Conditioned Passive Avoidance Response Research Articles

Peculiarities of Fear Memory Disturbance in Male and Female <i>Disc1-</i>Q31L Mice

Depressive disorder is the most common psychopathology that can coexist with other mental illnesses such as post-traumatic stress disorder. It has been shown that there are gender differences in susceptibility to these psychopathologies. Mice of the mutant strain Disc1-Q31L are characterized by depressive-like behavior and disruption of the molecular pathways involved in the processes associated with fear memory. Gender and interstrain differences in the processes of learning and extinction of the conditioned response of passive avoidance were studied in male and female Disc1-Q31L mice and control C57BL/6 mice. It was shown that male and female mice of both strains learned equally well the conditioned response of passive avoidance, but differed in fear memory extinction, the ability to form a new safety memory trace in the previously dangerous dark compartment of the setup. However, there was a deficit in the extinction of the conditioned response of passive avoidance in C57BL/6 females compared to males, as well as interstrain differences in the dynamics of extinction in both females and males. Disc1-Q31L males reached full extinction later than C57BL/6 males, while Disc1-Q31L females did not exhibit extinction during the 24 days of the test. Thus, this work shows the interaction of the effect of gender and the Disc1-Q31L mutation on the processes of fear memory extinction.

SEX AND STRAIN DIFFERENCES BETWEEN DISC1-L100P AND C57BL/6 MICE IN PASSIVE AVOIDANCE CONDITIONING DEFINATION

Sex and strain differences in the passive avoidance learning and extinction in male and female of DISC1-L100P mice and wild-type control mice were investigated. It was shown that male and female mice of both strains were well trained in the conditioned response of passive avoidance, but differed in fear memory extinction, the ability to form a new memory trace about the safety of the previously punished dark compartment of apparatus in the procedure of repeated testing. There was a deficit of fear memory extinction in females of DISC1-L100P and C57BL/6 mice compared to males. At the same time, interstrain differences in the dynamics of passive avoidance extinction in female mice of these strains were revealed. Results also showed that DISC1-L100P males differed from C57BL/6 in terms of the time to achieve complete extinction of this reaction.

Psychopharmacological Effects of Stimulation of the Functions of Neural Stem Cells by STAT3 Inhibitor under Conditions of Modeled Ethanol-Induced Encephalopathy.

The psychopharmacological effects of a stimulator of functions of progenitor cells of the nervous tissue STAT3 inhibitor (STAT3 Inhibitor XIV, LLL12) were studied under conditions of modeled alcoholic encephalopathy in C57BL/6 mice. The pharmacological agent corrected the parameters of exploratory behavior (characterizing predominantly cognitive activity) in the experimental animals at the late terms of observation. At the same time, the reproducibility of the conditioned passive avoidance response developed at the beginning of the course STAT3 inhibitor administration decreased. These effects developed against the background of a significant increase in the content of neural stem cells and their proliferative activity in the paraventricular zone of the brain.

Restoration of Parkinson's Disease-Like Deficits by Activating Autophagy through mTOR-Dependent and mTOR-Independent Mechanisms in Pharmacological and Transgenic Models of Parkinson's Disease in Mice.

We studied the possibilities of inhibition of neurodegeneration in MPTP-induced model of Parkinson's disease (PD) in C57Bl/6J mice and transgenic model of early PD stage (5-monthold B6.Cg-Tg(Prnp-SNCA*A53T)23Mkle/J mice) by autophagy activation through mTOR-dependent and mTOR-independent pathways with rapamycin and trehalose, respectively. Therapy with autophagy inducers in a "postponed" mode (7 days after MPTP intoxication) restored the expression of the dopaminergic neuron marker tyrosine hydroxylase and markedly improved cognitive function in the conditioned passive avoidance response (CPAR; fear memory). The transgenic model also showed an increase in the expression of tyrosine hydroxylase in the nigrostriatal system of the brain. An enhanced therapeutic effect of the combined treatment with the drugs was revealed on the expression of tyrosine hydroxylase, but not in the CPAR test. Thus, activation of both pathways of autophagy regulation in PD models with weakened neuroinflammation can restore the dopaminergic function of neurons and cognitive activity in mice.

Effect of Mild Traumatic Brain Injury on Behavioral Reactions and Neocortical Morphology in Rats.

The original weight-drop model was employed to examine the effect of mild traumatic brain injury (TBI) on behavioral phenotype and neocortical morphology in rats. The neurological examination of rats with moderate TBI revealed the focal symptoms corresponding to pronounced neurological disorders, whereas in rats after mild TBI, there were only minor coordination disorders. On day 7 after injury, the rats with mild TBI demonstrated enhanced anxiety assessed by conditioned passive avoidance response. The morphometric analysis of the brain tissues revealed narrowing of the capillaries and increased score of hyperchromic neocortical neurons, which attested to cerebral hypoxia. The manifestations of mild TBI in original rat model demonstrated a close similarity to the symptoms of TBI in humans.

Hippocampal Neurogenesis, Dopaminergic Neurons of the Substantia Nigra, and Behavior after Intranasal Administration of Native α-Synuclein Protein to Ageing Mice

Abstract—We studied the effects of intranasal administration of native α-synuclein, whose hyperproduction and misfolding are the key link in the pathogenesis of Parkinson’s disease and other age-related neurodegenerative diseases, on hippocampal neurogenesis, the number of dopaminergic neurons in the substantia nigra, locomotor activity, learning, memory, and anxiety level in ageing animals. The experiments were performed with 12-month-old C57BL/6 male mice which were treated for 14 days with a solution of recombinant native α-synuclein or physiological saline once every 24 hours. To evaluate behavioral indices, the following tests were used: open field, novel object recognition, conditioned passive avoidance response, and elevated plus maze. Proliferating cells, immature neurons, and dopaminergic nerve cells were immunohistochemically detected using antibodies against bromodeoxyuridine, doublecortin, and tyrosine hydroxylase. It was demonstrated that α-synuclein causes a considerable increase in the number of proliferating cells and a decrease in the number of immature neurons in the dentate gyrus the hippocampus, as well as a decrease in the density of dopaminergic neurons in the substantia nigra pars compacta. Mice that received native α-synuclein demonstrated lower locomotion speed, impairments in long-term associative memory, and changes in anxiety-like behavior. We compared the obtained data with the previous results on the effects of α-synuclein oligomers and fibrils under similar experimental protocols. The data are viewed as experimental evidence of the impairment of postnatal neurogenesis during the development of synucleinopathies and support the notion that different α-synuclein conformations cause the development of different neurodegenerative diseases.

Vascular, molecular and cellular mechanisms of development of cognitive impairments after occlusion of carotid arteries

The study was aimed at determining the role of vascular endothelial dysfunction in induction of molecular and cellular mechanisms of the development of cognitive impairments after occlusion of common carotid arteries. The experiments were carried out on Wistar rats. The animals were divided into 2 groups: group 1 - control, sham-operated animals; group 2 - rats with cerebral ischaemia-reperfusion. The model of cerebral ischaemia-reperfusion was reproduced by means of simultaneous occlusion of common carotid arteries (for 5 minutes). The degree of endothelial dysfunction was assessed by determining the amount of circulating endotheliocytes and concentration of nitrogen oxide products in blood plasma. In the brain, we measured the content of malonic dialdehyde, the activity of superoxide dismutase and catalase. Pathomorphological studies of cerebral structures and morphometric analysis of the number of damaged neurons in ischaemic exposure were carried out by means of light microscopy. Cognitive functions in rats were assessed by means of conditioned reaction of passive avoidance and the test of pattern identification. Common carotid arteries occlusion in rats induced an increase in the level of circulating endotheliocytes and a decrease in end products of nitrogen oxide - nitrites in blood plasma as compared with the control (p≤0.05); the content of malonic dialdehyde in the brain increased 2.6-fold, the activity of superoxide dismutase and catalase decreased 5.9-fold and 2.8-fold, respectively (p≤0.05). The findings of pathomorphological examination registered signs of vasoconstrictive reactions, endothelial oedema, an increase in the proportion of damaged neurons in the cortex of greater hemispheres and hippocampus (p≤0.05). It was determined that occlusion of common carotid arteries in rats resulted in memory disorders revealed in tests of conditioned passive avoidance response and pattern identification (p≤0.05). Occlusion of common carotid arteries in rats becomes a cause of endothelial dysfunction, neurometabolic alterations, damage of neurons in vulnerable regions of the cerebral cortex, hippocampus and cognitive impairments. Damaging factors of the neurovascular system are intensification of oxidative processes and a decrease in the level of antioxidant defence, presenting important targets of neuroprotection.

Влияние электромагнитных излучений на ориентировочно-исследовательскую активность и когнитивные функции крыс с контрастной возбудимостью нервной системы

In recent years, the mechanisms of influence that technogenic sources of electromagnetic radiation and the change in the natural electromagnetic field have on biological objects have been actively investigated. Nevertheless, the role of ancestral conditional characteristics of the nervous system in susceptibility towards and resilience against electromagnetic field oscillations has been virtually ignored. The objective of our research is to investigate the impact of the UHF band radiation emitted by a standard Wi-Fi router and the impact of external magnetic and electric fields weakened by shielding on animals’ innate behaviour which reflects orientational and exploratory activity and emotional response in an Open Field Test, and also on the cognitive functions (retention of the conditioned response of passive avoidance) of male rats from two selected strains: HT and LT — with high and low thresholds of nervous system excitability, and control Wistar rats. The outcomes of the study confirm the negative impact of weak external electric and magnetic fields, as well as EMR of the UHF band, on innate behaviour and memory in rats regardless of the selected strain, although HT rats were more sensitive to changes in magnetic and electric fields and electromagnetic radiation in comparison with LT rats.

Effects of quercetin on the neuromotor function and behavioral responses of Wistar and Zucker rats fed a high-fat and high-carbohydrate diet

Quercetin can affect some pathological manifestations in obesity. The mechanism underlying the presumed therapeutic effect of quercetin is probably related to the influence on the central processes regulating energy homeostasis. Thus, the purpose of this study was to examine the effect of quercetin on the neuromotor and behavioral functions in Zucker (Z) and Wistar (W) rats with genetically and/or diet-induced obesity. Rats of both strains received balanced or high fat and fructose diet (HFCD) in a 62-day experiment or the same diets supplemented with quercetin at the dose of 50 mg/kg body weight per day. The neuromotor function and behavioral responses were examined using the grip strength test, open field test, elevated plus maze test and conditioned passive avoidance response (CPAR) test. The quercetin potentiated a decrease in anxiety in W rats consumed HFCD and this effect was absent in Z rats with a defect in the leptin receptor gene. In contrast, quercetin increased locomotor activity and impaired short-term memory in the CPAR test only in Z rats with the absence of normal leptin reception. Against the background of the identified changes quercetin exerted significant effects on the lipid and nitrogen metabolism indices such as HDL cholesterol, AsAT/AlAT activities ratio, urea level as well as body and fat mass that were different in Z and W rats. The data obtained show that the effects of quercetin on behavior vary significantly between two strains of rat and consequently are mediated by processes of leptin reception.

Стан мнестичних процесів і рівень білків клітинної адгезії (NCAM) у гіпокампі щурів за умов введення S-аденозил-L-метіоніну та пре/пробіотиків на тлі медикаментозно-індукованого ураження печінки

Мета дослідження – вивчення впливу S-аденозил-L-метіоніну, пре/пробіотиків та їхнього сумісного застосування на мнестичні процеси та рівень білків клітинної адгезії (NCAM) у гіпокампі щурів за умов тривалого введення рифампіцину та ізоніазиду. Дослідження проводили на 68 білих щурах-самцях лінії Wistar масою 180–220 г. Експериментальну модель токсичного медикаментозно-індукованого ураження печінки (МІУП) відтворювали шляхом повторних внутрішньошлункових (в/ш) введень ізоніазиду та рифампіцину у дозах 50 мг/кг і 86 мг/кг маси тіла відповідно протягом 28 діб. Тварин було розподілено на п’ять груп (n = 8 у кожній). І – інтактна, ІІ – контроль (МІУП). Щурам ІІІ групи протягом останніх 14 діб експерименту за 1 год до введення туберкулостатиків внутрішньом’язово вводили S-аденозил-L-метіонін у дозі 35 мг/кг. Щури IV групи в/ш отримували комбіновану терапію, що поєднувала пребіотик Лактулозу в дозі 2680 мг/кг і пробіотик, що містить 4 млрд активних клітин (КУО): Lactobacillus acidophilus, Lactobacillus rhamnosus, Streptococcus thermophilus, Lactobacillus delbrueckii subsp. Bulgaricus у дозі 1 КУО/кг. Тваринам V групи проводили потрійну комбіновану фармакотерапію з усіма препаратами, які застосовували в ІІІ і ІV групах у відповідних дозах. Ноотропну активність оцінювали в тесті умовної реакції пасивного уникнення (УРПУ) за умов введення скополаміну. Для оцінки впливу скополаміну на процеси навчання використані значення показників груп тварин (інтактні, n = 10; МІУП, n = 18), які попередньо не отримували цей препарат. Кількісний уміст NCAM визначали за допомогою інгібіторного методу імуноферментного аналізу з використанням моноспецифічних антитіл щодо NCAM (Аbcam, Англія) і відповідного очищеного білка як стандарту (Аbnovo, США) у цитозольній фракції гомогенату гіпокампа. Отриманий цифровий матеріал обробляли методом варіаційної статистики за допомогою програми статистичного аналізу StatPlus, AnalystSoft Inc. Версія 6 (http://www.analystsoft.com/ru/). Результати експериментів свідчать про те, що курсове введення S-аденозил-L-метіоніну покращувало процеси навчання за умов введення скополаміну, що проявлялося суттєвим збільшенням латентного періоду в 3,14 разу (Р = 0,027) порівняно з групою тварин з МІУП, а число тварин з набутою навичкою складало 62,5 % (Р = 0,007). При цьому за умов введення цього препарату спостерігалося зростання на 38,7 % (Р = 0,004) рівнів цитоплазматичної форми NCAM у гіпокампі, яке ймовірно мало компенсаторний характер. Характерно, що корекція стану мікробіоти кишечника також може чинити позитивний вплив на мнестичні функції за умов тривалого введення туберкулостатиків, однак за своїм ноотропним потенціалом комбінація Лактулоза/Йогурт поступалася S-аденозил-Lметіоніну, хоча рівні NCAM у гіпокампі зростали на 39,6 % (Р = 0,004) порівняно з групою тварин з МІУП. Слід зазначити, що сумісне застосування пребіотика та пробіотика з S-аденозил-L-метіоніном не супроводжувалося підвищенням ноотропної активності. Отримані дані експериментально обґрунтовують застосування насамперед S-аденозил-Lметіоніну, а також пре/пробіотиків як перспективних засобів корекції когнітивних порушень за медикаментозних гепатитів. Необхідні подальші дослідження щодо можливості комбінованого використання зазначених засобів за умов вказаної патології.

Comparative analysis of the influence of a high-fat/high-carbohydrate diet on the level of anxiety and neuromotor and cognitive functions in Wistar and DAT-KO rats.

We compared anxiety, neuromotor, and cognitive functions in mutant rats with different allelic variants of dopamine transporter DAT knockout receiving balanced or excess in fat and fructose diet. The experiments were performed in DAT−/− homozygotes, DAT+/− heterozygotes, and DAT+/+ wild type rats. The genotype of DAT‐KO rats was confirmed by restriction analysis of DAT gene compared to behavioral responses in the open field test (OF). Animals in the first groups of each strain were fed a balanced AIN93M diet; and those in the second groups with a high‐fat/high‐fructose diet. Neuromotor function was studied as grip strength, and behavioral responses were assessed in the elevated plus maze and conditioned passive avoidance response tests. The mass of the internal organs and white and brown fat, as well as selected lipid and nitrogen metabolism parameters in blood plasma were determined at the end of the experiment. DAT−/− had the highest specific grip strength, and showed an increase in initial exploratory activity in comparison with DAT+/− and DAT +/+. The exploratory activity was significantly reduced in the second test compared to the first one in DAT−/− and DAT+/− of first but not second group. Anxiety decreased with age in the second groups of DAT+/− and DAT+/+ (but not in DAT−/−) and was higher in DAT+/+ than in DAT+/− and DAT−/−. Excess fat and fructose resulted in the deterioration of short‐term memory in DAT+/+. Lipidomic indices of blood plasma were less responsive to diet in DAT−/− and DAT−/+ in comparison to DAT+/+. The increased AsAT/AlAT activity ratio in DAT−/− compared with those in DAT+/+ suggests the activation of catabolism activity in the mutants. The consumption of excess fat and fructose significantly modified the effects produced by DAT gene allelic variants presumably due to the influence on the processes of dopamine metabolism.

Cognitive impairments and microvascular endothelial dysfunction in unilateral occlusion of the carotid artery

The study was aimed at determining cognitive impairments, vascular endothelial dysfunction and morphofunctional alterations of neurons in brain structures (neocortex and hippocampus) in unilateral occlusion of the common carotid artery in albino rats. The experiments were carried out on Wistar albino rats. The animals were divided into two groups: group 1 - the control group composed of sham-operated rats and group 2 - the study group consisting of rats with cerebral ischaemia. The model of cerebral ischaemia was reproduced by occlusion of the left common carotid artery. Cognitive functions in rats were assessed by means of the object recognition test and conditioned passive avoidance response (CPAR). The degree of vascular endothelial dysfunction was evaluated by the number of circulating endotheliocytes and concentration of end products of nitric oxide - nitrites in blood plasma. Pathomorphological studies of the brain and morphometric analysis of the number of damaged neurons after ischaemic exposure were carried out by means of light microscopy. It was determined that unilateral occlusion of the common carotid artery in rats resulted in memory impairment revealed by the object recognition test and CPAR (p≤0.05). Cerebral ischaemia induced an elevated level of circulating endotheliocytes and a decrease in end products of nitric oxide - nitrates as compared with the controls (p≤0.05). Morphological study demonstrated signs of vasoconstrictive reactions, microvascular endothelial oedema, as well as an increase in the proportion of damaged neurons localized in the fronto-bregmatic region of the cortex of the cerebral hemispheres and hippocampus on the ipsilateral side (p≤0.05). Unilateral occlusion of the common carotid artery in albino rats resulted in cognitive impairments, damage of neurons in the most vulnerable areas of the cortex of the cerebral hemispheres and hippocampus predominantly on the ipsilateral side. Cognitive impairments and ischaemic lesions of the brain structures are induced by endothelial dysfunction, enhanced desquamation of endotheliocytes and prevalence of vasoconstrictive reactions resulting from decreased production of the major vasorelaxing factor - nitric oxide.

Interspecific Differences in Behavioral Responses and Neuromotorics between Laboratory Rodents Receiving Rations with Easily Digested Carbohydrates.

We assessed the effect of intake of easily digested carbohydrates for 133 days on quantitative parameters of neuromotorics and cognitive function in Wistar rats and C57Bl/6J mice. Neuromotorics (muscle tone) was assessed in rats and mice by the forelimb muscle force (grip strength) over 4 months. Anxiety was assessed in the elevated plus-maze test and cognitive function (short-term and long-term memory) was evaluated by conditioned passive avoidance response (CPAR) test over 3 months. The mice, in contrast to rats, receiving the diet with easily digested sugars demonstrated suppression of neuromotorics. Anxiety increased with age in female mice, but not in rats, irrespective of the diet. Cognitive function in rats receiving experimental rations did not change significantly in comparison with the control. In mice, consumption of equimolar mixture of fructose and glucose impared short-term, but not long-term memory, in comparison with the group receiving glucose alone. We revealed a small (by 14-17%), but statistically significant increase in the brain weight in mice receiving fructose and sucrose. The study demonstrates sufficient interspecies differences in the influence of carbohydrate rations on neuromotorics and behavioral responses in the in vivo metabolic syndrome model.

Effect of carnosine on conditioned passive avoidance response in the norm and under hypoxia conditions

We studied the effect of the histidine-containing dipeptide carnosine in the norm and under hypoxia conditions on the conditioned passive avoidance response in a three-compartment chamber, which consisted of a light compartment, a dark dangerous compartment where the rat received a foot shock, and a dark safety compartment, where the rat did not receive a foot shock. We found that carnosine normalized physiological indices, which were impaired by acute exposure to hypobaric hypoxia, and increased the number of choices of the safety compartment in both intact rats and rats subjected to hypoxia.

Nootropic Effects of Filipendula Vulgaris Moench Water Extract Fractions.

Nootropic activity of water extract fractions from aerial parts of Filipendula vulgaris Moench was demonstrated on the models of hermetic volume hypoxia, conditioned passive avoidance response, open field test, and forced swimming with a load. The fractions stimulated hypoxic resistance, normalized orientation and exploratory behavior, improved conditioned response reproduction during testing after hypoxic injury, and increased exercise tolerance. Fractionation of the extract led to dissociation of the effect components, which suggests that individual constituents have specific characteristics. Ethylacetate fraction exhibited most pronounced nootropic activity and was superior to plant extract by some characteristics. The detected effects seemed to be caused by modulation of the hippocampus activity the under the effects of phenol and triterpene compounds.

Dynamics of cognitive disturbances in rats with acute cerebral ischemia on the background of introduction of 0.9 % solution NaCl

For today there are no clear recommendations on infusion therapy at the disease and injuries of brain, and infusion preparations are commonly used empirically. Within the framework of the complex study of an influence of the different infusion remedies on brain at an experimental ischemia of brain, an aim of this work is to investigate how the 0,9 % solution of NaCl influences on the dynamics of cognitive functions and neurological status of the rats with an acute cerebral ischemia at the course medical introduction into an animal organism. Methods . Experiments were carried out on 60 white rats-males. An acute disturbance of cerebral blood circulation (ADCBC) was modeled by means of the two-sided dressing of internal carotid arteries. The 0,9 % solution of NаСІ was injected intravenously in catheterized thigh vein 2,5 ml/kg 2 times/day (5 ml/kg for a day). The first introduction was carried out in 30 minutes after ADCBC and then every day in 12 hours during 7 days. The control groups consist of intact rats that received 0,9 % NаСІ and animals with a model ischemia without treatment. Neurological deficiency in animals was defined on the scale stroke-index McGrow C. P. The dynamics of position-finding activity was assessed in experiment “an open field”. An assessment of animal capacity to training and memorization of an aversive stimulus was studied in the test of conditioned response of passive avoidance. The results were processed using the program StatPlus 2009. Results . The study showed that bilateral carotid occlusion in rats without treatment is followed not only by the high animal lethality but also by the development of a hard neurological symptomatology and then by essential disturbance of mnestic functions in animals that survived during the recovery period of model insult (р<0,01). The study of lethality dynamics, neurological status, behavioral responses in rats with ADCBC on the model of bilateral carotid occlusion showed that the course therapeutic infusion of 0,9 % solution of NaCl had some advantages relative to the group of the control pathology on indicators of neurological deficiency, orientating and searching activity, capacity to training and memorization. Conclusions. Infusion therapy with 0,9 % solution of NaCl in rats with the model cerebral ischemia in some degree promotes the renewal of its cognitive functions (р<0,05) relative to the untreated animals. Therapeutic effect of infusion therapy obtained in experiment with isoosmolar 0,9 % solution of NaCl is a reason to study protective actions of infusion preparations of the other groups at an acute disturbance of cerebral blood circulation

Nootropic effect of meadowsweet (Filipendula vulgaris) extracts.

The effects of the extracts of the aboveground parts of Filipendula vulgaris Moench on the behavior and memory of mice after hypoxic injury and their physical performance in the open-field test were studied using the models of hypoxia in a sealed volume, conditioned passive avoidance response (CPAR), and forced swimming with a load. The extracts improved animal resistance to hypoxia, normalized orientation and exploration activities, promoted CPAR retention after hypoxic injury, and increased physical performance. Aqueous extract of meadowsweet had the most pronounced effect that corresponded to the effect of the reference drug piracetam. These effects were probably caused by modulation of hippocampal activity.

Dynamics of behavioral disorders in transgenic mice with modeled Alzheimer's disease.

Age-related development of behavioral disorders in transgenic mice with modeled Alzheimer's disease carrying V6S3-Tg(APP695)85Dbo Tg(PSENI)85Dbo) genotype was assessed at the age of 7.5, 10 and 20 months in the following tests: open-field, plus maze, T-maze, conditioned passive avoidance response, rotarod, conflict situation with water deprivation, behavioral despair, and arecoline tremor. The main behavioral disorder in transgenic mice at all observation terms was memory impairment in conditioning with positive (but not negative) reinforcement. At the age of 7.5 and 10 months, transgenic mice also showed signs of nonspecific excitement and anxiety, depression-like state, and symptoms of cholinergic deficit. Our results suggest that appropriate age for behavioral tests in studies of effects of potential anti-Alzheimer drugs in transgenic V6S3-Tg(APP695)85Dbo Tg(PSENI)85Dbo) mice is 7.5-10 months.

P.1.g.031 The neuroprotective and anti-amnestic effects of carbamylated erythropoietin derivates in ischemic brain injury

Purpose: To compare the neuroprotection of carbamylated erythropoietin (CEPO) and EPO fusion protein containing TR domain from glycoprotein MUC1 (CEPO-TR), and EPO fusion protein with modified Fc fragments of IgG1 (CEPO-Fc) against ischemic brain injury, including behavioral disturbances in a bilateral focal ischemic infarction of the rat prefrontal cortex. Methods: The carbamylated erythropoietin and erythropoietin derivates were produced by treatment of purified proteins with potassium cyanate in borate buffer. The resulting carbamylated erythropoietin and derivates exhibit no erythropoietic activity in UT-7/EPOR cell viability assay. Bilateral focal ischemic infarction of the prefrontal cortex (areas Frl and Fr2) was induced by the method of photochemical thrombosis in rats. After passive avoidance training and testing rats were treated, respectively, with following regimens: saline, 50mg/kg CEPO, 50mg/kg CEPO-TR, 50mg/kg CEPO-Fc. The substance was injected intraperitoneally in 1 h after operation. Neurological deficit scores and infarct volume were assessed at 4 and 7 days after operation. Functional state of CNS was determined by conditioned passive avoidance response (PA), i.e. by the latency of transition from light compartment to dark compartment (in sec). Morphometric measurements of the areas and volumes of the ischemic focus on serial slices were carried out on animal brain fixed by plunging into formalinethanol-acetic acid mixture (2:7:1). The data were statistically processed using Statistica 6.0 software. Summary of results: Additional oligosaccharides, linked to the erythropoietin, prolong its half-life and increase bioactivity in vivo. For this purpose we, for the first time, used TR domain from glycoprotein MUC1, bearing 5 additional sites for O-glycosylation. Our result indicated that ligation of TR-domain to the coding sequence of EPO did not affect secretion of the chimeric protein into the medium, receptor binding affinity in vitro bioactivity, compared with EPO wild type. However, both the in vitro potency and half-life in circulation of EPO bearing TR or Fc fragments were significantly enhanced. After photothrombosis and injection of saline, the latency of entry into the dark compartment decreased from 300 s to 76 s. Treatment of rats with CEPO, CEPOTR, and CEPO-Fc after photothrombosis restored latency of PA to 243, 258, 227 s, respectively, on day 4. Only injection of CEPOTR retains passive avoidance latency 207 s on the day 7. Ischemic damage volume in animals treated with CEPO-TR on 7th day after operation was 15.8±5.4 against 24.22±4.7 with control injection of saline (P< 0.05). This phenomenon on 7th day was observed only with CEPO-TR. Protection efficiency coefficient, calculated from these experiments represented 34%. Another erythropoietin derivate did not give statistically significant results. Conclusion: Treatment of rats with CEPO and CEPO fusion proteins after photothrombosis of cortex resulted in restoration of passive avoidance response and diminishing of volume of ischemic damage. Those, our study indicates that CEPO-Fc and CEPO-TR display neuroprotectionand anti-amnestic activity, while CEPO-TR demonstrates prolongation ability.

Latent Inhibition and Extinction of a Conditioned Passive Avoidance Reaction in C57BL/6J and DBA/2J Mice

Comparative analysis of the development of two interference processes – latent inhibition and the extinction of a conditioned passive avoidance reaction acquired using unconditioned stimuli with different levels of aversiveness (0.5 and 0.25 mA) – was performed in C57BL/6 J and DBA/2 J mice. In “strong” training, impairment of extinction to a new stimulus was seen only in DBA/2J mice. Decreases in punishment strength during training were accompanied by acceleration of extinction in C57BL/6J mice and the appearance of extinction in DBA/2J mice. Formation of the memory trace using both “strong” and “weak” reinforcement was identical for mice of both strains. Interstrain differences were also seen on analysis of latent inhibition. In “strong” and “weak” training, conditioned stimuli which had lost novelty due to eight pre-exposures in the experimental chamber showed latent inhibition in DBA/2J mice but not C57BL/6J mice. In “weak” training, DBA/2J mice also showed no extinction of the conditioned passive avoidance response to the irrelevant stimulus.