A. O. Tishkina

Chronic stress induces nonapoptotic neuronal death in the rat hippocampus.

403 Chronic stress results in functional disturbances in the CNS, possibly due to death of neuronal cells or their substantial structural changes. Hippocampal atrophy was found in patients with disorders related to repeated depression or posttraumatic stress syndrome [12]. Under experimental conditions, chronic three week immobilization stress or corticosterone adminis tration leads to death of pyramidal neurons in the hip pocampi of rats and monkeys [10, 13]. Chronic stress induced by inescapable electric paw skin irritation and asthenia inducing white noise is a basic factor leading to development of experimental neurosis [1, 2]. Increased numbers of dark neurons and acidophilic cells were observed in layer V of the sensorimotor cortex in rabbits after neurosis [3]. Other authors reported destructive changes in the sensorim otor cortex, such as formation of multiple lysosomes, lipid and lipofuscin bodies with foci of local degenera tion after neurotization [5, 7]. In contrast to impair ments of local cerebral blood flow and reorganization of the synaptic apparatus and cytoskeleton, neuronal death is not always found [9]. It was demonstrated that experimental neurosis activated free radical mediated processes in the rat brain, which resulted in neuronal death [2]. However, the activity of caspase 3, the main executive enzyme of apoptotic cell death, was decreased. In this study, we investigated morphologi cal changes in the rat hippocampus after chronic neu rosis. Male Wistar rats, supplied by the Stolbovaya animal farm (Moskow oblast), weighing 250–300 g, were used for the study. The animals were housed, five rats per cage, in a vivarium under natural illumination condi tions and free access to food and water. The experi mental protocol was approved by Ethical Commission of the Institute of Higher Nervous Activity and Neu rophysiology, Russian Academy of Sciences. Chronic stress was induced in one group of animals (n = 10) using a model of experimental neurosis. For this purpose, rats were kept for 4–5 h daily over the course of 14 days in a special device containing indi vidual cells, where they were treated with an intermit tent white noise of 65–70 dB combined with stimula tion by an electrical current of 1–1.5 mA regulated by the start of vocalization [2, 6]. Another group of rats (n = 5) was used as a control. One day after the end of stress, the rats were intra peritoneally anesthetized with chloral hydrate at a dose of 450 mg/kg. The brains were fixed with a solu tion of ethanol–formalin–glacial acetic acid at a ratio of 7 : 2 : 1 v/v, taken out and post fixed in the same fix ative for 2 h, and, after this, kept in 70% ethanol. Blocks were prepared from brain tissue, dehydrated, and paraffin embedded. Frontal brain sections were mounted onto slides and Nissl stained with cresyl vio let. Apoptotic changes in neurons were detected using Hoechst 33258 (Serva, Germany) fluorescent staining or TUNEL method with the use of an Apoptag kit (Chemicon, United States). For morphological analysis, three view fields were randomly chosen in the pyramidal layer of the CA1 hippocampal field in the left and right hemispheres. The following indices were counted in a fixed area of 0.01 mm2: the total number of neurons, number of neurons with pathological changes, and number of glial cells. Cells were counted using an Olympus CX 41 microscope (Olympus Optical, Japan) equipped with an oil immersion 100× lens with a digital aperture of 1.25. Statistical analysis was performed using the Sta tistica 6.0 software (StatSoft). Histological analysis of brain tissue from the exper imental animals after neurotization was performed using Nissl stained sections. In the hippocampi of the control rats, the main cellular fields were clearly seen (Figs. 1a, 1c, 1e, 1g), their cytoarchitectonics was not impaired, and the cell density in the pyramidal layer was normal. Cell boundaries, nuclei, and nucleoli were clearly observed, and pericellular edema was rarely found. Glial cells were seen very well. Blood ves PHYSIOLOGY

Region-specific changes in activities of cell death-related proteases and nitric oxide metabolism in rat brain in a chronic unpredictable stress model.

Effects of a chronic combined unpredictable stress on activities of two cell death-related proteases, calpain and cathepsin B, were studied along with indices of nitrergic system in rat brain structures. Male Wistar rats were subjected to a 2-week-long combined stress (combination of unpaired flash light and moderate footshock associated with a white noise session). Stress resulted in a significant loss in the body and thymus weight and increased defecation in the open field test, though neither motor and exploratory activity, nor plasma corticosterone differed from the respective control levels. Decreased calpain activity and increased cathepsin B activity were demonstrated in the hippocampus of stressed rats (previously we have shown that caspase-3 activity was significantly suppressed in the brain of rats subjected to same type of stress). A significant reduction in the number of NOS-containing neurons was accompanied by a chronic stressinduced decline in NOS activity in the neocortex. Similar changes were observed in the hippocampus. However, levels of NO metabolites were elevated in both structures. Thus, stress-induced structural modifications in the brain may be mediated by disturbances in the nitrergic system and increased lysosomal proteolysis.

Chronic combined stress induces selective and long-lasting inflammatory response evoked by changes in corticosterone accumulation and signaling in rat hippocampus.

Hippocampus is believed to be selectively vulnerable to stress. We hypothesized that this phenomenon may be mediated by relatively high vulnerability to neuroinflammation related to impairments of local glucocorticoid metabolism and signaling. We have evaluated inflammatory responses induced by acute or chronic combined stress in the cerebral cortex and hippocampus as well as circulating and brain corticosterone (CS) levels as well as expression of corticosterone target genes. The hippocampus showed higher stress-induced expression of the proinflammatory cytokine IL-1β as compared to the cerebral cortex. A month after the termination of the chronic stress, IL-1β mRNA in the cerebral cortex reached control level, while in the hippocampus it remained significantly increased. Under chronic stress, the maladaptive inflammatory response in hippocampus was accompanied by a significant increase in local CS levels, as compared to cerebral cortex. Under acute stress, the increased CS level induced changes in CS-regulated genes expression (CRF and IGF1), while this phenomenon was not observed after chronic stress. Thus, the hippocampus appears to be more vulnerable to stress-induced inflammation as compared to the neocortex and demonstrates persistent inflammatory response induced by chronic stress. Stress-induced maladaptive inflammatory response is associated with a selective increase in hippocampal CS accumulation and changes in CS signaling.

Panthenol as neuroprotectant: Study in a rat model of middle cerebral artery occlusion

In vivo study of neuroprotective effects of D-panthenol has been performed on a rat model of middle cerebral artery occlusion (MCAO). Administration of panthenol (i.p., 200 mg/kg 24 h, 0.5 h before and 0.5 h, 24 h, 48 h after MCAO) significantly ameliorated neurological deficit 1–5 days after MCAO, reduced body weight loss and cortical damage induced by 30 min MCAO. The results are the first in vivo evidence for neuroprotective properties of D-panthenol and suggest that it has a good potential for treatment of neurological diseases.

Neonatal proinflammatory stress induces accumulation of corticosterone and interleukin-6 in the hippocampus of juvenile rats: Potential mechanism of synaptic plasticity impairments

Infectious diseases in early postnatal ontogenesis can induce neuroinflammation, disrupt normal central nervous system development, and contribute to pathogenesis of cerebral pathologies in adults. To study long-term consequences of such early stress, we induced neonatal proinflammatory stress (NPS) by injecting bacterial lipopolysaccharide into rat pups on postnatal days 3 and 5 and then assessed the levels of corticosterone, proinflammatory cytokines and their mRNAs, and neurotrophins and their mRNAs in the hippocampus and neocortex of the one-month-old animals. Long-term potentiation (LTP) was studied in hippocampal slices as an index of synaptic plasticity. NPS-induced impairments of LTP were accompanied by the accumulation of corticosterone and IL-6 in the hippocampus. In the neocortex, a decrease in exon IV BDNF mRNA was detected. We suggest that excessive corticosterone delivery to hippocampal receptors and proinflammatory changes persisting during brain maturation are among the principal molecular mechanisms responsible for NPSinduced neuroplasticity impairments.

The glial response in the rodent hippocampus to systemic administration of bacterial lipopolysaccharide

Interaction of astrocytes and microglia is an important factor of the development of neuroinflammation because impairment of the mechanisms of this neuroprotective interaction may underlie the progression of neuropathology. We studied the changes in cell populations of astro- and microglia in the rodent hippocampus after intraperitoneal injection of bacterial lipopolysaccharide (LPS). Chronic administration of LPS elevated the number of microglial cells in the hilus of the dentate gyrus of the mouse hippocampus, whereas a single injection of a cumulative dose of LPS (5 mg/kg) did not induce a similar response. In rats, a single injection of LPS decreased the number of astrocytes in the hilus of the dentate gyrus of the hippocampus. We believe that in the hippocampus, in the cerebral structure, which is selectively vulnerable to neuroinflammation, glial cells of the dentate hilus are most responsive under the conditions of neuroinflammation induced by systemic administration of LPS.

Effects of early neonatal proinflammatory stress on the expression of BDNF transcripts in the brain regions of prepubertal male rats

Early postnatal proinflammatory stress may evoke behavioral impairments in adulthood; however, the underlying mechanisms are still elusive. The brain-derived neurotrophic factor (BDNF) plays a key role in neuroplastic changes in health, as well as in pathology. The BDNF gene is transcribed to exon-specific mRNAs and the pattern of their expression depends on stimulus. We suggested that disturbances of the exonspecific BDNF mRNA expression in the brain regions after stress induced by proinflammatory stimuli in the early postnatal period could be one of the underlying mechanisms of consequent behavioral impairments. Thus, the aim of the study was to examine the effects of proinflammatory stress in early postnatal ontogeny on the BDNF polypeptide content and the patterns of expression of the BDNF gene in the neocortex and hippocampus of prepubertal male rats. The proinflammatory stress was induced by the subcutaneous administration of bacterial lipopolysaccharide (LPS) to rat pups on postnatal days 3 and 5, while BDNF expression was studied in 36-day-old rats. The BDNF polypeptide content was estimated using an enzyme-linked immunosorbent assay, while a quantitative polymerase chain reaction followed by reverse transcription was used to detect the exon-specific BDNF mRNA expression. The levels of BDNF and its transcripts, containing the common exon IX were similar in the control and LPS-treated rats. In the rats treated with LPS, the level of BDNF mRNA containing exon IV was lower in the neocortex but not in the hippocampus. No changes in the expression of the transcripts containing exons I and VI were observed in any of the brain structures studied. We suggest that specific alterations in BDNF expression may be involved in susceptibility to the development of behavioral impairments of animals subjected to early proinflammatory stress.

Neuroimmune aspects of brain damage after focal ischemia

We have studied the level of products of nitrosative stress, immunoglobulins, and antibodies against nitrated proteins in the cerebrospinal fluid (CSF) and in the neocortex of rats 24 hours after focal ischemia. In the acute stage after ischemia, the level of metabolites of nitric oxide, nitrate, and nitrite, significantly increased in the CSF and in the ischemic neocortex of the animals. In addition, in the CSF of rats, the level of immunoglobulins (Ig) significantly increased and the nitrotyrosine-BSA binding was enhanced. Immunochemical staining of the sections of the brain by anti-rat Ig antibodies revealed their presence in the ischemic focus but not in the contralateral hemisphere. In the ischemic hemisphere in some Ig-positive cells, we observed colocalization of staining with the pro-apoptotic protein Bax. At this stage, in the ischemic hemisphere a membrane attack complex, the product of the terminal stage of complement activation, was detected by immunohistochemistry. Thus, the development of an immune response during the acute stage after experimental stroke is associated with some features of apoptotic cell death and seems to a certain extent to be modulated by nitrosative stress products, such as proteins modified by nitration.

Changes in the expression of astroglial and microglial markers in the hippocampus of rats adapted to chronic stress and the effects of panthenol

We studied the morphological changes and expression of the astroglial and microglial markers in the sensorimotor cortex and hippocampus of rats that were subjected to chronic stress. Chronic neurotization was associated with the appearance of a large number of damaged neurons in layer V of the sensorimotor cortex and the pyramidal layer of the hippocampus. These changes were considerably expressed in the CA3 field. A neurosis-like state in rats was accompanied by an increased expression of microglial markers in the hippocampus and this effect was evident even 1 month after the end of stress. The expression of the astroglial marker GFAP decreased in the CA3 hippocampal field. Treatment of animals with the pantothenic acid derivative panthenol stabilized the glial response to chronic stress; however, its effects were not long-lasting and did not prevent activation of microglia during the period after stress.

The Acute Post-Traumatic Period in Rats Is Accompanied by an Anxiety State and a Decrease in the Proportion of REM Sleep

The sequelae of craniocerebral trauma (CCT) were studied using a model based on severe (3–4 atm) lateral hydrodynamic percussion (liquid-percussive brain injury) in male Sprague–Dawley rats. With the aim of detecting the symptoms of anxiety states, the rats’ behavior was assessed in the dark-light box and the elevated plus maze test; sleep impairments were detected by recording the electrocorticogram (ECoG) before trauma and during the first week after trauma. The results provided evidence of the post-CCT development of signs of an anxiety state, accompanied by decreases in the proportion of REM sleep and decreases in the amplitude and frequency of the ECoG during this phase.