CA1 Zone Research Articles

Bring CLARITY to Temporal Lobe Epilepsy: 3D Visualization of p-Tau(Ser262) and 14-3-3 Zeta

CLARITY is one new technology which allows the brain tissue become transparent. It has successfully been combined with immunofluorescence staining to achieve the 3D visualization of some molecules or neuronal cells in some disease brains. The temporal lobe epilepsy (TLE) is one neuronal disease which is characterized by the sprouting of the massy fibers (MSF). Previous study has showed that MSF could be affected by the phosphorylation at site-262 of microtubule association protein Tau (p-tau (Ser262)). However, in TLE little useful information was reported concerning the 3D architecture of p-tau (Ser262) and its relationship with 14-3-3 zeta that regulated the phosphorylation of Tau in AD disease. In this paper, pilocarpine-induced epilepsy model was established and identified by Timm-staining. Double immunofluorescent staining results showed that the development of TLE gave rise to the colocalization of p-tau (Ser262) and 14-3-3 zeta protein in CA1 and CA3 zone in hippocampi. The mm-thick brain sections were passively clarified, and 3D reconstruction imaging of the immunofluorescent staining showed that p-tau (Ser262) was diverse cluster-like shape. These results proved that CLARITY could be used to study TLE, in which the 3D morphologic changes of p-tau (Ser262) and the role of 14-3-3 zeta in the regulation of Tau needed to be further investigated.

Neuroprotective effect of epidermal growth factor plus growth hormone-releasing peptide-6 resembles hypothermia in experimental stroke

Background: Combined therapy with epidermal growth factor (EGF) and growth hormone-releasing peptide 6 (GHRP-6) in stroke models has accumulated evidence of neuroprotective effects from several studies, but needs further support before clinical translation. Comparing EGF + GHRP-6 to hypothermia, a gold neuroprotection standard, may contribute to this purpose.Objectives: The aims of this study were to compare the neuroprotective effects of a combined therapy based on EGF + GHRP-6 with hypothermia in animal models of (a) global ischemia representing myocardial infarction and (b) focal brain ischemia representing ischemic stroke.Methods: (a) Global ischemia was induced in Mongolian gerbils by a 15-min occlusion of both carotid arteries, followed by reperfusion. (b) Focal brain ischemia was achieved by intracerebral injection of endothelin 1 in Wistar rats. In each experiment, three ischemic treatment groups – vehicle, EGF + GHRP-6, and hypothermia – were compared to each other and to a sham-operated control group. End points were survival, neurological scores, and infarct volume.Results: (a) In global ischemia, neurological score at 48–72 h, infarct volume, and neuronal density of hippocampal CA1 zone in gerbils treated with EGF + GHRP-6 were similar to the hypothermia-treated group. (b) In focal ischemia, the neurologic score and infarct volume of rats receiving EGF + GHRP-6 were also similar to animals in the hypothermia group.Discussion: With hypothermia being a good standard neuroprotectant reference, these results provide additional proof of principle for EGF and GHRP-6 co-administration as a potentially neuroprotective stroke therapy.

Functional hyaluronate collagen scaffolds induce NSCs differentiation into functional neurons in repairing the traumatic brain injury.

A sodium hyaluronate collagen scaffold was specifically functionalized with nutrient-bFGF which can induce the differentiation of neural stem cells (NSCs) into multi-type and mature functional neurons at a high percentage in two week. When jointly transplanting the bFGF-CRS and NSCs into the CA1 zone of the traumatic brain injured area of adult rats, the bFGF-CRS could provide an optimal microenvironment, which promoted survival, migration and neuronal differentiation of transplanted NSCs and functional synapse formation among iN cells, as well as between iN cells and host brain tissue in TBI rats, consequently leading to the cognitive function recovery of TBI rats.

Therapeutic Hypothermia Enhances Cold-Inducible RNA-Binding Protein Expression and Inhibits Mitochondrial Apoptosis in a Rat Model of Cardiac Arrest.

Therapeutic hypothermia is well known for its protective effect against brain injury after cardiac arrest, but the exact mechanism remains unclear. Cold-inducible RNA-binding protein (CIRP), a member of cold shock protein, enables mammalian cells to withstand decreased temperature by regulating gene translation. However, the role of CIRP in global cerebral ischemia after therapeutic hypothermia has not been clearly elucidated. In the present study, rats resuscitated from 4min of cardiac arrest were separately treated with therapeutic hypothermia (immediately after return of spontaneous circulation (ROSC); targeted temperature at 33°C) and therapeutic normothermia (targeted temperature at 36.8°C) for 6h. The hippocampus was harvested at 0h (baseline), 6h, 12h, 1day, 3days, and 7days after ROSC. The expression of CIRP messenger RNA (mRNA) was assessed by real-time PCR. CIRP and mitochondrial apoptosis-associated proteins were tested by Western blot. The histological changes and neurological function were respectively evaluated by hematoxylin-eosin staining and neurological deficit score (NDS). Compared with baseline, rats resuscitated from cardiac arrest showed increased expression of CIRP, Bax, Caspase 9, and Caspase 3 and decreased expression of Bcl-2 in hippocampus (P < 0.05). However, therapeutic hypothermia after ROSC alleviated the alterations of Bax, Caspase 9, Caspase 3, and Bcl-2, while further increased the hippocampal expression of CIRP mRNA and protein, when compared with the normothermia rats (P < 0.05). In addition, compared with the therapeutic normothermia rats, histopathological damage in CA1 zone and NDS were respectively decreased and increased in the hypothermia rats (P < 0.05). Our findings suggest that 32°C therapeutic hypothermia exerts an important neuroprotective effects by up-regulating CIRP expression and inhibiting mitochondrial apoptosis factor production in the cardiac arrest rat model.

Lamotrigine attenuates cerebral ischemia-induced cognitive impairment and decreases β-amyloid and phosphorylated tau in the hippocampus in rats.

Lamotrigine (LTG) has shown benefits in animal models of cerebral ischemia, but the mechanism involved was not fully studied. This study was carried out to examine the effects of LTG on cognitive dysfunction, β-amyloid1-42 accumulation, and tau protein hyperphosphorylation in the hippocampus of ischemic rats. Transient ischemic stroke was induced by middle cerebral artery occlusion. The Morris water maze test was used to evaluate the cognitive function of rats. We found that LTG significantly attenuated ischemia-induced cognitive deficits and decreased neuronal injury in the hippocampal CA1 zone. Moreover, LTG reduced β-amyloid1-42 and phosphorylated tau (AT8) in the hippocampus after ischemia. These results suggested that the cognition-protective effects of LTG after cerebral ischemia might involve inhibition of toxic β-amyloid accumulation and tau hyperphosphorylation in the hippocampus.

Involuntary, forced and voluntary exercises are equally capable of inducing hippocampal plasticity and the recovery of cognitive function after stroke

Objectives:Forced and voluntary exercises are known to improve cognition and induce neuroprotection after stroke, however, any effects of involuntary movement induced by functional electrical stimulation (FES) are unclear. The effects of involuntary exercise induced by FES, forced and voluntary exercise on the recovery of cognitive function in vascular dementia and the regional repair of ischaemic lesions were investigated using a rat model.Methods:Wistar rats were randomly assigned to a sham group, a vascular dementia control group (VD), an involuntary exercise group (I-Ex), a forced exercise group (F-Ex) or a voluntary exercise group (V-Ex). An object recognition test (ORT) and an object location test (OLT) were used to evaluate the recovery of cognitive function. Levels of synapsin I (SYN), synaptophysin (SYP), postsynaptic density 95 (PSD-95), microtubule-associated protein 2 (MAP-2) and Tau in the hippocampus were evaluated using western blotting and immunohistochemistry. Nissl staining was applied to visualise the loss of viable neurons from the hippocampus.Results:Involuntary exercise and voluntary exercise both improved cognition in terms of ORT and OLT results. Forced exercise only improved ORT results. The levels of SYN, PSD-95, MAP-2 and Tau in the hippocampus were enhanced by all three patterns of exercise training. Moreover, all three patterns reduced losses of dendrons and neurons in the hippocampal CA1 and CA2 zones, but without significant differences among the three exercise regimens.Conclusion:Involuntary exercise induced by FES has beneficial effects on cognitive function after vascular dementia comparable to those of forced and voluntary exercise.

Lipid emulsion mitigates local anesthesia-induced central nervous system toxicity in rats.

The aim of the present study was to investigate the effect of intravenously administered lipid emulsion on local anesthetic (LA)-induced central nervous system (CNS) toxicity. A total of 100 male Sprague Dawley rats were allocated at random into the following groups: Sham (A), lidocaine (B), levobupivacaine (C) and ropivacaine (D). Groups B-D were each subdivided into three subgroups: Toxic, post-conditioning and pre-conditioning. Intracerebroventricular injections of 0.9% normal saline (sham group) or LA were administered via microsyringe; in addition, a 20% lipid emulsion was injected into tail vein prior to the LA injection (pre-conditioning subgroups) or following rat respiratory arrest (post-conditioning subgroups). The heart rate, blood pressure, neurological behavior scores, neuronal density and time from LA injection to respiratory arrest, apnea and start of arrhythmia were measured. Rats in the toxic groups died due to respiratory arrest following the injection of LA into the lateral ventricle. Rats in the post-conditioning subgroups were resuscitated from the LA-induced respiratory arrest, while the pre-conditioning subgroup rats exhibited no respiratory arrest. No significant differences in heart rate were observed between the toxic and post-conditioning subgroups in the levobupivacaine and ropivacaine groups (P>0.05); however, a significant difference was observed between these treatment groups and the rats treated with lidocaine (P<0.01). A significant difference was also observed in the time from the LA injection to the onset of arrhythmia among the rats in groups B, C and D (P<0.01). No significant differences in the neurological behavior scores and neuronal density were observed in the hippocampal CA1 zone among group C and D rats in the post- and pre-conditioning subgroups at various time-points following treatment. Beyond that, the same phenomena regarding neurological behavior scores was observed in post- and pre-conditioning subgroups of group B at 12 and 24 h treatment, contrasting with the statistically significant difference between post- and pre-conditioning subgroups at 6 h treatment (P<0.01). The results of the present study therefore indicate that pre- and post-conditioning with lipid emulsion effectively mitigates LA-induced CNS toxicity in rats.

Нитрозирующий стресс и апоптоз нейронов са1-зоны гиппокампа в условиях моделирования хронической алкогольной интоксикации: нейропротективные эффекты тиоцетама

Fulfilled researches showed that modeling of chronic alcohol intoxication by daily intragastric ethanol administration to non-pedigreed male white rats (first 10 days – 15% ethanol solution in dose 4 g/kg, next 10 days – 15% ethanol solution in dose 6 g/kg, then 10 days – 25% ethanol solution in dose 4 g/kg) leads to activation of nitrosorbidi stress reactions and initiation of neuro-apoptosis. Thus, as compared with healthy animals group, in rats’ brain after chronic ethanol administration it was revealed significant increase of nitro-tyrosine (nitrosorbidi stress marker) in cytosolic and mitochondrial fractions of brain homogenate against the background of revealing of apoptosis molecular markers in hippocampal CA1 zone neurons (fragmentation of neuron&amp;#180;s nuclei of hippocampal CA1 zone, increase of density and ratio of apoptotic- and destructive-changed cells) as well as deprivation of anti-apoptotic mechanisms (decrease of density of Bcl-2-positive neurons in hippocampal CA1 zone and decrease of Bcl-2 protein concentration in hippocampus). Subsequent 14-day intragastric administration of complex neuro-metabolic cerebro-protector Thiocetam in dose 100 mg/kg to the animals after chronic alcoholization showed significant protective effect on hippocampal CA1 zone neurons. Thus, Thiocetam administration resulted in decrease of nitrotyrosine levels in mitochondrion and in cytosol of brain of animals with chronic alcoholization as compared with untreated animals and with animals receiving Piracetam. Thiocetam administration to experimental animals led to significant decrease of density of apoptotic- and destructive-changed neurons of hippocampal CA1 zone and ratio of apoptotic-changed cells as compared with control group and animals receiving Piracetam. Course of treatment with Thiocetam increases significantly the density of Bcl-2-positive neurons in CA1 zone of hippocampus and significantly increases concentration of Bcl-2 in brain tissue of experimental animals as compared with control group and animals which received Piracetam according the same schedule. The increase of Bcl-2 protein expression in animals received Thiocetam testifies to the activation of anti-apoptotic defense of damaged neurons under the influence of this preparation. We suppose that one of the molecular mechanisms of neuro-protective effect of Thiocetam in chronic intoxication is the breaking of NO-depended mechanisms of neuro-apoptosis.

Effect of rhynchophylline on the expression of p-CREB and sc-Fos in triatum and hippocampal CA1 area of methamphetamine-induced conditioned place preference rats

To explore the effect of rhynchophylline (Rhy) on the expression of p-CREB and c-Fos in the striatum and hippocampal CA1 area of methamphetamine-induced conditioned place preference (CPP) rat, methamphetamine (2mg/kg) was injected to rats and the conditioned place preference was observed in these rats treated with or without Rhy. An immunohistochemistry assay was used to determine the expression of p-CREB and c-Fos in the striatum and hippocampal CA1 area. Methamphetamine induced significant behavior alteration in CPP, while after pretreatment with rhynchophylline or ketamine, the time of staying in methamphetamine-paired compartment of rats was significantly reduced. Methamphetamine also increased the number of p-CREB positive cells in the striatum and hippocampal CA1 zone, as well as p-Fos positive cells. However, the compound Rhy could attenuate the effect. These findings show that Rhy can suppress the acquisition of CPP in rats induced by methamphetamine and the action may be related with the reduced expression of p-CREB and p-Fos in the striatum and hippocampus.

Effects of Electroacupuncture on Hippocampal and Cortical Apoptosis in A Mouse Model of Cerebral Ischemia-reperfusion Injury

To observe the effects of electroacupuncture on hippocampal and cortical apoptosis in a mouse model of cerebral ischemia-reperfusion injury. Mouse models established by repeated cerebral ischemia-reperfusion, followed by electroacupuncture at Shenshu, Geshu, and Baihui points. The control group mice were intragastrically administered Hydergine. On day 1 and 7 post-treatment, hippocampal and cortical apoptosis was detected by terminal-deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL), and apoptosis images in the hippocampal CA1 zone and cortical area were analyzed. In the model group, apoptotic cells were detected one day after treatment and some cellular fibers were disarrayed. By day 7 post-treatment, there was an increase in the number of apoptotic cells in the hippocampal CA1 region. In addition, there were apoptotic cells in the cortical area, the cortical layers were thinner with localized neuronal loss and sieve-like lymphocyte infiltration, as well as glial cell proliferation and visible infarct lesions. However, in the Hydergine and electroacupuncture groups, there was a small number of apoptotic cells. At 7 days post-treatment in the model group, field number, numerical density on area, and surface density were increased. However, in the Hydergine and electroacupuncture groups these parameters were decreased (P < 0.01), with a significant difference between the two treatment groups (P < 0.01). Electroacupuncture treatment inhibited apoptosis and provided neuroprotection.

β2 Adrenoreceptor-Mediated Noradrenergic Effect on GABA-ergic Transmission in the CA1 Zone of the Rat Hippocampus in vitro

Using extracellular recording of evoked potentials, we examined the effect of an agonist of β 2 adrenoreceptors, metaproterenol (MPT), on GABA-ergic transmission in the CA1 zone of slices of the rat hippocampus. Isolated application of GABA evoked in these slices rapid reversible suppression of orthodromic population discharges recorded from the pyramidal layer of the above hippocampal zone after electrical stimulation of Schaffer collaterals in the radial layer. In most cases (13 of 19 preparations), combined application of GABA and MPT interfered with the development of the inhibitory GABA effect. In the case of the action of both of the above agents, the amplitude and duration of evoked responses decreased, but these changes were significantly weaker than those observed upon isolated GABA application. Our experiments showed that the noradrenergic system is capable of modulating GABA-ergic inhibition via β 2 adrenoreceptors and, in such a way, is probably involved in regulation of the inhibition level in the hippocampus. Noradrenaline-induced effects on inhibitory neuronal networks in the hippocampus, similarly to those exerted by several other cerebral neurotransmitter systems, underlie the involvement of the noradrenergic cerebral system in a few physiological processes (emotions, attention, and memory) and are related to some pathological states (Alzheimer’s disease, schizophrenia, epilepsy, etc.).

Treatment of Alzheimer’s Disease with Anti-Homocysteic acid Antibody

Homocysteic acid (HA) may play an important role in Alzhiemer disease (AD) as we previously reported that HA induced accumulation of intraneuronal A[beta]42. In this study, we first analyzed HA levels in a mouse model of AD. 4-month old pre-pathologic 3xTg-AD mice exhibited higher levels of HA in the hippocampus as compared to age-matched nontransgenic, suggesting that HA accumulation may precede both A[beta] and tau pathologies. To further determine the pathogenic role of HA in AD, we treated young 3xTg-AD mice with vitamin B6-deficient food for 3 weeks to induce the production of HA in the brain. Concominantly, mice received either saline or anti-HA antibody intraventricularly using a guide cannula every 3 days. Mice received anti-HA antibody significantly rescued cognitive impairment induced by vitamin B6 deficiency. Pathologically, 3-week treatment with vitamin B-6 deficient food resulted in strong neurodegeneration in the hippocampal CA1 zone and decreased hippocampal volume. In contrast, anti-HA antibody treatment attenuated these pathological changes. Taken together, we conclude that increased brain HA triggers memory impairment whose condition was deteriorated by amyloid and subsequent neurodegeneration and reduction of neurogenesis. Our results indicate a pathogenic role of HA in AD.

Treatment of Alzheimer's Disease with Anti-Homocysteic acid Antibody

AbstractHomocysteic acid (HA) may play an important role in Alzhiemer disease (AD) as we previously reported that HA induced accumulation of intraneuronal A[beta]42. In this study, we first analyzed HA levels in a mouse model of AD. 4-month old pre-pathologic 3xTg-AD mice exhibited higher levels of HA in the hippocampus as compared to age-matched nontransgenic, suggesting that HA accumulation may precede both A[beta] and tau pathologies. To further determine the pathogenic role of HA in AD, we treated young 3xTg-AD mice with vitamin B6-deficient food for 3 weeks to induce the production of HA in the brain. Concominantly, mice received either saline or anti-HA antibody intraventricularly using a guide cannula every 3 days. Mice received anti-HA antibody significantly rescued cognitive impairment induced by vitamin B6 deficiency. Pathologically, 3-week treatment with vitamin B-6 deficient food resulted in strong neurodegeneration in the hippocampal CA1 zone and decreased hippocampal volume. In contrast, anti-HA antibody treatment attenuated these pathological changes. Taken together, we conclude that increased brain HA triggers memory impairment whose condition was deteriorated by amyloid and subsequent neurodegeneration and reduction of neurogenesis. Our results indicate a pathogenic role of HA in AD.

Treatment of Alzheimer's Disease with Anti-Homocysteic acid Antibody

AbstractHomocysteic acid (HA) may play an important role in Alzhiemer disease (AD) as we previously reported that HA induced accumulation of intraneuronal A[beta]42. In this study, we first analyzed HA levels in a mouse model of AD. 4-month old pre-pathologic 3xTg-AD mice exhibited higher levels of HA in the hippocampus as compared to age-matched nontransgenic, suggesting that HA accumulation may precede both A[beta] and tau pathologies. To further determine the pathogenic role of HA in AD, we treated young 3xTg-AD mice with vitamin B6-deficient food for 3 weeks to induce the production of HA in the brain. Concominantly, mice received either saline or anti-HA antibody intraventricularly using a guide cannula every 3 days. Mice received anti-HA antibody significantly rescued cognitive impairment induced by vitamin B6 deficiency. Pathologically, 3-week treatment with vitamin B-6 deficient food resulted in strong neurodegeneration in the hippocampal CA1 zone and decreased hippocampal volume. In contrast, anti-HA antibody treatment attenuated these pathological changes. Taken together, we conclude that increased brain HA triggers memory impairment whose condition was deteriorated by amyloid and subsequent neurodegeneration and reduction of neurogenesis. Our results indicate a pathogenic role of HA in AD.

Effects of cholinesterase inhibitor metrifonate on naive rats and rats with a model of hypoxia-induced impaired memory

Abstract Cholinesterase inhibitors are currently used in the therapy of different kind of dementia to improve brain memory functions. The acetylcholinesterase inhibitor metrifonate was studied in naive rats and in rats with a model of sodium nitrite-induced hypoxia. One active avoidance test and in two passive avoidance tests were used. In the active avoidance test metrifonate increased the number of avoidances during the learning session only. In both passive avoidance tests, metrifonate prolonged latency differently during the learning session and in short-term or in long-term memory retention. Hypoxic rats showed lower numbers of avoidances in learning and memory retention sessions. Metrifonate increased the number of avoidances during the learning session for hypoxic rats. In the step-through passive avoidance test, metrifonate increased the latency of reactions in the learning session and in long-term memory retention tests. In the step-down passive avoidance test, the groups with hypoxia and metrifonate did not change the latency of reaction in the learning and long-term memory retention sessions, but increased the latency of reactions in the short-term memory retention test. Morphological data showed a significant impaired neuronal structure in a CA1 zone of the hippocampus in hypoxic rats and a tendency to preserving in rats treated with metrifonate. Our results suggest that metrifonate improves cognitive functions in naive and in hypoxic rats.

Research on the relationship between brain anoxia at different regional oxygen saturations and brain damage using near-infrared spectroscopy

The objective of this paper is to investigate the difference in physiological parameters, EEG and morphology of brain tissues in newborn pigs with different regional oxygen saturations of brain (rSO2) and provide a basis for the determination of brain injury and degree of injury with the rSO2 in clinical practice. A noninvasive near-infrared spectroscopy (NIRS) technique was used to monitor the rSO2 of 27 newborn pigs. After mechanical ventilation and inhalation of 3–11% oxygen for 30 min by the newborn pigs, the pigs were grouped according to the rSO2 in the brain caused by inhalation of different concentrations of oxygen. There were six animals each in rSO2 < 30%, 30–35%, 35–40%, 40–50% groups and three animals in the rSO2 > 60% group (normal control). The physiological parameters and the EEG were monitored during the experiment. The animals were sacrificed by decollation at 72 hours after brain injury, and light microscope examination and pathological analysis of the ultrastructure were conducted on the brain tissues in the CA1 zone of hippocampi. In rSO2 > 40% groups, the mean arterial pressure (MAP) was stable and there were no significant changes in blood lactic acid, amplitudes of the EEG, light microscopic findings and ultrastructure after hypoxia. When the rSO2 was between 30% and 40%, the MAP was stable, the level of blood lactic acid increased, metabolic acidosis occurred, there was no significant change in the amplitudes of the EEG, there were ischemic changes in brain tissues under a light microscope and there was an injury of mitochondria in the neurons in the CA1 zone of hippocampi. When the rSO2 was less than 30%, circulatory failure occurred, the level of blood lactic acid increased, there was serious metabolic acidosis, the amplitudes of the EEG significantly decreased, there were vacuolization and broken fragments of cells under the light microscope and the mitochondria in the neurons in the CA1 zone of hippocampi were seriously injured. Under varying degrees of hypoxia, when the rSO2 is between 30% and 40%, brain injury occurs and the functional zones of mitochondria are injured in newborn pigs. When the rSO2 is less than 30%, the brain functions are seriously abnormal, and the serious morphological impairment in the functional zones of mitochondria is the basis for the disturbance of energy metabolism in brain neurocytes after hypoxia and the sequelae of the nervous system.

P-050: Distinct atrophy pattern in hippocampal subfields in Alzheimer’s disease (AD) and mild cognitive impairment (MCI)

The hippocampus is not a homogeneous structure but divided into several subfields with distinctive histological characteristics and functions, which are differently affected by aging and Alzheimer's disease. Therfore, volumetric measurements of individual hippocampal subfields might allow for a better distinction between aging and AD than measurement of hippocampus as a whole. High-resolution images at 4T depict details of the internal structure of the hippocampus sufficiently to allow for in vivo volumetry of different hippocampal subfields. To determine patterns of neuron loss in hippocampal subfields in AD, MCI. 58 subjects (37 cognitively elderly controls (mean age: 73.4±6.3), 8 MCI (mean age 76.7±6.9 and 13 AD (mean age 75.7±7.8) were studied on a 4T magnet with following sequences: 1. High resolution T2 weighted fast spin echo for manual marking of hippocampal subfields on five consecutive slices: ERC, subiculum, CA1, CA1&CA2 transition zone (CA1&CA2), CA& dentate gyrus (CA3&DG) and hippocampus (sum of CA1, CA1&CA2, CA3&DG) and 2. T2 weighted image for calculation of the total intracranial volume. ANOVA followed by post hoc analyses was used to test for group differences of subfield volumes normalized to total intracranial volume. Table 1 shows that AD had significantly (p<0.05) smaller volumes of ERC, subiculum, CA1, CA1&CA2 and hippocampus than controls but not of CA3&DG. Compared to MCI, AD had smaller volumes of ERC and subiculum but not of CA1, CA1&CA2 and hippocampus. When MCI were compared to controls, CA1&CA2 but none of the other subfields were significantly smaller (cf. Table 1). The finding that AD patients have significant volume loss in ERC, subiculum, CA1 and CA1&CA2 but not in CA3&DG is consistent with histopathology. In MCI, the volume loss was most pronounced in CA1&CA and was similar in magnitude to the change found in AD consistent with early pathology in this region. These preliminary findings suggest that measurements of hippocampal subfields might be a very sensitive measure for diagnosis of early AD and for monitoring of treatment effects.

Protective effects of iptakalim, a novel ATP-sensitive potassium channel opener, on global cerebral ischemia-evoked insult in gerbils1

To investigate the protective role of iptakalim, a novel ATP sensitive potassium channel opener, on global cerebral ischemia-evoked insult in gerbils and glutamate-induced PC12 cell injury. Global cerebral ischemia was induced by occluding the bilateral common carotid arteries in gerbils for 5 min. The open field maze and T-maze were employed to investigate the experimental therapeutic value of iptakalim on ischemic brain insult (n=8). The pyramidal cells in the hippocampal CA1 regions were counted to assess the protective effects of iptakalim. Glutamate released from the gerbil hippocampus and PC12 cells were determined by HPLC. Intracellular calcium was measured by Fluo-3 AM with A Bio-Rad Radiance 2100TM confocal system in conjunction with a Nikon TE300 microscope. Astrocyte glutamate uptake measurements were determined by liquid scintillation counting. Iptakalim (0.5-4.0 mg/kg per day, ip) could reduce the high locomotor activity evoked by ischemia and improve global cerebral ischemia-induced working memory impairments. Histological studies revealed that iptakalim could increase the survival neuron in the hippocampus CA1 zone in a dose-dependent manner. Moreover, iptakalim could reverse ischemia-evoked increases of glutamate in the hippocampus of gerbils. In an in vitro study, iptakalim protected PC12 cells against glutamate-induced excitotoxicity, reduced the [Ca(2+)](i) increases, and enhanced the glutamate uptake activity of primary cultured astrocytes. Iptakalim plays a key role in preventing global cerebral ischemia-evoked insults in gerbils and glutamate-induced PC12 cell injury by anti-excitotoxicity. Iptakalim might be a promising novel candidate for the prevention and/or treatment of stroke.

Diadenosine Polyphosphate Analog Controls Postsynaptic Excitation in CA3-CA1 Synapses via a Nitric Oxide-Dependent Mechanism

Previously, we have described the modulatory effect of diadenosine polyphosphates Ap4A and Ap5A on synaptic transmission in the rat hippocampal slices mediated by presynaptic receptors (Klishin et al., 1994). In contrast, we now describe how nonhydrolyzable Ap4A analog diadenosine-5',5'''-P1,P4-[beta,beta'-methylene]tetraphosphate (AppCH2ppA) at low micromolar concentrations exerts strong nondesensitizing inhibition of orthodromically evoked field potentials (OFPs) without affecting the amplitude of excitatory postsynaptic currents and antidromically evoked field potentials, as recorded in hippocampal CA1 zone. The effects of AppCH2ppA on OFPs are eliminated by a P2 receptor antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) but not mimicked by purinoceptor agonists alpha,beta-methylene-ATP and adenosine 5'-O-(3-thio)-triphosphate, indicating that a P2-like receptor is involved but not one belonging to the conventional P2X/P2Y receptor classes. Diadenosine polyphosphate receptor (P4) antagonist Ip4I (diinosine tetraphosphate) was unable to modulate AppCH2ppA effects. Thus, the PPADS-sensitive P2-like receptor for AppCH2ppA seems to control selectively dendritic excitation of the CA1 neurons. The specific nitric oxide (NO)-scavenger 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide is shown to significantly attenuate AppCH2ppA-mediated inhibitory effects, indicating that NO is involved in the cascade of events initiated by AppCH2ppA. Further downstream mediation by adenosine A1 receptors is also demonstrated. Hence, AppCH2ppA-mediated effects involve PPADS-sensitive P2-like receptor activation leading to the production of NO that stimulates intracellular synthesis of adenosine, causing in turn postsynaptic A1 receptor activation and subsequent postsynaptic CA1 dendritic inhibition. Such spatially selective postsynaptic dendritic inhibition may influence dendritic electrogenesis in pyramidal neurons and consequently mediate control of neuronal network activity.

Structural modifications of astrocytes in the hippocampus after experimental cerebral ischemia in gerbils

We studied reactions of astrocytes in the CA1 hippocampal zone of the mongolian gerbil (Meriones unguiculatus) after experimental short-lasting (7 min) cerebral ischemia resulting from bilateral occlusion of the carotid arteries. Immunocytochemical staining of hippocampal sections with antibodies against an astrocytes marker, glial fibrillary acidic protein (GFAP), was used. We measured the density of labelled cells in the layers of the CA1 zone at different time intervals (from 1 to 30 days) after cerebral ischemization. The number of labelled astrocytes within this period increased, and the dynamics of their density in different layers demonstrated significant dissimilarities. The earliest manifestations of reactive astrogliosis were observed in the hilus. The greatest rise in the number of astrocytes was found in the str. lacunosum-moleculare and str. moleculare on the 7th day, while in the str. pyramidale the maximum was reached only on the 14th day, which corresponded to the period of the highest intensity of delayed postischemic neuronal death. Thus, the intensity of morphological changes of the neurons and the level of reactivity of the astrocytes demonstrate a rather clear correlation; this fact can be one of the aspects of the dynamics of postischemic damage to the hippocampal neurons.