Effect of Glucocorticoid Receptor Antagonism on Alcohol Self-Administration in Genetically-Selected Marchigian Sardinian Alcohol-Preferring and Non-Preferring Wistar Rats

Alcoholism is a chronically relapsing disorder characterized by high alcohol intake and a negative emotional state during abstinence, which contributes to excessive drinking and susceptibility to relapse. Stress, dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis and alterations in glucocorticoid receptor (GR) function have been linked to transition from recreational consumption to alcohol use disorder (AUD). Here, we investigated the effect of pharmacological antagonisms of GR on alcohol self-administration (SA) using male and female Wistar and Marchigian Sardinian alcohol-preferring (msP) rats, a rodent line genetically selected for excessive alcohol drinking and highly sensitive to stress. Animals were trained to self-administer 10% (v/v) alcohol. Once a stable alcohol SA baseline was reached, we tested the effect of the GR antagonists mifepristone (0.0, 10, 30 and 60 mg/kg; i.p.) and CORT113176 (0.0, 10, 30 and 60 mg/kg) on alcohol SA. To evaluate whether the effects of the two compounds were specific for alcohol, the two drugs were tested on a similar saccharin SA regimen. Finally, basal blood corticosterone (CORT) levels before and after alcohol SA were determined. Systemic injection with mifepristone dose-dependently reduced alcohol SA in male and female Wistars but not in msPs. Administration of CORT113176 decreased alcohol SA in male and female Wistars as well as in female msPs but not in male msP rats. At the highest dose, mifepristone also reduced saccharin SA in male Wistars and female msPs, suggesting the occurrence of some nonspecific effects at 60 mg/kg of the drug. Similarly, the highest dose of CORT113176 (60 mg/kg) decreased saccharin intake in male Wistars. Analysis of CORT levels revealed that females of both rat lines had higher blood levels of CORT compared to males. Alcohol consumption reduced CORT in females but not in males. Overall, these findings indicate that selective blockade of GR selectively reduces alcohol SA, and genetically selected msP rats are less sensitive to this pharmacological manipulation compared to heterogeneous Wistars. Moreover, results suggest sex differences in response to GR antagonism and the ability of alcohol to regulate GR transmission.     

Role of Lateral Hypothalamus in Acupuncture Inhibition of Cocaine Psychomotor Activity

Acupuncture modulates the mesolimbic dopamine (DA) system; an area implicated in drug abuse. However, the mechanism by which peripheral sensory afferents, during acupuncture stimulation, modulate this system needs further investigation. The lateral hypothalamus (LH) has been implicated in reward processing and addictive behaviors. To investigate the role of the LH in mediating acupuncture effects, we evaluated the role of LH and spinohypothalamic neurons on cocaine-induced psychomotor activity and NAc DA release. Systemic injection of cocaine increased locomotor activity and 50 kHz ultrasonic vocalizations (USVs), which were attenuated by mechanical stimulation of needles inserted into HT7 but neither ST36 nor LI5. The acupuncture effects were blocked by chemical lesions of the LH or mimicked by activation of LH neurons. Single-unit extracellular recordings showed excitation of LH and spinohypothalamic neurons following acupuncture. Our results suggest that acupuncture recruits the LH to suppress the mesolimbic DA system and psychomotor responses following cocaine injection.     

Effects of Oral Administration of Chitin Nanofiber on Plasma Metabolites and Gut Microorganisms

The aim of this study was to examine the effects of oral administration of chitin nanofibers (CNFs) and surface-deacetylated (SDA) CNFs on plasma metabolites using metabolome analysis. Furthermore, we determined the changes in gut microbiota and fecal organic acid concentrations following oral administrations of CNFs and SDACNFs. Healthy female mice (six-week-old) were fed a normal diet and administered tap water with 0.1% (v/v) CNFs or SDACNFs for 28 days. Oral administration of CNFs increased plasma levels of adenosine triphosphate (ATP), adenosine diphosphate (ADP), and serotonin (5-hydroxytryptamine, 5-HT). Oral administration of SDACNFs affected the metabolisms of acyl-carnitines and fatty acids. The fecal organic level analysis indicated that oral administration of CNFs stimulated and activated the functions of microbiota. These results indicate that oral administration of CNFs increases plasma levels of ATP and 5-HT via activation of gut microbiota.     

Corticosterone Administration Alters White Matter Tract Structure and Reduces Gliosis in the Sub-Acute Phase of Experimental Stroke

White matter tract (WMT) degeneration has been reported to occur following a stroke, and it is associated with post-stroke functional disturbances. White matter pathology has been suggested to be an independent predictor of post-stroke recovery. However, the factors that influence WMT remodeling are poorly understood. Cortisol is a steroid hormone released in response to prolonged stress, and elevated levels of cortisol have been reported to interfere with brain recovery. The objective of this study was to investigate the influence of corticosterone (CORT; the rodent equivalent of cortisol) on WMT structure post-stroke. Photothrombotic stroke (or sham surgery) was induced in 8-week-old male C57BL/6 mice. At 72 h, mice were exposed to standard drinking water ± CORT (100 µg/mL). After two weeks of CORT administration, mice were euthanised and brain tissue collected for histological and biochemical analysis of WMT (particularly the corpus callosum and corticospinal tract). CORT administration was associated with increased tissue loss within the ipsilateral hemisphere, and modest and inconsistent WMT reorganization. Further, a structural and molecular analysis of the WMT components suggested that CORT exerted effects over axons and glial cells. Our findings highlight that CORT at stress-like levels can moderately influence the reorganization and microstructure of WMT post-stroke.     

Differences in the Relative Abundance of ProBDNF and Mature BDNF in A549 and H1299 Human Lung Cancer Cell Media

Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, has been linked to several human malignancies and shown to promote tumorigenesis. The purpose of this study was to explore the relative abundance of pro-brain-derived neurotrophic factor (proBDNF) and mature BDNF (mBDNF) in A549 (p53 wild-type) and H1299 (p53-null) lung cancer cell media. Higher levels of proBDNF were detected in the media of A549 cells than in H1299 cell media. Using inhibitors, we found that the levels of proBDNF and mBDNF in the media are likely regulated by PI3K, AKT, and NFκB. However, the largest change in these levels resulted from MMP2/9 inhibition. Blocking p53 function in A549 cells resulted in increased mBDNF and decreased proBDNF, suggesting a role for p53 in regulating these levels. The ratio of proBDNF/mBDNF was not affected by MMP2 knockdown but increased in the media of both cell lines upon knockdown of MMP9. Downregulation of either MMP2 or MMP9 by siRNA showed that MMP9 siRNA treatment of either A549 or H1299 cells resulted in decreased cell viability and increased apoptosis, an effect diminished upon the same treatment with proBDNF immunodepleted media, suggesting that MMP9 regulates the cytotoxic effects induced by proBDNF in lung cancer cells.     

Stress Adaptation and the Brainstem with Focus on Corticotropin-Releasing Hormone

Stress adaptation is of utmost importance for the maintenance of homeostasis and, therefore, of life itself. The prevalence of stress-related disorders is increasing, emphasizing the importance of exploratory research on stress adaptation. Two major regulatory pathways exist: the hypothalamic–pituitary–adrenocortical axis and the sympathetic adrenomedullary axis. They act in unison, ensured by the enormous bidirectional connection between their centers, the paraventricular nucleus of the hypothalamus (PVN), and the brainstem monoaminergic cell groups, respectively. PVN and especially their corticotropin-releasing hormone (CRH) producing neurons are considered to be the centrum of stress regulation. However, the brainstem seems to be equally important. Therefore, we aimed to summarize the present knowledge on the role of classical neurotransmitters of the brainstem (GABA, glutamate as well as serotonin, noradrenaline, adrenaline, and dopamine) in stress adaptation. Neuropeptides, including CRH, might be co-localized in the brainstem nuclei. Here we focused on CRH as its role in stress regulation is well-known and widely accepted and other CRH neurons scattered along the brain may also complement the function of the PVN. Although CRH-positive cells are present on some parts of the brainstem, sometimes even in comparable amounts as in the PVN, not much is known about their contribution to stress adaptation. Based on the role of the Barrington’s nucleus in micturition and the inferior olivary complex in the regulation of fine motoric—as the main CRH-containing brainstem areas—we might assume that these areas regulate stress-induced urination and locomotion, respectively. Further studies are necessary for the field.     

Effects of RU486 in Treatment of Traumatic Stress-Induced Glucocorticoid Dysregulation and Fear-Related Abnormalities: Early versus Late Intervention

Central glucocorticoid receptor (GR) activity is enhanced following traumatic events, playing a key role in the stress-related cognitive abnormalities of posttraumatic stress disorder (PTSD). GR antagonists are expected to have potential as pharmacological agents to treat PTSD-related symptoms such as anxiety and fear memory disruption. However, an incubation period is usually required and stress-induced abnormalities do not develop immediately following the trauma; thus, the optimal intervention timing should be considered. Single prolonged stress (SPS) was employed as a rodent PTSD model to examine the effects of early or late (1–7 versus 8–14 days after the SPS) sub-chronic RU486 (a GR antagonist) administration. Behaviorally, fear conditioning and anxiety behavior were assessed using the fear-conditioning test and elevated T-maze (ETM), respectively. Neurochemically, the expressions of GR, FK506-binding proteins 4 and 5 (FKBP4 and FKBP5), and early growth response-1 (Egr-1) were assessed in the hippocampus, medial prefrontal cortex (mPFC), amygdala, and hypothalamus, together with the level of plasma corticosterone. Early RU486 administration could inhibit SPS-induced behavioral abnormalities and glucocorticoid system dysregulation by reversing the SPS-induced fear extinction deficit, and preventing SPS-reduced plasma corticosterone levels and SPS-induced Egr-1 overexpression in the hippocampus. Early RU486 administration following SPS also increased the FKBP5 level in the hippocampus and hypothalamus. Finally, both early and late RU486 administration inhibited the elevated hippocampal FKBP4 level and hypothalamus GR level in the SPS rats. Early intervention with a GR antagonist aids in the correction of traumatic stress-induced fear and anxiety dysregulation.     

A Rat Model of Post-Traumatic Stress Syndrome Causes Phenotype-Associated Morphological Changes and Hypofunction of the Adrenal Gland

Background: Rats exposed to chronic predator scent stress mimic the phenotype of complex post-traumatic stress disorder (PTSD) in humans, including altered adrenal morphology and function. High- and low-anxiety phenotypes have been described in rats exposed to predator scent stress (PSS). This study aimed to determine whether these high- and low-anxiety phenotypes correlate with changes in adrenal histomorphology and corticosteroid production. Methods: Rats were exposed to PSS for ten days. Thirty days later, the rats’ anxiety index (AI) was assessed with an elevated plus-maze test. Based on differences in AI, the rats were segregated into low- (AI ≤ 0.8, n = 9) and high- (AI > 0.8, n = 10) anxiety phenotypes. Plasma corticosterone (CORT) concentrations were measured by ELISA. Adrenal CORT, desoxyCORT, and 11-dehydroCORT were measured by high-performance liquid chromatography. After staining with hematoxylin and eosin, adrenal histomorphometric changes were evaluated by measuring the thickness of the functional zones of the adrenal cortex. Results: Decreased plasma CORT concentrations, as well as decreased adrenal CORT, desoxyCORT and 11-dehydroCORT concentrations, were observed in high- but not in low-anxiety phenotypes. These decreases were associated with increases in AI. PSS led to a significant decrease in the thickness of the zona fasciculata and an increase in the thickness of the zona intermedia. The increase in the thickness of the zona intermedia was more pronounced in low-anxiety than in high-anxiety rats. A decrease in the adrenal capsule thickness was observed only in low-anxiety rats. The nucleus diameter of cells in the zona fasciculata of high-anxiety rats was significantly smaller than that of control or low-anxiety rats. Conclusion: Phenotype-associated changes in adrenal function and histomorphology were observed in a rat model of complex post-traumatic stress disorder.     

Acupuncture Modulates Intracranial Self-Stimulation of the Medial Forebrain Bundle in Rats

Acupuncture affects the central nervous system via the regulation of neurotransmitter transmission. We previously showed that Shemen (HT7) acupoint stimulation decreased cocaine-induced dopamine release in the nucleus accumbens. Here, we used the intracranial self-stimulation (ICSS) paradigm to evaluate whether HT stimulation regulates the brain reward function of rats. We found that HT stimulation triggered a rightward shift of the frequency–rate curve and elevated the ICSS thresholds. However, HT7 stimulation did not affect the threshold-lowering effects produced by cocaine. These results indicate that HT7 points only effectively regulates the ICSS thresholds of the medial forebrain bundle in drug-naïve rats.     

Time-Course Changes of Steroidogenic Gene Expression and Steroidogenesis of Rat Leydig Cells after Acute Immobilization Stress

Leydig cells secrete testosterone, which is essential for male fertility and reproductive health. Stress increases the secretion of glucocorticoid (corticosterone, CORT; in rats), which decreases circulating testosterone levels in part through a direct action by binding to the glucocorticoid receptors (NR3C1) in Leydig cells. The intratesticular CORT level is dependent on oxidative inactivation of glucocorticoid by 11β-hydroxysteroid dehydrogenase 1 (HSD11B1) in Leydig cells. In the present study, we investigated the time-course changes of steroidogenic gene expression levels after acute immobilization stress in rats. The plasma CORT levels were significantly increased 0.5, 1, 3 and 6 h after immobilization stress, while plasma testosterone levels were significantly reduced 3 and 6 h, after stress and luteinizing hormone (LH) did not change. Immobilization stress caused the down-regulation of Scarb1, Star and Cyp17a1 expression levels in the rat testis starting at the first hour of stress, ahead of the significant decreases of plasma testosterone levels. Other mRNA levels, including Cyp11a1, Hsd3b1 and Hsd17b3, began to decline after 3 h. Hsd11b1 and Nos2 mRNA levels did not change during the course of stress. Administration of glucocorticoid antagonist RU486 significantly restored plasma testosterone levels. In conclusion, Scarb1, Star and Cyp17a1 expression levels are more sensitive to acute stress, and acute immobilization stress causes the decline of the steroidogenic pathway via elevating the levels of glucocorticoid, which binds to NR3C1 in Leydig cells to inhibit steroidogenic gene expression.     

Effects of Growth Hormone Receptor Ablation in Corticotropin-Releasing Hormone Cells

Corticotropin-releasing hormone (CRH) cells are the dominant neuronal population responsive to the growth hormone (GH) in the paraventricular nucleus of the hypothalamus (PVH). However, the physiological importance of GH receptor (GHR) signaling in CRH neurons is currently unknown. Thus, the main objective of the present study was to investigate the consequences of GHR ablation in CRH-expressing cells of male and female mice. GHR ablation in CRH cells did not cause significant changes in body weight, body composition, food intake, substrate oxidation, locomotor activity, glucose tolerance, insulin sensitivity, counterregulatory response to 2-deoxy-D-glucose and ghrelin-induced food intake. However, reduced energy expenditure was observed in female mice carrying GHR ablation in CRH cells. The absence of GHR in CRH cells did not affect anxiety, circadian glucocorticoid levels or restraint-stress-induced corticosterone secretion and activation of PVH neurons in both male and female mice. In summary, GHR ablation, specifically in CRH-expressing neurons, does not lead to major alterations in metabolism, hypothalamic–pituitary–adrenal axis, acute stress response or anxiety in mice. Considering the previous studies showing that central GHR signaling regulates homeostasis in situations of metabolic stress, future studies are still necessary to identify the potential physiological importance of GH action on CRH neurons.     

Stress and Nasal Allergy: Corticotropin-Releasing Hormone Stimulates Mast Cell Degranulation and Proliferation in Human Nasal Mucosa

Psychological stress exacerbates mast cell (MC)-dependent inflammation, including nasal allergy, but the underlying mechanisms are not thoroughly understood. Because the key stress-mediating neurohormone, corticotropin-releasing hormone (CRH), induces human skin MC degranulation, we hypothesized that CRH may be a key player in stress-aggravated nasal allergy. In the current study, we probed this hypothesis in human nasal mucosa MCs (hM-MCs) in situ using nasal polyp organ culture and tested whether CRH is required for murine M-MC activation by perceived stress in vivo. CRH stimulation significantly increased the number of hM-MCs, stimulated both their degranulation and proliferation ex vivo, and increased stem cell factor (SCF) expression in human nasal mucosa epithelium. CRH also sensitized hM-MCs to further CRH stimulation and promoted a pro-inflammatory hM-MC phenotype. The CRH-induced increase in hM-MCs was mitigated by co-administration of CRH receptor type 1 (CRH-R1)-specific antagonist antalarmin, CRH-R1 small interfering RNA (siRNA), or SCF-neutralizing antibody. In vivo, restraint stress significantly increased the number and degranulation of murine M-MCs compared with sham-stressed mice. This effect was mitigated by intranasal antalarmin. Our data suggest that CRH is a major activator of hM-MC in nasal mucosa, in part via promoting SCF production, and that CRH-R1 antagonists such as antalarmin are promising candidate therapeutics for nasal mucosa neuroinflammation induced by perceived stress.     

The FKBP5 Gene Affects Alcohol Drinking in Knockout Mice and Is Implicated in Alcohol Drinking in Humans

FKBP5 encodes FK506-binding protein 5, a glucocorticoid receptor (GR)-binding protein implicated in various psychiatric disorders and alcohol withdrawal severity. The purpose of this study is to characterize alcohol preference and related phenotypes in Fkbp5 knockout (KO) mice and to examine the role of FKBP5 in human alcohol consumption. The following experiments were performed to characterize Fkpb5 KO mice. (1) Fkbp5 KO and wild-type (WT) EtOH consumption was tested using a two-bottle choice paradigm; (2) The EtOH elimination rate was measured after intraperitoneal (IP) injection of 2.0 g/kg EtOH; (3) Blood alcohol concentration (BAC) was measured after 3 h limited access of alcohol; (4) Brain region expression of Fkbp5 was identified using LacZ staining; (5) Baseline corticosterone (CORT) was assessed. Additionally, two SNPs, rs1360780 (C/T) and rs3800373 (T/G), were selected to study the association of FKBP5 with alcohol consumption in humans. Participants were college students (n = 1162) from 21–26 years of age with Chinese, Korean or Caucasian ethnicity. The results, compared to WT mice, for KO mice exhibited an increase in alcohol consumption that was not due to differences in taste sensitivity or alcohol metabolism. Higher BAC was found in KO mice after 3 h of EtOH access. Fkbp5 was highly expressed in brain regions involved in the regulation of the stress response, such as the hippocampus, amygdala, dorsal raphe and locus coeruleus. Both genotypes exhibited similar basal levels of plasma corticosterone (CORT). Finally, single nucleotide polymorphisms (SNPs) in FKBP5 were found to be associated with alcohol drinking in humans. These results suggest that the association between FKBP5 and alcohol consumption is conserved in both mice and humans.     

Anxiety-like Behavior and GABAAR/BDZ Binding Site Response to Progesterone Withdrawal in a Stress-Vulnerable Strain,the Wistar Kyoto Rats

Stress susceptibility could play a role in developing premenstrual anxiety due to abnormalities in the hypothalamus–pituitary–adrenal (HPA) axis and impairments in the GABAA receptors’ benzodiazepine (BDZ) site. Hence, we studied the stress-vulnerable Wistar Kyoto rat strain (WKY) to evaluate progesterone withdrawal (PW) effects on anxiety, HPA axis response, and to explore indicators of GABAA functionality in the BDZ site. For five days, ovariectomized WKY rats were administered 2.0 mg/kg of progesterone. Twenty-four hours after the last administration, rats were tested in the anxiety-like burying behavior test (BBT) or elevated plus maze test (EPM), and corticosterone was determined. [3H]Flunitrazepam binding autoradiography served as the BDZ binding site index of the GABAA receptor in amygdala nuclei and hippocampus’s dentate gyrus (DG). Finally, different doses of diazepam in PW-WKY rats were tested in the BBT. PW induced anxiety-like behaviors in both BBT and EPM compared with No-PW rats. PW increased corticosterone, but was blunted when combined with PW and BBT. PW increased [3H]Flunitrazepam binding in the DG and central amygdala compared with No-PW rats. Diazepam at a low dose induced an anxiogenic-like response in PW rats, suggesting a paradoxical response to benzodiazepines. Overall, PW induced anxiety-like behavior, a blunted HPA axis response, and higher GABAAR/BZD binding site sensitivity in a stress-vulnerable rat strain. These findings demonstrate the role of stress-susceptibility in GABAAR functionality in a preclinical approximation of PMDD.     

Radiation detector made of a high-quality polycrystalline diamond

Radiation detector was made of a high-quality CVD polycrystalline diamond composed of frost column like structure diamond grains, and induced charge distribution spectra and drift velocities were measured by using alpha particles. As a result, the CVD polycrystalline achieved maximum induced charge of 83% of HP/HT type IIa diamond. Moreover, the CVD crystal had lower charge loss on electrons compared with the HP/HT type IIa diamond. Drift velocities of electrons and holes were v_e = 7.7 × 10⁴ and v_h = 7.3 × 10⁴cm/s at an electric field of 20 kV/cm, respectively. In addition, response function measurement for 14 MeV neutrons was carried out.     

Transient Focal Cerebral Ischemia Leads to miRNA Alterations in Different Brain Regions,Blood Serum,Liver, and Spleen

Ischemic stroke is characterized by an occlusion of a cerebral blood vessel resulting in neuronal cell death due to nutritional and oxygen deficiency. Additionally, post-ischemic cell death is augmented after reperfusion. These events are paralleled by dysregulated miRNA expression profiles in the peri-infarct area. Understanding the underlying molecular mechanism in the peri-infarct region is crucial for developing promising therapeutics. Utilizing a tMCAo (transient Middle Cerebral Artery occlusion) model in rats, we studied the expression levels of the miRNAs (miR) 223-3p, 155-5p, 3473, and 448-5p in the cortex, amygdala, thalamus, and hippocampus of both the ipsi- and contralateral hemispheres. Additionally, the levels in the blood serum, spleen, and liver and the expression of their target genes, namely, Nlrp3, Socs1, Socs3, and Vegfa, were assessed. We observed an increase in all miRNAs on the ipsilateral side of the cerebral cortex in a time-dependent manner and increased miRNAs levels (miR-223-3p, miR-3473, and miR-448-5p) in the contralateral hemisphere after 72 h. Besides the cerebral cortex, the amygdala presented increased expression levels, whereas the thalamus and hippocampus showed no alterations. Different levels of the investigated miRNAs were detected in blood serum, liver, and spleen. The gene targets were altered not only in the peri-infarct area of the cortex but selectively increased in the investigated non-affected brain regions along with the spleen and liver during the reperfusion time up to 72 h. Our results suggest a supra-regional influence of miRNAs following ischemic stroke, which should be studied to further identify whether miRNAs are transported or locally upregulated.     

7H-Dibenzo(c,g)carbazole-DNA Adducts in Target and Non-target Organs of Mice Exposed by Acute and Chronic Administration

Carcinogen-DNA adducts appear to be biomarkers of effective dose and/or early effect. The relationship between these biomarker levels in target and non-target organs under acute and chronic exposures is important. 7H-Dibenzo(c,g)carbazole(DBC) has been shown to induce tumors in experimental animals. The time courses of DBC-DNA adduct formation in skin, liver and lung following both single (i.p.) and repeated (topical) administration of DBC were investigated. In the acute study, DNA binding levels increased rapidly in the lung over 3–7 days at all doses and then decreased slowly over 21 days; levels in the liver were 10 times that in the lung with similar increases in levels over time. In the chronic study, levels increased with time over 15 weeks with the liver binding levels 8–10 times that seen in skin and lung. In both studies the major adduct in liver was adduct 6, and in the lung adduct 3, while in skin adducts 2 and 3 were the major adducts. These data indicate that the adduct pattern is target organ specific, irrespective of the route, dose of administration or exposure regimen.     

Astaxanthin,a Carotenoid,Stimulates Immune Responses by Enhancing IFN-γ and IL-2 Secretion in Primary Cultured Lymphocytes in Vitro and ex Vivo

Astaxanthin, a potent antioxidant carotenoid, plays a major role in modulating the immune response. In this study, we examined the immunomodulatory effects of astaxanthin on cytokine production in primary cultured lymphocytes both in vitro and ex vivo. Direct administration of astaxanthin (70–300 nM) did not produce cytotoxicity in lipopolysaccharide (LPS, 100 µg/ mL)- or concanavalin A (Con A, 10 µg/ mL)-activated lymphocytes, whereas astaxanthin alone at 300 nM induced proliferation of splenic lymphocytes (p < 0.05) in vitro. Although astaxanthin, alone or with Con A, had no apparent effect on interferon (INF-γ) and interleukin (IL-2) production in primary cultured lymphocytes, it enhanced LPS-induced INF-γ production. In an ex vivo experiment, oral administration of astaxanthin (0.28, 1.4 and 7 mg/kg/day) for 14 days did not cause alterations in the body or spleen weights of mice and also was not toxic to lymphocyte cells derived from the mice. Moreover, treatment with astaxanthin significantly increased LPS-induced lymphocyte proliferation ex vivo but not Con A-stimulated lymphocyte proliferation ex vivo. Enzyme linked immunosorbent assay (ELISA) analysis revealed that administration of astaxanthin significantly enhanced INF-γ production in response to both LPS and Con A stimulation, whereas IL-2 production increased only in response to Con A stimulation. Also, astaxanthin treatment alone significantly increased IL-2 production in lymphocytes derived from mice, but did not significantly change production of INF-γ. These findings suggest that astaxanthin modulates lymphocytic immune responses in vitro, and that it partly exerts its ex vivo immunomodulatory effects by increasing INF-γ and IL-2 production without inducing cytotoxicity.