Sex steroid hormones in depressive disorders as a basis for new potential treatment strategies.

The sex steroid hormones (SSHs) such as testosterone, estradiol, progesterone, and their metabolites have important organizational and activational impacts on the brain during critical periods of brain development and in adulthood. A variety of slow and rapid mechanisms mediate both organizational and activational processes via intracellular or membrane receptors for SSHs. Physiological concentrations and distribution of SSHs in the brain result in normal brain development. Nevertheless, dysregulation of hormonal equilibrium may result in several mood disorders, including depressive disorders, later in adolescence or adulthood. Gender differences in cognitive abilities, emotions as well as the 2-3 times higher prevalence of depressive disorders in females, were already described. This implies that SSHs may play a role in the development of depressive disorders. In this review, we discuss preclinical and clinical studies linked to SSHs and development of depressive disorders. Our secondary aim includes a review of up-to-date knowledge about molecular mechanisms in the pathogenesis of depressive disorders. Understanding these molecular mechanisms might lead to significant treatment adjustments for patients with depressive disorders and to an amelioration of clinical outcomes for these patients. Nevertheless, the impact of SSHs on the brain in the context of the development of depressive disorders, progression, and treatment responsiveness is complex in nature, and depends upon several factors in concert such as gender, age, comorbidities, and general health conditions.

Perinatal hypoxic-ischemic damage: review of the current treatment possibilities.

Neonatal hypoxic-ischemic encephalopathy is a disorder with heterogeneous manifestation due to asphyxia during perinatal period. It affects approximately 3-12 children per 1000 live births and cause death of 1 million neonates worldwide per year. Besides, motor disabilities, seizures, impaired muscle tone and epilepsy are few of the consequences of hypoxic-ischemic encephalopathy. Despite an extensive research effort regarding various treatment strategies, therapeutic hypothermia with intensive care unit supportive treatment remains the only approved method for neonates who have suffered from moderate to severe hypoxic-ischemic encephalopathy. However, these protocols are only partially effective given that many infants still suffer from severe brain damage. Thus, further research to systematically test promising neuroprotective treatments in combination with hypothermia is essential. In this review, we discussed the pathophysiology of hypoxic-ischemic encephalopathy and delved into different promising treatment modalities, such as melatonin and erythropoietin. However, preclinical studies and clinical trials are still needed to further elucidate the mechanisms of action of these modalities.

Neonatal hypoxic-ischemic brain injury leads to sex-specific deficits in rearing and climbing in adult mice.

The study examined the morphological and long-term behavioral impacts of neonatal hypoxic-ischemic brain injury in a mouse model. We investigated the modification of different behavioral domains, such as spontaneous climbing, which represents fine motor skills. We also focused on sex-dependent differences during hypoxic-ischemic encephalopathy. The Rice-Vannucci model of hypoxia-ischemia was used, adjusted and adapted to 7-day-old C57BL/6NTac mice. The effects of induced hypoxia and ischemia were also studied separately. At postnatal day 60, mice underwent behavioral testing using the LABORAS apparatus. The perfusion for histological evaluation was performed one day after the behavioral analyses. In groups with separately induced hypoxia or ischemia, the observed alterations in behavior were not accompanied by morphological changes in the cortex or hippocampal formation. Female mice naturally climbed significantly more and hypoxic females reared less than hypoxic males (p<0.05). Male mice postnatally exposed to hypoxia-ischemia exhibited significantly lower vertical activity and higher horizontal activity (p<0.05). Mild hypoxic damage may not be morphologically detectable but may induce substantial behavioral changes in adult mice. There were significant differences between horizontal and vertical activity in reaction to hypoxia-ischemia. Our study indicates that the importance of behavioral testing is irreplaceable and may be reflected in neonatal medicine.

Early postnatal hypoxia induces behavioral deficits but not morphological damage in the hippocampus in adolescent rats.

Hypoxia is one of the major pathological factors affecting brain function. The aim of the present study was to describe the effect of neonatal hypobaric hypoxia on the behavior of rats and to analyze its effect on hippocampal neurodegeneration. Hypobaric hypoxia at a simulated altitude of 9000 m was induced for one hour in neonatal rat pups (PND7 and PND9) of both sexes. Subsequently, the rats underwent behavioral testing on PND25 and PND35 using a LABORAS apparatus to assess spontaneous behavior. Hypoxia did not cause any morphological damage in the hippocampus of rats. However, hypoxia on PND7 led to less horizontal locomotor activity both, in males (on PND25) and females (on PND35). Hypoxia on PND9 led to higher rearing in females on PND25. Hypoxic males exhibited higher grooming activity, while females lower grooming activity on PND35 following hypoxia induced on PND7. In females, hypoxia on PND9 resulted in higher grooming activity on PND25. Sex differences in the effect of hypoxia was observed on PND35, when hypoxic males compared to hypoxic females displayed more locomotor, rearing and grooming activity. Our data suggest that hypoxia on PND7 versus PND9 differentially affects locomotion and grooming later in adolescence and these effects are sex-dependent.

Gender differences involved in the pathophysiology of the perinatal hypoxic-ischemic damage.

Hypoxic-ischemic encephalopathy (HIE) is a neonatal condition that occurs as a consequence of perinatal asphyxia, which is caused by a number of factors, commonly via compression of the umbilical cord, placental abruption, severe meconium aspiration, congenital cardiac or pulmonary anomalies and birth trauma. Experimental studies have confirmed that male rat pups show a higher resistance to HIE treatment. Moreover, the long-term consequences of hypoxia in male are more severe in comparison to female rat pups. These sex differences can be attributed to the pathophysiology of hypoxia-ischemia, whereby studies are beginning to establish such gender-specific distinctions. The current and sole treatment for HIE is hypothermia, in which a reduction in temperature prevents long-term effects, such as cerebral palsy or seizures. However, in most cases hypothermia is not a sufficient treatment as indicated by a high mortality rate. In the present review, we discuss the gender differences within the pathophysiology of hypoxia-ischemia and delve into the role of gender in the incidence, progression and severity of the disease. Furthermore, this may result in the development of potential novel treatment approaches for targeting and preventing the long-term consequences of HIE.

Sex-related differences in locomotion and climbing of C57Bl/6NTac mice in a novel environment.

Laboratory mice in standard laboratory cages, besides horizontal and vertical locomotor activity, spontaneously display cage-bar related activities such as cage-grid climbing. Although, grid-climbing activity is one of the major components of spontaneous home-cage behavior of mice, its exact role is not fully understood. This study aimed to describe the sex-differences in coping with novelty and in spontaneous behavior of laboratory mice concerning the cage-climbing activity in an observer-independent open field test. Adult mice of both sexes (C57Bl/6NTac) underwent behavioral testing in LABORAS system. Female mice travelled significantly longer distance (by 30 %, p<0.05) and showed higher grid-climbing activity (by 50 %, p<0.05) than males. Based on our results, the grid-climbing is a sex-dependent activity of mice, however, its exact role remains to be elucidated.

Pathophysiology of perinatal hypoxic-ischemic encephalopathy - biomarkers, animal models and treatment perspectives.

Hypoxic-ischemic encephalopathy (HIE) is one of the leading pediatric neurological conditions causing long-term disabilities and socio-economical burdens. Nearly 20-50 % of asphyxiated newborns with HIE die within the newborn period and another third will develop severe health consequences and permanent handicaps. HIE is the result of severe systemic oxygen deprivation and reduced cerebral blood flow, commonly occurring in full-term infants. Hypoxic-ischemic changes trigger several molecular and cellular processes leading to cell death and inflammation. Generated reactive oxygen species attack surrounding cellular components resulting in functional deficits and mitochondrial dysfunction. The aim of the present paper is to review present knowledge about the pathophysiology of perinatal hypoxic-ischemic encephalopathy, especially with respect to novel treatment strategies and biomarkers that might enhance early detection of this disorder and thus improve the general outcome of patients.

Subconvulsive dose of kainic acid transiently increases the locomotor activity of adult Wistar rats.

Kainic acid (KA) is a potent neurotoxic substance valuable in research of temporal lobe epilepsy. We tested how subconvulsive dose of KA influences spontaneous behavior of adult Wistar rats. Animals were treated with 5 mg/kg of KA and tested in Laboras open field test for one hour in order to evaluate various behavioral parameters. Week after the KA treatment animals were tested again in Laboras open field test. Finally, rat's brains were sliced and stained with Fluoro-Jade B to detect possible neuronal degeneration. Treatment with KA increased the time spent by locomotion (p<0.01), exploratory rearing (p<0.05) and animals traveled longer distance (p<0.01). These parameters tended to increase thirty minutes after KA administration. Week after the treatment we did not found differences in any measured behavioral parameter. Histology in terms of Fluoro-Jade B staining did not reveal any obvious neuronal damage in hippocampus. These results demonstrate that subconvulsive KA dose changes the behavioral parameters only transiently. Clarification of timing of the KA induced changes may contribute to understand mutual relationship between non-convulsive seizures and behavioral/cognitive consequences.

Low dose domoic acid influences spontaneous behavior in adult rats.

Domoic acid (DA) is a potent marine neurotoxine present in seafood. Intoxication by DA causes gastrointestinal symptoms like vomiting and diarrhoea and also the so-called amnesic shellfish poisoning (inflicting memory impairment and seizures). Since exposure to non-convulsive doses is relevant to the human health, we investigated the effect of low dose DA administration in adult Wistar rats. Rats were administered with DA at the dose 1.0 mg/kg and their behavior was monitored for one hour in three sessions. The first session started immediately after DA administration. The second and third session started one and two weeks later. After the third session, the histochemical analysis of the hippocampi of the animals was conducted (Fluoro-Jade B, bis-benzimide). DA increased time spent by locomotion and distance travelled in the second half of the first session and this effect was pronounced during the second and third session. Exploratory rearing was decreased by DA administration in the first half of the first session. DA influenced the grooming in biphasic manner (decrease followed by an increase of time spent by grooming). This biphasic trend was observed even two weeks after the DA administration. Histochemistry of DA treated rats did not confirm the presence of apoptotic bodies, Fluoro-Jade B positive cells were not found neither in CA1 nor CA3 area of the hippocampi. Our study revealed that a low dose of DA affect short and long-term the spontaneous behavior of rats without inducing neuronal damage.

Influence of low-dose neonatal domoic acid on the spontaneous behavior of rats in early adulthood.

Consumption of seafood containing toxin domoic acid (DA) causes an alteration of glutamatergic signaling pathways and could lead to various signs of neurotoxicity in animals and humans. Neonatal treatment with domoic acid was suggested as valuable model of schizophrenia and epilepsy. We tested how repeated early postnatal DA administration influences the spontaneous behavior of rats in adulthood. Rats were injected with 30 microg DA/kg from postnatal day (PND) 10 until PND 14. Their behavior was observed in the open field test for one hour (Laboras, Metris) at PND 35, PND 42 and PND 112. We did not find any difference between DA treated rats and animals injected with equivalent volume of saline in both test sessions at PND 35 and PND 42. DA rats at PND 112 exhibited significantly higher vertical and horizontal exploratory activity (tested parameters: locomotion, distance travelled, average speed reached during test, grooming and rearing) between the 30th-40th min of the test session and habituated over 10 min later. We conclude that at least in the given experimental design, neonatal DA treatment results in alteration of the spontaneous behavior of rats in adulthood.

CT imaging and spontaneous behavior analysis after osmotic blood-brain barrier opening in Wistar rat.

In our previous experiments we demonstrated that osmotic opening of the blood brain barrier (BBB) in rats by administration of mannitol into the internal carotid artery leads to cerebral edema. The aim of this study was to confirm objectively the development of brain edema and determine whether it affects spontaneous locomotor activity in rats (SLA). Brain edema was verified by computer tomography (CT) examination of the brain and SLA was observed during open field test. Twenty four adult male rats were divided into four groups of six: (1) control animals (C), (2) controls with anesthesia (CA), (3) controls with sham surgery (CS), (4) experimental - osmotic opening of the BBB (MA). Osmotic BBB disruption manifested by reducing the density of brain tissue (hypodensity), suggesting a higher water content in the brain tissue. SLA was compared between C, CA, CS and MA groups and between MA and CA groups. Significant difference was found only between the control group and MA group. In the first 30 min of the examination, rats after the mannitol administration revealed a marked limitation of spontaneous locomotor activity. Experimental results demonstrated reduction of spontaneous locomotor activity in rats with induced brain edema.

Both water intoxication and osmotic BBB disruption increase brain water content in rats.

Our previous experiments revealed that water intoxication and osmotic BBB disruption in the rat allow penetration of high-molecular substances into the brain and that resulting changes in the internal environment of the CNS lead to pathological development, such as the loss of integrity of myelin. The aim of the present study was to determine whether the previously described phenomena are associated with increased water content in the brain. To answer the question following methods were used: a) water intoxication: intraperitoneal administration of distilled water, b) osmotic BBB disruption: application of mannitol (20 %) selectively into the internal carotid artery, c) brain wet weight was measured after decapitation, and subsequently (after six days in thermostat set at 86 °C) the dry weight were estimated d) in animals with 20 % and 30 % hyperhydration the degree of myelin deterioration was estimated e) animal locomotor activity was tested by continuous behavior tracking and analysis. Brain water content after water intoxication and following the administration of mannitol was higher than in the control group. Different degrees of hyperhydration led to different levels of brain water content and to different degrees of myelin impairment. Hyperhydration corresponding to 20 % of the body weight brought about lower locomotor activity. Increased water content in the brain after the BBB osmotic disruption is surprising because this method is frequently used in the clinical practice.

Influence of allopurinol on evoked cortical afterdischarges during early ontogenesis.

The aim of our study was to test the hypothesis, whether repeated allopurinol pre-treatment (in dose of 135 mg/kg s.c.) can influence changes of brain excitability caused by long-term hypoxia exposition in young immature rats. Rat pups were exposed together with their mother in to an intermittent hypobaric hypoxia (simulated altitude of 7 000 m) since the day of birth till the 11th day (youngest experimental group) or 17th day for 8 hours a day. Allopurinol was administered daily immediately before each hypoxia exposition. The duration of evoked afterdischarges (ADs) and the shape of evoked graphoelements were evaluated in 12, 18, 25 and 35-day-old freely moving male pups. Hypobaric hypoxia prolonged the duration of ADs in 12, 18 and 25-day-old rats. The ADs were prolonged in 35-day-old rats only after the first stimulation. Allopurinol shorted the duration of ADs only in 12-day-old pups. In older experimental group the effect of allopurinol treatment was less pronounced.

Nicotine and kainic acid effects on cortical epileptic afterdischarges in immature rats.

Aim of the study was to test the effect of nicotine (NIC) and kainic acid (KA) co-treatment in immature rats. Male Wistar albino rats (two different age groups) were chosen for the study. Experiments started on postnatal day (PD) 8 or 21 and animals were treated twice a day for three days with nicotine, fourth day KA was administered. Animals at PD12 (PD25 respectively) were examined electrophysiologically for cortical epileptic afterdischarges (ADs). First cortical ADs in PD12 animals were longer, when compared to PD25 rats (group treated with both substances). Nor NIC or KA treatment affected the length of discharges in PD12 rats. Older experimental group exhibited the shortening of the first ADs (group treated with NIC and KA, compared with groups exposed to single treatment). Few changes were found in KA treated group - 2(nd) and 4(th) ADs were shorter when compared with first ADs. These results demonstrate that NIC treatment played minor role in seizure susceptibility of PD12 rats, sensitivity to NIC differs during ontogenesis and subconvulsive dose of KA influenced the length of discharges only in PD25 animals.

Nicotine influences the motor performance of immature rats in two different sensorimotor tasks.

The aim of this study was to assess the effect of nicotine on motor performance of immature (12-day-old) rats. We used two sensorimotor tasks (surface righting response and negative geotaxis test) to evaluate the influence of nicotine on animal's motor activity in course of 24 hours. Animals were treated intraperitoneally with two different nicotine doses (0.5 mg/kg and 1.0 mg/kg) and tested in four sessions (1 minute, 10 minutes, 1 hour and 24 hours after the injection). We concluded that nicotine significantly influences the motor behaviour in 12-day-old rats and this effect is dose dependent.

Is learning ability and spatial memory in rats influenced by single dose of nicotine?

A lot of studies have been concentrated on an effect of a short or a long-term administration of nicotine in humans or in animals. The negative effects on the human organism have been known for a long time, but these health problems are known especially from observing smokers. The number of tasks in human and in animals with accent on positive effect of nicotine has increased especially with regard to improvement of cognitive functions. The aim of this study was to investigate, how much a single dose of nicotine can influence the learning ability in rats. Male Wistar albino rats, 25-day-old, were studied. Two groups of animals received an intraperitoneal (i.p.) injection of nicotine in two different doses (0.75 mg/kg and 1.00 mg/kg b.w.). The third group received a single i.p. injection of saline in the equal volume (the control group). After the drug application the escape latency and the path length were measured and assessed in two periods of sessions in the Morris water maze. In our study no explicit changes of learning ability after a single nicotine injection was confirmed. Only at the third day of the task the trajectory was shorter (p<0.05) but this result appears too isolated. So we cannot exclude that such improvement was caused by other factors than by the nicotine administration.

Effect of the single-dose of nicotine-administration on the brain bioelectrical activity and on behaviour in immature 12-day-old rats.

A variety of current studies is concentrated on the effect of short-term or long-term administration of nicotine in humans and in animals. The aim of this study was to investigate the effect of nicotine after a single administration in two different doses on the brain bioelectrical activity and on behaviour and motor activity in young, immature rats. Male Wistar albino rats, 12-day-old, were used in the experiment. Two groups were administrated by one intraperitoneal (i.p.) injection of nicotine in two various doses. The last (the third) group, which was given one i.p. saline injection, served as a control group. The group with lower dose of nicotine (0.75 mg/kg body weight) showed only mild alteration of the electrocorticogram (ECoG), and no behavioural or motor changes. In the second group (with higher dose of nicotine--1.00 mg/kg), epileptiform discharges manifested in about 50% of animals. Those animals showed also changes in motor activity (tremor of hindlimbs), but only slightly expressed within the time when epileptiform changes occurred in the ECoG. Routine behaviour and locomotion was observed only in a part of animals. In the third group (control group) no changes in bioelectrical activity, in behaviour or in motor activity were observed. We conclude that even a single dose of nicotine can evoke alteration in the ECoG, in behaviour and in motor activity of immature rats. On the other hand, the quantity, quality and length of ECoG abnormalities as well as parameters of behaviour were closely related to the dose of nicotine.

Magnesium and posthypoxic changes of nitrergic population in rat hippocampus.

We used NADPH-diaphorase staining to study effects of magnesium pre-treatment during long-lasting hypoxia on the brain structure of rats. NADPH-diaphorase is an enzyme co-localized in neurons with NO-synthase that is responsible for NO synthesis. NO participates in hypoxic-ischaemic injury of the brain. Hypoxia was induced in consecutive days from the 2nd till the 11th day of postnatal life in a hypobaric chamber (for 8 hours per day). Magnesium was administered before each hypoxia exposition. At the age of 12 days, the animals were transcardially perfused with 4% buffered neutral paraformaldehyde under the deep thiopental anaesthesia. Cryostat sections were stained to identify NADPH-diaphorase positive neurons that were then quantified in five hippocampal regions. In comparison to the control animals, intermittent hypoxia brought about higher density of NADPH-diaphorase positive neurons in all studied areas of the hippocampal structure: in CA1 and CA3 areas of the hippocampus and in hilus, in the dorsal and ventral blades of the dentate gyrus. Magnesium pre-treatment during hypoxia reduced number of NADPH-diaphorase positive neurons in all studied areas.

Changes of hippocampal neurons after perinatal exposure to ethanol.

The effect of ethanol on the structural development of the central nervous system was studied in offspring of Wistar rats, drinking 20 % ethanol during pregnancy and till the 28th day of their postnatal life. The structural changes in the hippocampus and dentate gyrus were analyzed at the age of 18, 35 and 90 days. A lower width of pyramidal and granular cell layers, cell extinction and fragmentation of numerous nuclei were found in all experimental animals compared to control animals. The extent of neural cell loss was similar in all monitored areas and in all age groups. At the age of 18 and 35 days, the degenerating cells were observed in the CA1 and CA3 area of the hippocampus and in the ventral and dorsal blade of the dentate gyrus. Numerous glial cells replaced the neuronal population of this region. Some degenerating cells with fragmented nuclei were observed at the age of 90 days. Our experiments confirmed the vulnerability of the developing central nervous system by ethanol intake during the perinatal period and revealed a long-lasting degeneration process in the hippocampus and dentate gyrus.

Pentylentetrazol associated changes of hippocampal neurons in immature rats.

Using histochemical analysis, the NADPH-diaphorase, Fluoro-Jade B and bis-benzimide (Hoechst 33342) the effect of intraperitoneal administration of pentylentetrazol (PTZ) on hippocampal neurons was studied. 18-day-old male rats of the Wistar strain received PTZ (60 mg/kg) in one dose. The next day, the 19-day-old animals were transcardially perfused with 4% paraformaldehyde under deep thiopental anaesthesia. Cryostat sections were stained to identify NADPH-diaphorase positive neurons that were then quantified in the CA1 and CA3 areas of the hippocampus, in the dorsal and ventral blades of the dentate gyrus and in the hilus of the dentate gyrus. Combination of the Fluoro-Jade B and bis-benzimide (Hoechst 33342) staining was used in the same areas, to identify possible neurodegeneration. Number of NADPH-d positive neurons was higher after pentylentetrazol administration in CA1 and CA3 areas of the hippocampus and in the hilus of the dentate gyrus, compared to the control group which we consider as baseline. Morphological alterations (cell loss) in CA3 area of the hippocampus and in the hilus of the dentate gyrus only (evaluated by Hoechst 33342) were found in animals receiving PTZ; no FJ-B positive cells were found and we can conclude that neurons were destroyed by the PTZ insult.