Multi-walled carbon nanotubes functionalized with pyrene-PEG via π-π interactions: toxicological assessment in zebrafish embryos.

Multi-walled carbon nanotubes (MWCNT) have many promising biological applications, even though functionalization is needed for better biocompatibility. Functionalization of MWCNT with polyethylene glycol (PEG) is a promising and widely studied approach, but the best PEGylation method is still under investigation. In this work, we have tested the biological implications of MWCNT functionalized via π-stacking with pyrene-PEG (MWCNT-Pyr-PEG) in zebrafish embryos. As Pyr toxicity is well documented and represents a major concern for the safety of the proposed approach, we have also tested the effects of the exposure to the isolated conjugate (Pyr-PEG). The resulting suspensions were stable in saline medium and well dispersed. Zebrafish embryos at 24 h post-fertilization (hpf) were dechorionated and randomly assigned to seven experimental groups (n = 50 per group): control, MWCNT-Pyr-PEG at 0.2, 2.0, and 20.0 mg l-1, and Pyr-PEG at the same concentrations, and exposures were performed in 96-well plates. Specimens were observed for heart rate, malformations, body length, mortality, traveled distance, and number of new movements. Heart rate was reduced in embryos exposed to any tested concentration of MWCNT-Pyr-PEG, while this effect was observed with Pyr-PEG from 2 mg l-1. The highest concentration of MWCNT-Pyr-PEG also led to increased occurrence of malformations, shortened body length and reduced traveled distance. The functionalization approach shows promise due to the stability in saline media, even though toxic effects were observed in the highest tested concentrations, being the MWCNT the main actors underlying these outcomes.

Toxicological assessment of PEGylated single-walled carbon nanotubes in early developing zebrafish.

Functionalization of single-walled carbon nanotubes (SWCNT) with polyethylene glycol (PEG) is among the most promising strategies to avoid SWCNT aggregation in aqueous media, improving its interactions with biological systems. However, the best molecular PEG weight and functionalization strategy remain under investigation. In this work we assessed the toxicological effects of SWCNT functionalized with PEG at 600 Da in zebrafish embryos. Embryos were exposed to SWCNT at 0.01, 0.1 and 1 mg/L from 3 to 96 h post-fertilization (hpf). At the highest concentration, SWCNT led to toxic effects at several endpoints, including mortality, delayed hatching, malformations, reduced body length, increased ROS production and DNA damage. Even with these effects, SWCNT could not be detected within the bodily tissues of the larvae. Our results give evidence that the tested PEGylation approach was unsuitable to avoid SWCNT aggregation in aqueous media, and that SWCNT can induce toxicity even without being absorbed by the organism by obstructing the chorion pores.

Enhanced cholinergic-tone during the stress induce a depressive-like state in mice.

Major depressive disorder has a heterogeneous etiology, since it arises from the interaction of multiple factors and different pathophysiological mechanisms are involved in the symptomatology. This study aimed to investigate the role of the cholinergic system in the susceptibility to stress and, consequently, in the depression-like behavior. C57BL/6 mice were treated with Physostigmine (PHYS), an acetylcholinesterase (AChE) inhibitor, and were submitted to the social defeat stress. For the behavioral evaluation of the locomotor activity, anxiety-like and depression-like behaviors the open field, elevated plus maze, sucrose preference, social interaction and forced swim were used. Hippocampus and prefrontal cortex samples were collected for evaluation of AChE activity, as well as blood samples for analysis of serum cortisol levels. Our results showed that 15 min after the injection of PHYS there was a significant inhibition of AChE activity in the hippocampus and in the prefrontal cortex. On the other hand, in the end of the experimental design, day 12, there was no difference in AChE activity levels. Inhibition of AChE and exposure to the stress led to an increase in cortisol levels. Animals that received PHYS and were exposed to stress showed less social interaction and greater learned helplessness, anhedonia and anxious-like behavior. Taken together, our findings suggest that increasing the cholinergic tone shortly before stress induction impacts on the ability to cope with upcoming stressful situations, leading to a depressive-like state.

Implications of PEGylation of Carbon Nanotubes for Central Nervous System Bioavailability.

BACKGROUND AND OBJECTIVE: In this mini-review, we have compiled the most recent and comparable information to shed light on the action of PEGylation in the biodistribution of carbon nanotubes (CNT) in the central nervous system (CNS). It is well known that due to the complexity of the CNS and the severity of the outcome following changes in this system, this is one of the areas where there are more investments in research to develop new technologies and approaches for more effective and less invasive treatments. The CNS is highly protected against toxic and invasive microorganisms thanks to the blood brain barrier (BBB), but this protection also prevents the passage of potentially beneficial molecules for the treatment of neurological disorders. Nanotechnology attempts to develop nanocompounds that are biocompatible and non-immunogenic, and that are able to cross the BBB in therapeutic amounts without causing damage and to diffuse through nerve tissue. These compounds should also be cleared and biodistributed properly, being capable of performing drug delivery exclusively for CNS pathologies, such as neurodegenerative diseases (Parkinson's and Alzheimer's) and brain tumors. CONCLUSION: In this way, this review focuses on CNT PEGylation, aiming to help in the development of viable and effective nanomedicines for neuroscience applications.

Hippocampal neurogenesis and pattern separation: A meta-analysis of behavioral data.

The generation of new neurons in the hippocampus of adult mammals has become a widely accepted phenomenon, but the functional significance of the adult neurogenesis in the hippocampus is not fully understood. One of the main hypotheses currently investigated suggests that neurogenesis contributes to pattern separation in the dentate gyrus. Many behavioral studies were conducted aiming to test this hypothesis using rodents as animal model. In those studies, researches ablated neurogenesis in the animals and subsequently evaluate them in tests of behavioral pattern separation, that is, behaviors that are thought to rely on the computational process of pattern separation. The results of these studies are varied, with most supporting a role for neurogenesis in pattern separation, but some others not. To address this controversy we performed a systematic review and meta-analysis of studies evaluating the effect of neurogenesis ablation on behavioral pattern separation. Analysis results indicated that most of the literature in the topic is surprisingly consistent and, although there are two studies with divergent results, the bulk of the literature supports an effect of hippocampal neurogenesis on behavioral pattern separation. We discuss those findings in light of other behavioral effects of hippocampal neurogenesis ablation, limitations of behavioral data and other lines of evidence about the effect of hippocampal neurogenesis in the dentate gyrus.

Production of Isotonic, Sterile, and Kinetically Stable Lipid-Core Nanocapsules for Injectable Administration.

Lipid-core nanocapsules (LNC) were designed and prepared as a colloidal system for drug targeting to improve the stability of drugs and allow their controlled release. For parenteral administration, it is necessary to ensure formulation sterility. However, sterilization of nanotechnological devices using an appropriate technique that keeps the supramolecular structure intact remains a challenge. This work aimed to evaluate the effect of autoclaving on the physicochemical characteristics of LNC. Formulations were prepared by the self-assembling method, followed by isotonization and sterilization at varying times and temperatures. The isotonicity was confirmed by determining the freezing temperature, which was -0.51°C. The formulation was broadly characterized, and the diameter of the particles was determined utilizing complementary methods. To evaluate the chemical stability of poly(ε-caprolactone), its molecular weight was determined by size exclusion chromatography. The physicochemical characteristics (average diameter, viscosity, and physical stability) of the formulation were similar before and after adding glycerol and conducting the sterilization at the highest temperature (134°C) and the shorter exposure time (10 min). After autoclaving, the sterility test was performed and showed no detectable microbial growth. Multiple light scattering demonstrated that the formulations were kinetically stable, and the mean diameter was constant for 6 months, corroborating this result. The polymer was chemically stable in the sterilized formulation. Isotonic and sterile LNC aqueous suspensions were produced using glycerol and autoclaving. Briefly, the results open an opportunity to produce an isotonic and sterile LNC aqueous dispersion applicable as nanomedicine for intravenous administration in clinical trials.

Toxicity of single-wall carbon nanotubes functionalized with polyethylene glycol in zebrafish (Danio rerio) embryos.

Single-wall carbon nanotubes functionalized with polyethylene glycol (SWCNT-PEG) are promising materials for biomedical applications such as diagnostic devices and controlled drug-release systems. However, several questions about their toxicological profile remain unanswered. Thus, the aim of this study was to investigate the action of SWCNT-PEG in Danio rerio zebrafish embryos at the molecular, physiological and morphological levels. The SWCNT used in this study were synthesized by the high-pressure carbon monoxide process, purified and then functionalized with distearoyl phosphatidylethanolamine block copolymer-PEG (molecular weight 2 kDa). The characterization process was carried out with low-resolution transmission electron microscopy, thermogravimetric analysis and Raman spectroscopy. Individual zebrafish embryos were exposed to the SWCNT-PEG. Toxic effects occurred only at the highest concentration tested (1 ppm) and included high mortality rates, delayed hatching and decreased total larval length. For all the concentrations tested, the alkaline comet assay revealed no genotoxicity, and Raman spectroscopy measurements on the histological slices revealed no intracellular nanotubes. The results shown here demonstrate that SWCNT-PEG has low toxicity in zebrafish embryos, but more studies are needed to understand what mechanisms are involved. However, the presence of residual metals is possibly among the primary mechanisms responsible for the toxic effects observed, because the purification process was not able to remove all metal contamination, as demonstrated by the thermogravimetric analysis. More attention must be given to the toxicity of these nanomaterials before they are used in biomedical applications. Copyright © 2016 John Wiley & Sons, Ltd.

Biopersistence of PEGylated Carbon Nanotubes Promotes a Delayed Antioxidant Response after Infusion into the Rat Hippocampus.

Carbon nanotubes are promising nanomaterials for the diagnosis and treatment of brain disorders. However, the ability of these nanomaterials to cross cell membranes and interact with neural cells brings the need for the assessment of their potential adverse effects on the nervous system. This study aimed to investigate the biopersistence of single-walled carbon nanotubes functionalized with polyethylene glycol (SWCNT-PEG) directly infused into the rat hippocampus. Contextual fear conditioning, Y-maze and open field tasks were performed to evaluate the effects of SWCNT-PEG on memory and locomotor activity. The effects of SWCNT-PEG on oxidative stress and morphology of the hippocampus were assessed 1 and 7 days after infusion of the dispersions at 0.5, 1.0 and 2.1 mg/mL. Raman analysis of the hippocampal homogenates indicates the biopersistence of SWCNT-PEG in the hippocampus 7 days post-injection. The infusion of the dispersions had no effect on the acquisition or persistence of the contextual fear memory; likewise, the spatial recognition memory and locomotor activity were not affected by SWCNT-PEG. Histological examination revealed no remarkable morphological alterations after nanomaterial exposure. One day after the infusion, SWCNT-PEG dispersions at 0.5 and 1.0 mg/mL were able to decrease total antioxidant capacity without modifying the levels of reactive oxygen species or lipid hydroperoxides in the hippocampus. Moreover, SWCNT-PEG dispersions at all concentrations induced antioxidant defenses and reduced reactive oxygen species production in the hippocampus at 7 days post-injection. In this work, we found a time-dependent change in antioxidant defenses after the exposure to SWCNT-PEG. We hypothesized that the persistence of the nanomaterial in the tissue can induce an antioxidant response that might have provided resistance to an initial insult. Such antioxidant delayed response may constitute an adaptive response to the biopersistence of SWCNT-PEG in the hippocampus.

PEGylated carbon nanotubes impair retrieval of contextual fear memory and alter oxidative stress parameters in the rat hippocampus.

Carbon nanotubes (CNT) are promising materials for biomedical applications, especially in the field of neuroscience; therefore, it is essential to evaluate the neurotoxicity of these nanomaterials. The present work assessed the effects of single-walled CNT functionalized with polyethylene glycol (SWCNT-PEG) on the consolidation and retrieval of contextual fear memory in rats and on oxidative stress parameters in the hippocampus. SWCNT-PEG were dispersed in water at concentrations of 0.5, 1.0, and 2.1 mg/mL and infused into the rat hippocampus. The infusion was completed immediately after training and 30 min before testing of a contextual fear conditioning task, resulting in exposure times of 24 h and 30 min, respectively. The results showed that a short exposure to SWCNT-PEG impaired fear memory retrieval and caused lipid peroxidation in the hippocampus. This response was transient and overcome by the mobilization of antioxidant defenses at 24 h. These effects occurred at low and intermediate but not high concentration of SWCNT-PEG, suggesting that the observed biological response may be related to the concentration-dependent increase in particle size in SWCNT-PEG dispersions.

Participation of group I p21-activated kinases in neuroplasticity.

PAKs are a family of serine/threonine protein kinases activated by small GTPases of the Rho family, including Rac and Cdc42, and are categorized into group I (isoforms 1, 2 and 3) and group II (isoforms 4, 5 and 6). PAK1 and PAK3 are critically involved in biological mechanisms associated with neurodevelopment, neuroplasticity and maturation of the nervous system, and changes in their activity have been detected in pathological disorders, such as Alzheimer's disease, Huntington's disease and mental retardation. The group I PAKs have been associated with neurological processes due to their involvement in intracellular mechanisms that result in molecular and cellular morphological alterations that promote cytoskeletal outgrowth, increasing the efficiency of synaptic transmission. Their substrates in these processes include other intracellular signaling molecules, such as Raf, Mek and LIMK, as well as other components of the cytoskeleton, such as MLC and FLNa. In this review, we describe the characteristics of group I PAKs, such as their molecular structure, mechanisms of activation and importance in the neurobiological processes involved in synaptic plasticity.

Biodistribution and toxicological study of PEGylated single-wall carbon nanotubes in the zebrafish (Danio rerio) nervous system.

Nanotechnology has been proven to be increasingly compatible with pharmacological and biomedical applications. Therefore, we evaluated the biological interactions of single-wall carbon nanotubes functionalized with polyethylene glycol (SWNT-PEG). For this purpose, we analyzed biochemical, histological, behavioral and biodistribution parameters to understand how this material behaves in vitro and in vivo using the fish Danio rerio (zebrafish) as a biological model. The in vitro results for fish brain homogenates indicated that SWNT-PEG had an effect on lipid peroxidation and GSH (reduced glutathione) content. However, after intraperitoneal exposure, SWNT-PEG proved to be less biocompatible and formed aggregates, suggesting that the PEG used for the nanoparticle functionalization was of an inappropriate size for maintaining product stability in a biological environment. This problem with functionalization may have contributed to the low or practically absent biodistribution of SWNT-PEG in zebrafish tissues, as verified by Raman spectroscopy. There was an accumulation of material in the abdominal cavity that led to inflammation and behavioral disturbances, as evaluated by a histological analysis and an open field test, respectively. These results provide evidence of a lack of biocompatibility of SWNTs modified with short chain PEGs, which leads to the accumulation of the material, tissue damage and behavioral alterations in the tested subjects.

The value of intraocular pressure asymmetry in diagnosing glaucoma.

PURPOSE: To investigate the amount of intraocular pressure (IOP) asymmetry in a large group of ethnically diverse patients with and without glaucoma, and to delineate the risk for glaucoma which increasing amounts of IOP asymmetry confer upon the patient. PATIENTS AND METHODS: Collaborative retrospective study of 326 glaucoma patients and 326 controls. Former Wills Eye Institute fellows collected single pre-treatment measurements of IOP on patients diagnosed as having definite glaucoma based on characteristic optic nerve damage and confirmatory visual field damage. Patients with a normal eye examination who had normal-appearing optic discs and no apparent glaucoma, or who had a normal eye examination in association with refractive error or cataract, were used as controls. RESULTS: Intraocular pressure asymmetry is a significant risk factor for having glaucoma (odds ratio, 2.14; 95% confidence interval, 1.86-2.47; P<0.001). Absence of IOP asymmetry between the fellow eyes is associated with a 1% probability of having glaucoma. A difference of 3 mm Hg is associated with a 6% probability of having glaucoma, and a difference of >6 mm Hg with a 57% probability of having glaucoma. The association between IOP asymmetry and glaucoma status is significant for subjects with both elevated IOP (P=0.014) and statistically normal IOP (maximum IOP ≤ 21 mm Hg; P<0.001). CONCLUSIONS: Inter-eye asymmetry of IOP is a common finding in patients with glaucoma. There is a direct relationship between the amount of IOP asymmetry between the fellow eyes and the likelihood of having glaucoma.

In vitro exposure to fullerene C(60) influences redox state and lipid peroxidation in brain and gills from Cyprinus carpio (Cyprinidae).

Studies concerning the impact of nanomaterials, especially fullerene (C(60) ), in fresh water environments and their effects on the physiology of aquatic organisms are still scarce and conflicting. We aimed to assess in vitro effects of fullerene in brain and gill homogenates of carp Cyprinus carpio, evaluating redox parameters. A fullerene suspension was prepared by continued stirring under fluorescent light during two months. The suspension concentration was measured by total carbon content and ultraviolet-visible spectroscopy nephelometry. Characterization of C(60) aggregates was performed with an enhanced dark-field microscopy system and transmission electronic microscopy. Organ homogenates were exposed during 1, 2, and 4 h under fluorescent light. Redox parameters evaluated were reduced glutathione and oxidized glutathione, cysteine and cystine, total antioxidant capacity; activity of the antioxidant enzymes glutathione S-transferase and glutathione reductase (GR), and lipid peroxidation (TBARS assay). Fullerene induced a significant increase (p < 0.05) in lipid peroxidation after 2 h in both organs and reduced GR activity after 1 h (gills) and 4 h (brain) and antioxidant capacity after 4 h (brain). Levels of oxidized glutathione increased in the brain at 1 h and decreased at 2 h as well. Given these results, it can be concluded that C(60) can induce redox disruption via thiol/disulfide pathway, leading to oxidative damage (higher TBARS values) and loss of antioxidant competence.

Participation of hippocampal cholinergic system in memory persistence for inhibitory avoidance in rats.

Memory persistence needs a new event of consolidation 12h after the acquisition. We investigated the role of the cholinergic activity on the persistence of memory. For this purpose, we performed the treatments 9 or 12h after acquisition and the memory tested 2 or 7 days after inhibitory avoidance (IA) training. Here we report that activity of medial septum, by transitorily inactivating this structure with lidocaine 12h after IA training, is essential for memory persistence at the 7th day, but not for the formation at the 2nd day. We also report that muscarinic and nicotinic cholinergic receptors of CA1 area are engaged on memory persistence. Since scopolamine (mAChRs antagonist) and mecamylamine (nAChRs blocker) infusions, 12h post-training, demonstrated impairment on long term memory (LTM), persistence on the 7th day but no effect on LTM formation was found on the 2nd day in the IA test. The same effects were found with pirenzepine, an M1 antagonist. No effects on the formation and persistence of memory on the 2nd and 7th days were demonstrated after DHßE infusions (nAChRs subtype antagonist α4ß2, α3ß2). These findings suggest that mAChR and nAChR at the CA1 area, and also MS activation, are required for the persistence of memory.

A link between role of two prefrontal areas in immediate memory and in long-term memory consolidation.

The dorsolateral and medial prefrontal cortex are critical for immediate memory processing. The possibility has been raised that those two areas may also contribute to long-term memory formation. Here, we studied the role of specific receptors in dorsolateral and medial prefrontal cortex in immediate and in long-term memory formation of one-trial inhibitory avoidance. Four different specific receptor ligands were infused into these two areas: the dopamine D1 receptor antagonist, SCH23390, the GABA(A) receptor agonist, muscimol, the AMPA glutamatergic receptor antagonist, ciano-nitro-quinoxaline-dione (CNQX), and the NMDA glutamatergic receptor antagonist, aminophosphonovaleric acid (AP5). In all cases the doses used had been previously shown to affect immediate or long-term memory. In the experiments on immediate memory the drugs were given 5 min before training and the animals were tested 3s post-training. These animals were then also tested 24h later for long-term memory. The effect of the treatments on long-term memory was studied by their infusion 0, 90, 180 or 270 min post-training, testing the animals 24h after training. Immediate memory was inhibited by SCH23390, muscimol and CNQX, but not by AP5, given into any of the two subregions. Long-term memory formation was inhibited by SCH23390, muscimol and CNQX, but not by AP5, given pre-training or 0, 90 or 180 but not 270 min post-training into the dorsolateral region; or 90 but not 0 or 180 min post-training into the medial region. Thus, there is a time- and receptor-dependent correlation in the two areas between their role in immediate and in long-term memory processing. Both roles require intact glutamate AMPA and dopamine D1 receptors, are inhibited by GABAergic synapses, and are unaffected by AP5. In the dorsolateral prefrontal cortex the link between immediate and long-term memory appears to be direct; in the medial area the link suffers a 90 min delay.

Mice deficient for the vesicular acetylcholine transporter are myasthenic and have deficits in object and social recognition.

An important step for cholinergic transmission involves the vesicular storage of acetylcholine (ACh), a process mediated by the vesicular acetylcholine transporter (VAChT). In order to understand the physiological roles of the VAChT, we developed a genetically altered strain of mice with reduced expression of this transporter. Heterozygous and homozygous VAChT knockdown mice have a 45% and 65% decrease in VAChT protein expression, respectively. VAChT deficiency alters synaptic vesicle filling and affects ACh release. Whereas VAChT homozygous mutant mice demonstrate major neuromuscular deficits, VAChT heterozygous mice appear normal in that respect and could be used for analysis of central cholinergic function. Behavioral analyses revealed that aversive learning and memory are not altered in mutant mice; however, performance in cognitive tasks involving object and social recognition is severely impaired. These observations suggest a critical role of VAChT in the regulation of ACh release and physiological functions in the peripheral and central nervous system.

Retrieval and the extinction of memory.

1. Memory is assessed by measuring retrieval which is often elicited by the solely presentation of the conditioned stimulus (CS). However, as known since Pavlov, presentation of the CS alone generates extinction. 2. One-trial avoidance (IA) is a much used conditioned fear paradigm in which the CS is the safe part of a training apparatus, the unconditioned stimulus (US) is a footshock and the conditioned response (CR) is to stay in the safe area. Retrieval of the memory for the step-down version of this task is measured in the absence of the US, as latency to step-down from the safe area (i.e., a platform). 3. Extinction of the IA response is installed at the moment of the first non-reinforced test session, as clearly shown by the fact that many drugs, including PKA, ERK and protein synthesis inhibitors as well as NMDA receptor antagonists, hinder extinction when infused into the hippocampus or the basolateral amygdala at the moment of the first test session but not later. 4. Some, but not all the molecular systems required for extinction are also activated by retrieval, further endorsing the hypothesis that although retrieval is necessary for the generation of extinction this last process constitutes a new learning secondary to the non-reinforced expression of the original trace.

The transition from memory retrieval to extinction

A retenção das memórias é avaliada através da sua expressão. A expressão do traço mnemônico é iniciada freqüentemente pelo estímulo condicionado (CS); porém, como definido por Pavlov, a apresentação apenas do CS induz extinção. A esquiva inibitória de apenas uma sessão (IA) é um paradigma de condicionamento ao medo muito utilizado, no qual o CS é a parte segura da caixa de treinamento (plataforma), o estímulo incondicionado (US) é um choque aplicado nas patas do animal quando o mesmo desce da plataforma e a resposta condicionada é permanecer na área segura. Na IA, a expressão da memória é medida na ausência do US, sendo definida como a latência para descer da área segura. A extinção é instalada no momento da primeira sessão de teste, tal como fica claramente demonstrado pelo fato de que várias drogas, entre elas inibidores de síntese protéica, de PKA e de ERK e antagonistas dos receptores NMDA, impedem a extinção quando administrados no hipocampo ou na amígdala basolateral no momento da primeira sessão de teste, mas não mais tardiamente. Alguns, mas não todos os sistemas moleculares requeridos para a extinção, também são ativados pela expressão das memórias, fortalecendo a hipótese de que mesmo que a expressão seja comportamental e bioquimicamente necessária para a ocorrência da extinção, este último processo constitui um novo aprendizado, secundário a expressão do traço original.

The transition from memory retrieval to extinction.

Memory is measured by measuring retrieval. Retrieval is often triggered by the conditioned stimulus (CS); however, as known since Pavlov, presentation of the CS alone generates extinction. One-trial avoidance (IA) is a much used conditioned fear paradigm in which the CS is the safe part of a training apparatus, the unconditioned stimulus (US) is a footshock and the conditioned response is to stay in the safe area. In IA, retrieval is measured without the US, as latency to step-down from the safe area (i.e., a platform). Extinction is installed at the moment of the first unreinforced test session, as clearly shown by the fact that many drugs, including PKA, ERK and protein synthesis inhibitors as well as NMDA receptor antagonists, hinder extinction when infused into the hippocampus or the basolateral amygdala at the moment of the first test session but not later. Some, but not all the molecular systems required for extinction are also activated by retrieval, further endorsing the hypothesis that although retrieval is behaviorally and biochemically necessary for the generation of extinction, this last process constitutes a new learning secondary to the unreinforced expression of the original trace.

The effects of aging on leukocyte glucocorticoid receptor concentration and response to dexamethasone in dogs.

Glucocorticoid receptor concentration (GR) was determined in mononuclear (MNS) and polymorphonuclear (PMNS) cells isolated from 8 aged and 9 young male dogs. In addition, leukocyte responses to dexamethasone (0.1 mg kg(-1) i.v.) and plasma glucose concentration were also measured. The GR in MNS and PMNS was higher (p<0.05) in young dogs (6.64 +/- 0.57 and 7.04 +/- 0.29 fmolesx10(6) cells, respectively, versus 4.40 +/- 0.24 and 5.06 +/- 0.33 fmolesx10(6) cells, respectively, in aged dogs). The maximum increase in neutrophils (DeltaNEU) was lower (p<0.05) in aged dogs 6003.38 +/- 1398.5x10(6) versus 11168.67 +/- 1863.16x10(6) cells l(-1) in young dogs. The maximum decrease in lymphocytes (DeltaLYM) was lower (p<0.05) in aged dogs 550 +/- 56.75x10(6) cells l(-1) versus 1825.89 +/- 313.1x10(6) cells l(-1) in young dogs. In young dogs, significant (p<0.05) correlations between GR in PMNS and DeltaNEU (r=0.80) and between GR in MNS and DeltaLYM (r=0.76) were observed. In aged dogs, these correlations were not significant. The lower GR value and the lack of correlation between this parameter and its respective Delta in aged dogs suggest that changes in leukocytes responsiveness to glucocorticoids is occurring during the aging process.