Significance of IL-6 Deficiency in Recognition Memory in Young Adult and Aged Mice.

Chronic peripheral elevation of interleukin 6 (IL-6) in humans is associated with cognitive deficits. 4- and 24-month-old IL-6-deficient C57BL/6J (IL-6KO) and reference wild-type (WT) mice were tested in an object recognition test. Discrimination ratios and recognition indexes were significantly lower in 4-month-old IL-6KO and in 24-month-old WT mice vs 4-month-old WT animals. Their discrimination ratios had negative values and recognition indexes were below 50% indicating inability to differentiate the novel from the familiar object after 1-hour delay. In 24-month-old IL-6KO mice recognition index reached 53.17% indicating that their recognition memory was not worsened with age in comparison with younger IL-6-deficient animals. Results of holeboard and elevated plus maze indicated that this effect was memory specific. Inborn IL-6 deficiency attenuated recognition memory in 4-month-old mice and did not altered recognition memory in aged animals. IL-6 signalling may constitute a target for development of the protection against memory disturbances connected with IL-6 overexpression.

Expression of collagens type I and V in healing rat's tympanic membrane.

OBJECTIVES: Our preliminary study performed on perforated rat's tympanic membrane (TM) using Rat Wound Healing RT2 Profiler PCR Array showed significantly increased levels of mRNA for collagens type I and V. Enhanced expression of those genes does not assure that their protein products are indeed present, and in what quantity. Therefore, this study was undertaken to analyze the collagen type I and V content in the healing TM. METHODS: Sixty rats were used, of which 10 served as controls and the others had their TM perforated. The experimental animals were divided into five subgroups on the basis of time points (03, 06, 09, 14, 20 day after injury). Videootoscopy and histology were employed to assess the morphology of the healing process. The expression of collagen type I and V was evaluated using Western blot analysis. Tissue localization of collagens was determined by the immunofluorescence method. RESULTS: The collagen type I expression was three times higher on the third day after injury and remained on that level for whole period of observation, up to day 20. The increase of the collagen type V expression was gradual, reaching the highest level on day 14 following injury. In comparison to the control TM statistically significant increase in the level of expression was observed starting from day 09 to the end of observation period. In healing TM immunofluorescent labeling of collagen type I and V was seen on the surface of remnants of previous lamina propria and in the loose proliferating fibrous tissue. On day 20 immunofluorescence was present mainly on the surface of thin connective tissue layers forming the scar in the place of previous perforation. CONCLUSION: Although the collagens type I and V are present only in subepithelial layer in the normal rat's TM they play significant role in TM healing process.

IL-6 deficiency alters spatial memory in 4- and 24-month-old mice.

Significance of interleukin 6 (IL-6) deficiency in cognitive processes was evaluated in 4- and 24-month-old C57BL/6J IL-6-deficient (IL-6 KO) and control (WT) mice in Morris water maze (MWM), holeboard test (HB) and elevated plus maze (EPM). During 3-day learning escape latency time (ELT) was longer in IL-6 KO than in WT mice, however their swimming was slower, floating longer, and path length did not differ. The comparison of ELT and the distance traveled between the first and the third learning day within each group revealed significant decrease of ELT in all groups with the highest difference in 4-month-old WT mice, and significant decrease of distance traveled only in both groups of WT mice. In a single probe trial, performed 24 h after the last learning session, there were no major differences in the absolute values of ELT, but ELT turned out to be significantly shorter in both IL-6 KO groups, when it was compared to the ELT on the last learning day, indicating on better memory retrieval. In HB test only significant increase in number of rearings in aged WT mice, and in EPM significant prolongation of open arm time and higher number of open arm entries in 4-month-old IL-6 KO mice were observed. Results of HB and EPM tests showed that alterations of learning and reference memory observed in MWM were specific to cognition. Attenuation of learning ability in young adult IL-6-deficient mice assessed in MWM suggests that physiological level of IL-6 is involved in mechanisms engaged in proper memory formation, and it may also indicate on the importance of IL-6 signaling in brain development. Maintained on similar level in both 4- and 24-month-old IL-6 KO mice learning ability and its attenuation in 24-month-old vs 4-month-old WT mice indicates on slower age-related memory decline in mice not expressing IL-6. Better performance of IL-6 KO mice in the probe trial points to their reference memory improvement and may also indicate that IL-6 plays a role in mechanism responsible for cognitive flexibility.

Facilitatory Effect of IL-6 Deficiency on Long-Term Spatial Memory in Young Adult Mice.

The significance of interleukin 6 (IL-6) in long-term reference memory was tested in the Morris water maze (MWM) in 4-month-old C57BL/6J IL-6-deficient (IL-6 KO) and control mice. Three-day learning measured by escape latency time to find the hidden platform was comparable in both genotypes. In a single probe trial performed 7 days later, without the platform, latency to the platform site and path length to the target place were significantly shorter (p < 0.05 and p < 0.02, respectively), and platform-site crossovers more frequent (p < 0.05) in IL-6 KO mice. The swimming speed in IL-6 KO mice was significantly lower during learning (p = 0.0025) but not in the probe trial. Lack of differences between genotypes in a hole-board and in an elevated plus maze indicates that the observed effects were memory specific. The facilitatory effect of IL-6 deficiency on long-term reference memory in MWM indicates that IL-6 plays a role in consolidation process.

Facilitatory effect of AM281 on recognition memory in rats.

BACKGROUND: Our approach was to determine the influence of a single systemic administration of AM281, synthetic cannabinoid structurally similar to SR141716A, on recognition memory in rats. METHODS: To assess the influence of AM281 on acquisition of information the compound was given intraperitoneally once, at the doses of 0.1, 0.5, 1.0 or 2.0mg/kg, 15min before learning trial (T1) and in order to evaluate its influence on consolidation process AM281 was given at indicated doses, immediately after T1 trial in an object recognition test. Since cannabinoids may alter motor function and affect anxiety, the influence of AM281 on psychomotor activity and anxiety was evaluated in an open-field and elevated plus maze test, respectively. RESULTS: Administration of AM281 at the doses: 0.1, 0.5 and 1.0mg/kg significantly improved acquisition of information, while 0.1 and 0.5mg/kg of AM281 significantly facilitated consolidation process. Not only did AM281 not affect locomotor and exploratory activity, but also anxiety. CONCLUSION: This is the first evidence that AM281 exerts facilitatory effect on recognition memory in rats. This effect seems to be memory specific because no alterations in animals' psychomotor activity and anxiety were observed.

Enhanced expression of hepatocyte growth factor in the healing of experimental acute tympanic membrane perforation.

OBJECTIVES: The present study was performed to investigate the expression of hepatocyte (HGF), epidermal (EGF) and vascular endothelial (VEGF) growth factors in the course of healing of experimental tympanic membrane (TM) perforations in rats. The goal was to explain the role of these growth factors in the healing process of TM and to assess the possibility of their future application as healing promoters. METHODS: Seventy rats were used, of which 10 served as controls and the others had their TM perforated. The experimental animals were divided into six subgroups on the basis of time points (01, 03, 05, 07, 09, 15 day after injury). Videootoscopy and histology were employed to assess the morphology of the healing process. The expressions of HGF, EGF and VEGF were evaluated using Western blot analysis. Tissue localization of HGF was determined by the immunofluorescence method. RESULTS: HGF was hardly detectable in normal TM; however, a significant increase was noted in its expression starting from the third day after injury throughout the follow-up period, with the highest level on day 05. The analysis of HGF tissue localization with immunofluorescence revealed diffuse staining in the cytoplasm of proliferating epithelial cells. The expression of EGF was elevated on the first day after injury, not reaching statistical significance, and then returned to the level observed in the control TM. No significant differences were noted in the expression of VEGF. CONCLUSION: High expression of HGF during the healing process of acute TM perforations makes it a promising candidate for further studies oriented towards its possible use in augmentation of TM healing.

CP55,940 attenuates spatial memory retrieval in mice.

BACKGROUND: Cannabinoids constitute a varied group of lipophilic substances able to infiltrate the blood-brain barrier and influence neuronal processes. Clinical observations supported by experimental data have revealed that these compounds exert a deleterious effect on cognitive processes. The present study was carried out to determine the influence of a single systemic administration of CP55,940, a potent synthetic agonist of cannabinoid receptors, on spatial memory retrieval assessed in a Morris water maze. METHODS: C57BL/6J male mice were submitted to three consecutive days of training to find a hidden platform in the water maze. CP55,940 was given intraperitoneally once, at doses of 0.025, 0.125 or 0.25mg/kg on the fourth day, 30min before testing memory retrieval, and in separate groups before testing psychomotor activity and anxiety level in a hole-board test. RESULTS: CP55,940 only at the highest dose of 0.25mg/kg significantly altered all parameters used to assess spatial memory. It increased the latency in the first crossing of the former platform location (target area), decreased the number of target area crossings and shortened the time spent in the target quadrant. Moreover, CP55,940 at doses of 0.25 and 0.125mg/kg attenuated motor and exploratory activity in hole-board test. CONCLUSION: Since the attenuated psychomotor activity after a dose of 0.125mg/kg did not interfere with memory retrieval, we assume that the impairment of spatial memory observed after the highest dose of CP55,940 (0.25mg/kg) was exerted by its influence on cognitive processes, however, the impact on locomotion could not be excluded.

[The healing process of tympanic membrane perforations in rats]. / Proces gojenia perforacji blon bebenkowych u szczurów.

INTRODUCTION: Tympanic membrane (TM) perforations are commonly seen in clinical practice as a result of trauma or in the course of otitis media. The TM is a unique structure suspended in air which makes its healing processes different than in the skin wounds. The aim of the study was otoscopical and histological evaluation of the rat's TM healing process. MATERIAL AND METHODS: 56 male Wistar rats were used for the study. Fifty of them had TMs perforated bilaterally using CO2 laser, additional 6 served as a controls. The animals were sacrificed on either day 1, 2, 3, 6 and 10 post injury. Process of healing was assessed otoscopicaly, subsequently TM were dissected and processed for histological evaluation. RESULTS: At day 6 after perforation half and on day 10 all of TM were healed. On the first day, in histological evaluation, focal thickening of the epithelial layer was observed at some distance from the edge of perforation, on the side of annulus. On the following day proliferation of epithelium covering outer surface of TM on the side of the malleus handle and annulus was clearly visible. An eosinophilic mass containing macrophages and granulocytes was seen in front of the migrating epithelium. On day 3-6 migrating epithelium reached the edge of perforation. Proliferation of the connective tissue layer followed the epithelium. CONCLUSIONS: The present results indicate that the squamous epithelium covering the outer surface of TM constitutes the first layer which restores continuity of TM. The proliferation of the connective tissue occurs in the direct vicinity of the proliferating and migrating epithelium.

Analysis of gene expression profiles in tympanic membrane following perforation using PCR Array in rats--preliminary investigation.

OBJECTIVES: The goal of this work was to identify genes, known to be involved in the skin wound healing, that express differentially in the healthy and injured tympanic membrane (TM), and designate the molecules potentially beneficial for treatment of TM perforation. The molecular mechanisms controlling the course of TM regeneration are far from being elucidated. METHODS: Twenty rats had their tympanic membranes perforated, while four served as a control. Animals were sacrificed on either days 1, 2, 3, 5 and 10 post injury, and TMs were immediately dissected and frozen in liquid nitrogen. Total TM RNA was isolated and reversely transcribed. qPCR was performed using Rat Wound Healing RT(2) Profiler PCR Array (QIAGEN) containing primers for 84 genes. RESULTS: Statistically significant changes in the expression of 42 genes were found in various stages of TM healing. The increased expression of genes taking part in the inflammatory reaction (interleukin 6, granulocyte and macrophage chemotactic proteins) was observed from day 2. The expression of several genes of extracellular matrix components and their remodeling enzymes was also changed. Among growth factor genes: Vegfa, Igf1 and Hbegf showed increased expression at the beginning of the healing process, while Hgf expression was highest on day 3. CONCLUSIONS: Several changes in the expression of genes involved in remodeling of extracellular matrix point to important role of connective tissue in TM healing. The molecules accelerating this process, like HbEGF and HGF, seem to be good candidates for further evaluation of their possible use in clinical treatment.

CCN1 expression in interleukin-6 deficient mouse kidney in experimental model of heart failure.

Chronic heart failure often leads to worsening of the renal function. Mediators of this process include inflammatory and neuroendocrine factors. CCN1 (Cyr 61), a member of growth factor-inducible immediate early genes, which modulates inflammation and fibrogenesis, is excreted with urine in the early phase of acute renal injury and may be involved in the pathogenesis of the cardiorenal syndrome. The aim of the study was to evaluate CCN1 protein abundance and localization in the kidney of IL-6-deficient C57BL/6J (IL-6 KO) mice and respective wild-type (WT) animals in basal conditions and in animals with chronic heart failure twelve weeks after myocardial infarction. Age- and sex-matched mice from both strains subjected to sham operation served as controls. One group of WT animals subjected to myocardial infarction was treated with antagonist of AT1 receptor telmisartan over 12 weeks. Abundance and localization of CCN1 protein in kidney were assessed with Western blotting and immunohistochemistry, respectively. In all groups the strongest immunohistochemical reaction for CCN1 was observed in distal convoluted tubules and in smaller arteries, however, the total expression of CCN1 protein was lower in IL-6 KO mice in comparison to WT animals. The main difference in CCN1 distribution between the examined genotypes was lack of reaction in internal renal medulla and very weak reaction in proximal convoluted tubules in IL-6 KO mice. Experimental heart failure only slightly attenuated the expression of CCN1 protein in the kidney of WT mice and had no effect in IL-6 KO mice. Although, blockade of AT1 receptor did not alter CCN1 protein expression in kidneys of WT mice after myocardial infarction, it significantly changed its CCN1 distribution in the renal tubular system.

Effects of CP 55,940--agonist of CB1 cannabinoid receptors on ghrelin and somatostatin producing cells in the rat pancreas.

Cannabinoids participate in the modulation of numerous functions in the human organism, increasing the sense of hunger, affecting carbohydrate and lipid metabolism, and controlling systemic energy balance mechanisms. Moreover, they influence the endocrine system functions, acting via two types of receptors, CB1 and CB2. The aim of the present study was to examine the number, distribution and activity of ghrelin and somatostatin producing endocrine cells in the pancreas of rats after a single administration of selective CP 55,940 agonist of CB1 receptor. The study was performed on 20 rats. Neuroendocrine cells were identified by immunohistochemical reactions, involving specific antibodies against ghrelin and somatostatin. The distribution and number of ghrelin- and somatostatin-immunoreactive cells were separately studied in five pancreas islets of each section. A performed analysis showed a decreased number of somatostatin-immunoreactive cells and a weak immunoreactivity of ghrelin and somatostatin containing neuroendocrine cells in the pancreatic islets of experimental rats, compared to control animals. The obtained results suggest that a single administration of a selective CP 55,940 agonist of CB1 receptor influences the immunoreactivity of endocrine cells with ghrelin and somatostatin expression in the pancreas islets.

Cannabinoids enhance gastric X/A-like cells activity.

It has been reported that cannabinoids may cause overeating in humans and in laboratory animals. Although, endogenous cannabinoids and their receptors (CB1) have been found in the hypothalamus, and recently also in gastrointestinal tract, the precise mechanism of appetite control by cannabinoids remains unknown. Recently, ghrelin--a hormone secreted mainly from the stomach X/A-like cells was proposed to be an appetite stimulating agent. The aim of this study was the evaluation of the influence of a single ip injection of a stable analogue of endogenous cannabinoid--anandamide, R-(+)-methanandamide (2.5 mg/kg) and CP 55,940 (0.25 mg/kg), an exogenous agonist of CB1 receptors, on ghrelin plasma concentration and on ghrelin immunoreactivity in the gastric mucosa of male Wistar rats. Four hours after a single injection of both cannabinoids or vehicle, the animals were anaesthetized and blood was taken from the abdominal aorta to determinate plasma ghrelin concentration by RIA. Subsequently, the animals underwent resection of distal part of stomach. Immunohistochemical study of gastric mucosa, using the EnVision method and specific monoclonal antibodies against ghrelin was performed. The intensity of ghrelin immunoreactivity in X/A-like cells was analyzed using Olympus Cell D image analysis system. The attenuation of ghrelin-immunoreactivity of gastric mucosa, after a single injection of R-(+)-methanandamide and CP 55,940 was accompanied by a significant increase of ghrelin plasma concentration. These results indicate that stimulation of appetite exerted by cannabinoids may be connected with an increase of ghrelin secretion from gastric X/A-like cells.

Alteration of parafollicular (C) cells activity in the experimental model of hypothyroidism in rats.

Our previous study has shown the alteration of C cells activity in rats with experimental model of hyperthyroidism. The aim of the present study was the evaluation of parafollicular cells activity in rats with hypothyroidism evoked by propylthiouracil (PTU) given in drinking water over 21 days. Histological, ultrastructural and immunocytochemical studies using specific antibodies against calcitonin and CGRP were performed on thyroid glands taken from experimental and control groups of rats. Moreover, in all animals the calcitonin plasma levels were evaluated by radioimmunoassay. After chronic administration of PTU, thyroid image showed predominant microfollicular hyperplasia and attenuated density of parafollicular cells. The intensity of immunocytochemical reactions for CT and CGRP were weaker in the majority of C cells in comparison to the control rats, in which strong immunocytochemical reaction was observed. Examination in the electron microscope reveals the features of hypoactivity both in follicular and parafollicular cells, in which the quantity and electron density of secretory granules were smaller in comparison to the control group. These microscopic changes were accompanied by a significant decrease of calcitonin plasma concentration. Alteration of C cells activity in the experimental model of hypothyroidism, accompanied by microfollicular hypertrophy, may point to the mutual cooperation between parafollicular and follicular cells.