a subconvulsive dose of pilocarpine in lithium chloride-pretreated rats. METHODS: Pilocarpine (15 mg/kg) was administered intraperitoneally to adult rats pretreated with lithium chloride (3 mEq/kg, i.p.). Behavior was observed for 2 h and videotaped in three consecutive sessions. At the same time, EEG was recorded from the sensorimotor cortex and the dorsal hippocampus. Threshold intensities of currents necessary to elicit hippocampal afterdischarges were determined 24 h and 1 week after the pilocarpine administration. The brains were histologically examined 1 week after pilocarpine administration using Nissl stain. RESULTS: Pilocarpine induced time-limited nonconvulsive status epilepticus (NCSE). Epileptic EEG activity concurrent with prominent behavioral features was observed both in the neocortex and, predominantly, in the hippocampus. No changes in afterdischarge thresholds were observed in the dorsal hippocampus 24 h and 1 week after NCSE. One week after NCSE, seizure-related brain damage was found mainly in the motor neocortical fields. CONCLUSIONS: Pilocarpine-induced NCSE in rats strongly resembles a short-term human complex partial status epilepticus. Our animal model is suitable for studying the possible adverse effects of prolonged nonconvulsive seizures.

Nonconvulsive status epilepticus in rats: impaired responsiveness to exteroceptive stimuli.

An animal model of human complex partial status epilepticus induced by lithium chloride and pilocarpine administration was developed in our laboratory. The objective of the study was to provide a detailed analysis of both ictal and postictal behavior and to quantify seizure-related morphological damage. In order to determine the animal's responsiveness to either visual or olfactory stimuli, adult male rats were submitted to the following behavioral paradigms: the object response test, the social interaction test, and the elevated plus-maze test. The rotorod test was used to evaluate motor performance. Two weeks after status epilepticus, brains were morphologically examined and quantification of the brain damage was performed. Profound impairment of behavior as well as responsiveness to exteroceptive stimuli correlated with the occurrence of epileptic EEG activity. When the epileptic EEG activity ceased, responsiveness of the pilocarpine-treated animals was renewed. However, remarkable morphological damage persisted in the cortical regions two weeks later. This experimental study provides support for the clinical evidence that even nonconvulsive epileptic activity may cause brain damage. We suggest that the model can be used for the study of both functional and morphological consequences of prolonged nonconvulsive seizures.

The benzodiazepine receptor partial agonist Ro 19-8022 suppresses generalized seizures without impairing motor functions in developing rats.

The action of a partial benzodiazepine receptor agonist Ro 19-8022 [(R)-1-[(10-chloro-4-oxo-3-phenyl-4H-benzo[a]quinolizin-1-yl)carbo nyl] -2-pyrrolidine-methanol] in doses from 0.01 to 150 mg/kg was studied using motor seizures induced by pentylenetetrazol (PTZ) in rats 7, 12, 18, 25 and 90 days old. Generalized tonic-clonic seizures were suppressed in all age groups, the three youngest groups being more sensitive than older animals. The highest dose of Ro 19-8022 did not exhibit anticonvulsant action in the 25-day-old rats. The other types of seizures (minimal clonic) induced by PTZ were suppressed by Ro 19-8022 only in 18-day-old and older rats in which PTZ reliably elicited these types of seizures. The doses of 50, 100 and 150 mg/kg were ineffective against minimal seizures. On the contrary, incidence of minimal seizures was significantly increased by Ro 19-8022 in 7-and 12-day-old rat pups. Motor performance of rat pups was not compromised by Ro 19-8022 (0.5 and 50 mg/kg) in contrast to the full agonist midazolam (1 and/or 2 mg/kg). The duration of anticonvulsant effects studied with a dose of 0.5 mg/kg was longer in 12-day-old than in adult rats. This dose of Ro 19-8022 resulted in a rebound proconvulsant effect 24 and 48 h after the administration in 12-day-old rats only. Ro 19-8022 exhibited an excellent anticonvulsant action especially against generalized tonic-clonic seizures; this action was lost with very high doses. It did not compromise the motor system, but in some tests motivation was probably lost.

Behavioural effects of a subconvulsive dose of kainic acid in rats.

Kainic acid can induce a continuum of non-convulsive seizures characterised by epileptic automatisms and convulsive motor seizures depending on the dose. There are scarce data on the behavioural effects of low doses of kainate inducing only non-convulsive seizures. Therefore, we studied spontaneous behaviour of adult male rats using a method of positive habituation based on a detailed analysis of patterns and attention of animals to a stimulus object. Twenty-three animals were individually tested in the experimental arena on two consecutive days. Comparing the data from the first two exposures, a conspicuous habituation in all animals was observed. On experimental day 3, 12 rats received kainate (6 mg/kg intraperitoneally) and the remaining 11 animals received a physiological saline. After 1 h, animals were put into the arena with an object localised in the centre. It was found that both kainate and saline treated animals exhibited a significant increase in the total number of central area visits, and both the total and mean time spent in the vicinity of the object. However, the mean time spent was significantly shorter in kainate treated rats. Furthermore. kainate rats exhibited a significant decrease in rearing as compared with the controls. In addition, an epileptic automatism (wet dog shakes) was observed in seven out of 12 animals given kainate. The comparison of transition matrices between consecutive behavioural categories showed significant differences between the kainate and control groups. Our results demonstrate that a non-convulsive dose of kainate induced changes in the structure of spontaneous behaviour and impaired the processes related to maintenance of attention.

Non-NMDA receptor antagonist GYKI 52466 suppresses cortical afterdischarges in immature rats.

GYKI 52466 (1-(4-aminophenyl)-4-methyl-7,8-methylendioxy-5H-2,3-benzo-diaz epi ne), a non-competitive non-NMDA receptor antagonist, was tested against epileptic afterdischarges elicited by cortical stimulation in 12-, 18- and 25-day-old rats with implanted electrodes. Shortening of afterdischarges and a decrease in intensity of clonic movements accompanying both stimulation and afterdischarges were induced by the 20 mg/kg dose of GYKI 52466 in 18- and 25-day-old animals, whereas 12-day-old rat pups exhibited only shortening of electroencephalographic afterdischarges. The 10 mg/kg dose of GYKI 52466 did not significantly change afterdischarges in any age group. Motor skills were compromised after the 20 mg/kg dose of GYKI 52466. This effect was again more marked in 18- and 25-day-old animals than in the youngest group. In addition, anxiolytic-like action was observed in the jumping down test in 25-day-old rats. This effect was not influenced by a benzodiazepine antagonist flumazenil. On the contrary, the anticonvulsant action of GYKI 52466 was partly blocked by flumazenil, indicating thus multiple mechanisms of action of GYKI 52466.

Anticonvulsant action of 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline in immature rats: comparison with the effects on motor performance.

Anticonvulsant action of 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)quinoxaline (NBQX), a competitive antagonist at non-N-methyl-D-aspartate receptors for excitatory amino acids, was studied in a model of cortical epileptic afterdischarges (ADs) in 12-, 18- and 25-day-old rat pups with implanted electrodes. Electrical stimulation of sensorimotor cortex was repeated four times with 20-min intervals, NBQX (in doses of 10, 30, 60 or 90 mg/kg i.p.) or solvent (dimethyl sulfoxide, 1 ml/kg i.p.) were injected 10 min after the first afterdischarge. Dimethyl sulfoxide did not change the phenomena recorded; NBQX shortened ADs or at least blocked progressive prolongation observed under control conditions. Intensity of movements accompanying stimulation decreased after NBQX, and clonic movements accompanying ADs were suppressed in a dose-dependent manner. The highest dose of NBQX disabled the animals; therefore, the action of this drug on motor skills was studied in another group of animals. Even the dose of 30 mg/kg NBQX interfered with motor performance in 12- and 18-day-old rat pups, 25-day-old rat pups were more resistant to this action. NBQX exhibited only moderate antiepileptic action (suppression of progressive lengthening of ADs) at doses where unwanted side effects were absent.

Ontogeny of social behavior of pups of laboratory rats genetically selected for activity level.

In pups of Wistar laboratory rats genetically selected for high (+A) and low (-A) locomotor activity in an open field social behavior was tested in a situation of paired interactions in a novel environment. Tests were performed on postnatal Days 16, 19, 23, and 27. They consisted of four five-minute trials with 50 min, intertrial intervals. Duration of active contacts, passive contacts, mutual grooming, following (chasing), and playful fighting was recorded. In pups of the +A line total duration of all social interactions increased between Days 16 and 23, while in the -A pairs preweaning maximum in most types of social activities was achieved already on Day 16, after this day the time spent in mutual contacts decreased. Analysis of dynamics of social behavior over trials in the -A line showed the tendency to habituate, maximum duration of paired interactions appeared in the first trial contrary to +A groups, in which duration of social contacts tended to increase with repeated trials. In both lines, duration of following responses increased from Day 16 to Day 27. In +A pairs following often led to playful fighting, while in -A pairs fighting was practically absent. The experiment demonstrated that differences between lines +A and -A appeared in those social contacts which required higher energy expenditure and which realized themselves in the larger space of the testing box.

Spontaneous wheel running in laboratory rats genetically selected for activity level in a novel environment.

Wistar rats were genetically selected for either high (+A) or low (-A) locomotor activity level in a new environment. The response to the novelty was tested in all filial generations at the age between 75-80 days of life. Groups of 15 +A male rats selected from the 11th and 12th generations were placed in the individual cages connected with rotation wheels at the age 149 +/- 9 days. For a period of 32 days, the spontaneous wheel running activity was recorded for the time intervals 0.00-6.00 a.m., 6.00-12.00, 12.00-18.00, and 18.00-24.00 h. The -A group exhibited a generally low level of running activity over the entire experimental period, whereas the time of running in the +A tended to increase. Inside the +A group, homogeneous as to the short- term activity level, there gradually differentiated two sub-groups, one with relatively constant medium values and the other which showed an enormous prolongation of the time spent with running. The +A and -A groups differed also in their daily activity rhythm. Whereas the +A rats reached maximum of their activity between 18-24 h., followed by a rapid decrease in the morning hours, the -A animals showed a tendency to a later onset of night-type activity, and a higher level continued to morning hours.