Zebrafish embryonically exposed to valproic acid present impaired retinal development and sleep behavior.

Prenatal exposure to valproic acid (VPA), a drug widely used to treat epilepsy and bipolar disorder, is an environmental risk factor for autism spectrum disorder (ASD). VPA has been used to reproduce the core symptoms of ASD in animal model organisms, including zebrafish. Visual system functioning is essential in the interpretation of social conditions and plays an important role of several behavioral responses. We hypothesized that behavioral deficits displayed by ASD patients may involve impaired visual processing. We used zebrafish as model organism to investigate the visual system after embryonic exposure to VPA using histological, behavioral and gene expression analysis. We analyzed the pineal gland of zebrafish and sleep-like behavior to study how VPA exposure alters photo-sensibility of zebrafish. VPA-exposed zebrafish showed a delay in the development of the retina and optic nerve, which normalized at five days post fertilization. At larval stage, VPA-exposed zebrafish showed sleep disturbances associated with a reduced number of serotonin-producing cells of the pineal gland. In addition, the number of hypocretin/orexin (hcrt) expressing neurons in the rostral hypothalamus at 6 and 14 days post fertilization was reduced. In conclusion, we demonstrated that although VPA exposure leads to a delay in visual system development, it does not affect larval visual function. The novel finding that VPA alters significantly cells involved in sleep regulation and the sleep-like state itself may be relevant for understanding sleep disturbances in ASD patients.

The Roles of Histamine Receptor 1 (hrh1) in Neurotransmitter System Regulation, Behavior, and Neurogenesis in Zebrafish.

Histamine receptors mediate important physiological processes and take part in the pathophysiology of different brain disorders. Histamine receptor 1 (HRH1) is involved in the development of neurotransmitter systems, and its role in neurogenesis has been proposed. Altered HRH1 binding and expression have been detected in the brains of patients with schizophrenia, depression, and autism. Our goal was to assess the role of hrh1 in zebrafish development and neurotransmitter system regulation through the characterization of hrh1-/- fish generated by the CRISPR/Cas9 system. Quantitative PCR, in situ hybridization, and immunocytochemistry were used to study neurotransmitter systems and genes essential for brain development. Additionally, we wanted to reveal the role of this histamine receptor in larval and adult fish behavior using several quantitative behavioral methods including locomotion, thigmotaxis, dark flash and startle response, novel tank diving, and shoaling behavior. Hrh1-/- larvae displayed normal behavior in comparison with hrh1+/+ siblings. Interestingly, a transient abnormal expression of important neurodevelopmental markers was evident in these larvae, as well as a reduction in the number of tyrosine hydroxylase 1 (Th1)-positive cells, th1 mRNA, and hypocretin (hcrt)-positive cells. These abnormalities were not detected in adulthood. In summary, we verified that zebrafish lacking hrh1 present deficits in the dopaminergic and hypocretin systems during early development, but those are compensated by the time fish reach adulthood. However, impaired sociability and anxious-like behavior, along with downregulation of choline O-acetyltransferase a and LIM homeodomain transcription factor Islet1, were displayed by adult fish.

Vesicular monoamine transporter 2 (SLC18A2) regulates monoamine turnover and brain development in zebrafish.

AIM: We aimed at identifying potential roles of vesicular monoamine transporter 2, also known as Solute Carrier protein 18 A2 (SLC18A2) (hereafter, Vmat2), in brain monoamine regulation, their turnover, behaviour and brain development using a novel zebrafish model. METHODS: A zebrafish strain lacking functional Vmat2 was generated with the CRISPR/Cas9 system. Larval behaviour and heart rate were monitored. Monoamines and their metabolites were analysed with high-pressure liquid chromatography. Amine synthesising and degrading enzymes, and genes essential for brain development, were analysed with quantitative PCR, in situ hybridisation and immunocytochemistry. RESULTS: The 5-bp deletion in exon 3 caused an early frameshift and was lethal within 2 weeks post-fertilisation. Homozygous mutants (hereafter, mutants) displayed normal low locomotor activity during night-time but aberrant response to illumination changes. In mutants dopamine, noradrenaline, 5-hydroxytryptamine and histamine levels were reduced, whereas levels of dopamine and 5-hydroxytryptamine metabolites were increased, implying elevated monoamine turnover. Consistently, there were fewer histamine, 5-hydroxytryptamine and dopamine immunoreactive cells. Cellular dopamine immunostaining, in wild-type larvae more prominent in tyrosine hydroxylase 1 (Th1)-expressing than in Th2-expressing neurons, was absent in mutants. Despite reduced dopamine levels, mutants presented upregulated dopamine-synthesising enzymes. Further, in mutants the number of histidine decarboxylase-expressing neurons was increased, notch1a and pax2a were downregulated in brain proliferative zones. CONCLUSION: Lack of Vmat2 increases monoamine turnover and upregulates genes encoding amine-synthesising enzymes, including histidine decarboxylase. Notch1a and pax2a, genes implicated in stem cell development, are downregulated in mutants. The zebrafish vmat2 mutant strain may be a useful model to study how monoamine transport affects brain development and function, and for use in drug screening.

The Histamine System in Zebrafish Brain: Organization, Receptors, and Behavioral Roles.

Three of the four histamine receptors have been identified in zebrafish. Whereas only one histamine receptor 1 gene (hrh1) is known, two copies of histamine receptor 2 (hrh2a and hrh2b) have been identified. Although initially only one gene encoding for histamine receptor 3 (hrh3) was recognized in zebrafish, the genome database contains information for two more hrh3-like genes, whereas no genes corresponding for histamine receptor 4 with expression mainly in the immune system have been identified. Hrh1 and hrh3 show prominent uneven expression in the zebrafish brain, with the strongest expression in the dorsal telencephalon. Quantitatively significant expression of hrh1, hrh2, and hrh3 can also be found in several peripheral organs. Whereas antagonists of hrh1, hrh2, and hrh3 all affect the locomotor activity of zebrafish larvae, interpretation of the data is hampered by a lack of information on receptor binding and signaling characteristics. Zebrafish mutants lacking any of the three histamine receptors have shown modest behavioral phenotypes, possibly due to genetic compensation. None of the receptor mutant fish have shown significant sleep phenotypes. Adult zebrafish lacking hrh3 display decreased locomotor activity. The zebrafish histamine system shows significant life-long plasticity: presenilin 1 mutant zebrafish develop an abnormally large number of histamine neurons and increased thigmotaxis and anxiety-related phenotype. Overexpression of histidine decarboxylase (hdc) in larval zebrafish is associated with an increased number of hypocretin neurons, whereas translation inhibition of hdc or exposure to α-fluoromethylhistidine leads to decreased numbers of hypocretin neurons. Current pharmacological evidence suggests that this may be mediated by hrh1. Further studies using acute, e.g., pharmacogenetic or optogenetic manipulation of selected components of brain circuits, are required to understand the full range of physiological functions of zebrafish histamine receptors.

Abnormal brain development of monoamine oxidase mutant zebrafish and impaired social interaction of heterozygous fish.

Monoamine oxidase (MAO) deficiency and imbalanced levels of brain monoamines have been associated with developmental delay, neuropsychiatric disorders and aggressive behavior. Animal models are valuable tools to gain mechanistic insight into outcomes associated with MAO deficiency. Here, we report a novel genetic model to study the effects of mao loss of function in zebrafish. Quantitative PCR, in situ hybridization and immunocytochemistry were used to study neurotransmitter systems and expression of relevant genes for brain development in zebrafish mao mutants. Larval and adult fish behavior was evaluated through different tests. Stronger serotonin immunoreactivity was detected in mao+/- and mao-/- larvae compared with their mao+/+ siblings. mao-/- larvae were hypoactive, and presented decreased reactions to visual and acoustic stimuli. They also had impaired histaminergic and dopaminergic systems, abnormal expression of developmental markers and died within 20 days post-fertilization. mao+/- fish were viable, grew until adulthood, and demonstrated anxiety-like behavior and impaired social interactions compared with adult mao+/+ siblings. Our results indicate that mao-/- and mao+/- mutants could be promising tools to study the roles of MAO in brain development and behavior. This article has an associated First Person interview with the first author of the paper.

Cerebral Dopamine Neurotrophic Factor Regulates Multiple Neuronal Subtypes and Behavior.

Cerebral dopamine neurotrophic factor (CDNF) protects dopaminergic neurons against toxic damage in the rodent brain and is in clinical trials to treat Parkinson's disease patients. Yet the underlying mechanism is poorly understood. To examine its significance for neural circuits and behavior, we examined the development of neurotransmitter systems from larval to male adult mutant zebrafish lacking cdnf Although a lack of cdnf did not affect overall brain dopamine levels, dopaminergic neuronal clusters showed significant abnormalities. The number of histamine neurons that surround the dopaminergic neurons was significantly reduced. Expression of tyrosine hydroxylase 2 in the brain was elevated in cdnf mutants throughout their lifespan. There were abnormally few GABA neurons in the hypothalamus in the mutant larvae, and expression of glutamate decarboxylase was reduced throughout the brain. cdnf mutant adults showed a range of behavioral phenotypes, including increased sensitivity to pentylenetetrazole-induced seizures. Shoaling behavior of mutant adults was abnormal, and they did not display social attraction to conspecifics. CDNF plays a profound role in shaping the neurotransmitter circuit structure, seizure susceptibility, and complex behaviors in zebrafish. These findings are informative for dissecting the diverse functions of this poorly understood factor in human conditions related to Parkinson's disease and complex behaviors.SIGNIFICANCE STATEMENT A zebrafish lacking cdnf grows normally and shows no overt morphologic phenotype throughout the life span. Remarkably, impaired social cohesion and increased seizure susceptibility were found in adult cdnf KO fish conceivably associated with significant changes of dopaminergic, GABAergic, and histaminergic systems in selective brain areas. These findings suggest that cdnf has broad effects on regulating neurogenesis and maturation of transmitter-specific neuronal types during development and throughout adulthood, rather than ones restricted to the dopaminergic systems.

Reduced CD4 T Lymphocytes in Lymph Nodes of the Mouse Model of Autism Induced by Valproic Acid.

OBJECTIVE: Considering the potential role of lymphocytes in the pathophysiology of autism spectrum disorder (ASD), we aimed to evaluate possible alterations of T cell pools in the lymphoid organs of an animal model of autism induced by valproic acid (VPA). Pregnant Swiss mice received a single intraperitoneal injection of 600 mg/kg of VPA (VPA group) or saline (control group) on day 11 of gestation. Male offspring were euthanized on postnatal day 60 for removal of thy-muses, spleens, and a pool of inguinal, axillary and brachial lymph nodes. Cellularity was evaluated, and flow cytometry analysis was performed on cell suspensions incubated with the mouse antibodies anti-CD3-FITC, anti-CD4-PE, and anti-CD8-PE-Cy7. We observed that the prenatal exposure to VPA induced a reduction in the numbers of CD3+CD4+ T cells in their lymph nodes when compared to the control animals. This was specific since it was not seen in the thymus or spleen. The consistent decrease in the number of CD4+ T cells in subcutaneous lymph nodes of mice from the animal model of autism may be related to the allergic symptoms frequently observed in ASD. Further research is necessary to characterize the immunological patterns in ASD and the connection with the pathophysiology of this disorder.

Embryonic exposure to valproic acid affects the histaminergic system and the social behaviour of adult zebrafish (Danio rerio).

BACKGROUND AND PURPOSE: Histamine modulates several behaviours and physiological functions, and its deficiency is associated with neuropsychiatric disorders. Gestational intake of valproic acid (VPA) is linked to autism spectrum disorder (ASD), characterized by impaired sociability and stereotypies. VPA effects on the neurochemistry and functional morphology of the histaminergic system in ASD are unclear. Zebrafish are highly social, and given the similarities between zebrafish and human neurotransmitter systems, we have studied the effects of VPA on histamine in zebrafish. EXPERIMENTAL APPROACH: Histaminergic, dopaminergic and noradrenergic systems of larval and adult zebrafish exposed to VPA from the end of gastrulation until neural tube formation were studied using HPLC, quantitative PCR, immunocytochemistry and in situ hybridization. Sociability, dark-flash response and locomotion were also studied. KEY RESULTS: Zebrafish larvae exposed to VPA showed decreased locomotion and an abnormal dark-flash response. Additionally, a reduced number of histaminergic neurons, low histamine and altered mRNA expression of key genes of the monoaminergic systems were also detected. The reduced mRNA expression of genes of the studied systems persisted until adulthood. Furthermore, adult VPA-exposed animals presented lower brain levels of noradrenaline and 3,4-dihydroxyphenylacetic acid, along with impaired sociability. CONCLUSIONS AND IMPLICATIONS: VPA exposure in early development causes molecular and neurochemical alterations in zebrafish, which persist into adulthood and accompany impaired sociability. These findings will highlight the possible involvement of the histaminergic system in outcomes related to neuropsychiatric disorders. Furthermore, it supports zebrafish as a tool to investigate mechanisms underlying these disorders.

Body composition of patients with autism spectrum disorder through bioelectrical impedance.

BACKGROUND: Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by deficits in sociability, communication, and limited repertoire of interests and behaviors. OBJECTIVES: We aimed to investigate the nutritional status through bioelectrical impedance analysis (BIA) and antrophometrics variables in 63 ASD children and adolescents (10.5 ± 4.1 years; 81% male). MATERIALS AND METHODS: Anthropometric variables were weight, height, and waist circumference (WC); body composition (fat mass, fat free mass) and phase angle (PA) were obtained through BIA. RESULTS: The body mass index showed a prevalence of overweight, obesity and underweight of 38.9, 36.5 and 15.8%, respectively. According to the body fat percentual, obesity prevalence was 49.2%, and 49.2% showed WC > 80th percentile for age. Eleven patients presented lower PA values than references. CONCLUSION: According to the these parameters, a large percentual of ASD children and adolescents in this sample had total overweight and obesity and truncal adiposity, which causes concern, as well as the percentage of underweight participants.

The effect of ketogenic diet in an animal model of autism induced by prenatal exposure to valproic acid.

OBJECTIVES: Autism spectrum disorder (ASD) is characterized by impairments in social interaction and communication, and by restricted repetitive behaviors and interests. Its etiology is still unknown, but different environmental factors during pregnancy, such as exposure to valproic acid (VPA), are associated with high incidence of ASD in children. In this context, prenatal exposure to VPA in rodents has been used as a reliable model of ASD. Ketogenic diet (KD) is an alternative therapeutic option for refractory epilepsy; however, the effects of this approach in ASD-like behavior need to be evaluated. We conducted a behavioral assessment of the effects of KD in the VPA model of autism. METHODS: Pregnant animals received a single-intraperitoneal injection of 600 mg/kg VPA, and their offspring were separated into four groups: (1) control group with standard diet (C-SD), (2) control group with ketogenic diet (C-KD), (3) VPA group with standard diet (VPA-SD), and (4) VPA group with ketogenic diet (VPA-KD). RESULTS: When compared with the control group, VPA animals presented increased social impairment, repetitive behavior and higher nociceptive threshold. Interestingly, the VPA group fed with KD presented improvements in social behavior. These mice displayed higher scores in sociability index and social novelty index when compared with the SD-fed VPA mice. DISCUSSION: VPA mice chronically exposed to a KD presented behavioral improvements; however, the mechanism by which KD improves ASD-like features needs to be further investigated. In conclusion, the present study reinforces the potential use of KD as a treatment for the core deficits of ASD.

Feeding behavior and dietary intake of male children and adolescents with autism spectrum disorder: A case-control study.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder associated with restrictive or repetitive behaviors and difficulties with verbal and interpersonal communication, in which some problems involving nutrition may be present. This study aims to evaluate dietary intake and identify feeding behavioral problems in male children and adolescents with ASD when compared to matched controls, as well as parents or caregivers' feelings about strategies for dealing with eating problems. A 3-day food record was performed and nutrient intake was compared to the Dietary Reference Intake according to age. To evaluate children feeding behavior and parents or caregivers' feelings, the Behavior Pediatrics Feeding Assessment Scale (BPFA) was used. ASD patients consumed in average more calories than controls (though with a high patient's frequency above and below calorie range references), had a limited food repertoire, high prevalence of children with inadequate calcium, sodium, iron vitamin B5, folate, and vitamin C intake. BPFA scores were also higher in the ASD group when compared to controls for all frequencies (child behavior, parents and total). These findings lead us to endorse the importance of evaluating feeding problems in the clinical routine, considering also the singular features of the patients.

Folic acid and autism: What do we know?

Autism spectrum disorders (ASD) consist in a range of neurodevelopmental conditions that share common features with autism, such as impairments in communication and social interaction, repetitive behaviors, stereotypies, and a limited repertoire of interests and activities. Some studies have reported that folic acid supplementation could be associated with a higher incidence of autism, and therefore, we aimed to conduct a systematic review of studies involving relationships between this molecule and ASD. The MEDLINE database was searched for studies written in English which evaluated the relationship between autism and folate. The initial search yielded 60 potentially relevant articles, of which 11 met the inclusion criteria. The agreement between reviewers was κ = 0.808. The articles included in the present study addressed topics related to the prescription of vitamins, the association between folic acid intake/supplementation during pregnancy and the incidence of autism, food intake, and/or nutrient supplementation in children/adolescents with autism, the evaluation of serum nutrient levels, and nutritional interventions targeting ASD. Regarding our main issue, namely the effect of folic acid supplementation, especially in pregnancy, the few and contradictory studies present inconsistent conclusions. Epidemiological associations are not reproduced in most of the other types of studies. Although some studies have reported lower folate levels in patients with ASD, the effects of folate-enhancing interventions on the clinical symptoms have yet to be confirmed.

Effects of an H3R antagonist on the animal model of autism induced by prenatal exposure to valproic acid.

Autism spectrum disorders (ASD) are a group of neurodevelopmental disorders primarily characterized by impaired social interaction and communication, and by restricted repetitive behaviors and interests. Ligands of histamine receptor 3 (H3R) are considered potential therapeutic agents for the treatment of different brain disorders and cognitive impairments. Considering this, the aim of the present study is to evaluate the actions of ciproxifan (CPX), an H3R antagonist, on the animal model of autism induced by prenatal exposure to valproic acid (VPA). Swiss mice were prenatally exposed to VPA on embryonic day 11 and assessed for social behavior, nociceptive threshold and repetitive behavior at 50 days of life. The treatment with CPX (3 mg/kg) or saline was administered 30 minutes before each behavioral test. The VPA group presented lower sociability index compared to VPA animals that were treated with CPX. Compared to the Control group, VPA animals presented a significantly higher nociceptive threshold, and treatment with CPX was not able to modify this parameter. In the marble burying test, the number of marbles buried by VPA animals was consistent with markedly repetitive behavior. VPA animals that received CPX buried a reduced amount of marbles. In summary, we report that an acute dose of CPX is able to attenuate sociability deficits and stereotypies present in the VPA model of autism. Our findings have the potential to help the investigations of both the molecular underpinnings of ASD and of possible treatments to ameliorate the ASD symptomatology, although more research is still necessary to corroborate and expand this initial data.

Hypomyelination, memory impairment, and blood-brain barrier permeability in a model of sleep apnea.

We investigated the effect of intermittent hypoxia, mimicking sleep apnea, on axonal integrity, blood-brain barrier permeability, and cognitive function of mice. Forty-seven C57BL mice were exposed to intermittent or sham hypoxia, alternating 30s of progressive hypoxia and 30s of reoxigenation, during 8h/day. The axonal integrity in cerebellum was evaluated by transmission electron microscopy. Short- and long-term memories were assessed by novel object recognition test. The levels of endothelin-1 were measured by ELISA. Blood-brain barrier permeability was quantified by Evans Blue dye. After 14 days, animals exposed to intermittent hypoxia showed hypomyelination in cerebellum white matter and higher serum levels of endothelin-1. The short and long-term memories in novel object recognition test was impaired in the group exposed to intermittent hypoxia as compared to controls. Blood-brain barrier permeability was similar between the groups. These results indicated that hypomyelination and impairment of short- and long-term working memories occurred in C57BL mice after 14 days of intermittent hypoxia mimicking sleep apnea.

Histaminergic system in brain disorders: lessons from the translational approach and future perspectives.

Histamine and its receptors were first described as part of immune and gastrointestinal systems, but their presence in the central nervous system and importance in behavior are gaining more attention. The histaminergic system modulates different processes including wakefulness, feeding, and learning and memory consolidation. Histamine receptors (H1R, H2R, H3R, and H4R) belong to the rhodopsin-like family of G protein-coupled receptors, present constitutive activity, and are subjected to inverse agonist action. The involvement of the histaminergic system in brain disorders, such as Alzheimer's disease, schizophrenia, sleep disorders, drug dependence, and Parkinson's disease, is largely studied. Data obtained from preclinical studies point antagonists of histamine receptors as promising alternatives to treat brain disorders. Thus, clinical trials are currently ongoing to assess the effects of these drugs on humans. This review summarizes the role of histaminergic system in brain disorders, as well as the effects of different histamine antagonists on animal models and humans.

Downregulation of uncoupling protein-1 mRNA expression and hypoadiponectinemia in a mouse model of sleep apnea.

PURPOSE: The knowledge on the effect of intermittent hypoxia on adipose tissue-mediated processes is incipient. The aim of the present study was to assess the effect of a sleep apnea model on a limited set of specific molecular, biochemical, histological, and behavioral parameters of adipose tissue function. METHODS: Mice were exposed to either intermittent hypoxia or sham hypoxia during 8 h a day for 37 days. Uncoupling protein-1 expression in brown adipose tissue was measured by real-time PCR and immunohistochemistry. Digital quantification of adipose cells and immunohistochemistry of uncoupling protein-1 were performed to determine cell dimensions, positive area, and staining intensity. Serum levels of leptin, adiponectin, and cortisol were measured by ELISA. RESULTS: In comparison with the control group, animals in the hypoxia group had significantly lower chow ingestion, weight gain, and smaller white and brown adipocytes on histological examination. Adiponectin levels were also lower in the hypoxia group. Uncoupling protein-1 mRNA was abolished in the mice exposed to hypoxia; accordingly, fewer cells positive for uncoupling protein-1 and lighter staining intensity were observed in brown adipocytes. CONCLUSIONS: An experimental model of sleep apnea produced changes in uncoupling protein-1 expression and adiponectin levels. These results confirm previous findings on the response of brown adipose tissue to intermittent hypoxia and indicate a yet-unknown interference of intermittent hypoxia on energy control, which may participate in the propensity to weight gain observed in patients with sleep apnea. Brown adipose tissue activity in this patient population needs to be further investigated.

Altered aquaporins in the brains of mice submitted to intermittent hypoxia model of sleep apnea.

Rostral fluid displacement has been proposed as a pathophysiologic mechanism of both central and obstructive sleep apnea. Aquaporins are membrane proteins that regulate water transport across the cell membrane and are involved in brain edema formation and resolution. The present study investigated the effect of intermittent hypoxia (IH), a model of sleep apnea, on brain aquaporins. Mice were exposed to intermittent hypoxia to a nadir of 7% oxygen fraction. Brain water content, Aquaporin-1 and Aquaporin-3 were measured in the cerebellum and hippocampus. Hematoxylin-eosin and immunohistochemistry stainings were performed to evaluate cell damage. Compared to the sham group, the hypoxia group presented higher brain water content, lower levels of Aquaporin-1 and similar levels of Aquaporin-3. Immunoreactivity to GFAP and S100B was stronger in the hypoxia group in areas of extensive gliosis, compatible with cytotoxic edema. These findings, although preliminary, indicate an effect of IH on aquaporins levels. Further investigation about the relevance of these data on the pathophysiology of OSA is warranted.

Simulating sleep apnea by exposure to intermittent hypoxia induces inflammation in the lung and liver.

Sleep apnea is a breathing disorder that results from momentary and cyclic collapse of the upper airway, leading to intermittent hypoxia (IH). IH can lead to the formation of free radicals that increase oxidative stress, and this mechanism may explain the association between central sleep apnea and nonalcoholic steatohepatitis. We assessed the level of inflammation in the lung and liver tissue from animals subjected to intermittent hypoxia and simulated sleep apnea. A total of 12 C57BL/6 mice were divided into two groups and then exposed to IH (n = 6) or a simulated IH (SIH) (n = 6) for 35 days. We observed an increase in oxidative damage and other changes to endogenous antioxidant enzymes in mice exposed to IH. Specifically, the expression of multiple transcription factors, including hypoxia inducible factor (HIF-1α), nuclear factor kappa B (NF-κB), and tumor necrosis factor (TNF-α), inducible NO synthase (iNOS), vascular endothelial growth factor (VEGF), and cleaved caspase 3 were shown to be increased in the IH group. Overall, we found that exposure to intermittent hypoxia for 35 days by simulating sleep apnea leads to oxidative stress, inflammation, and increased activity of caspase 3 in the liver and lung.

Brown adipose tissue: is it affected by intermittent hypoxia?

BACKGROUND: Intermittent hypoxia (IH), a model of sleep apnea, produces weight loss in animals. We hypothesized that changes in brown adipose tissue (BAT) function are involved in such phenomenon. We investigated the effect of IH, during 35 days, on body weight, brown adipose tissue wet weight (BATww) and total protein concentration (TPC) of BAT. METHODS: We exposed Balb/c mice to 35 days of IH (n = 12) or sham intermittent hypoxia (SIH; n = 12), alternating 30 seconds of progressive hypoxia to a nadir of 6%, followed by 30 seconds of normoxia. During 8 hours, the rodents underwent a total of 480 cycles of hypoxia/reoxygenation, equivalent to an apnea index of 60/hour. BAT was dissected and weighed while wet. Protein was measured using the Lowry protein assay. RESULTS: Body weight was significantly reduced in animals exposed to IH, at day 35, from 24.4 ± 3.3 to 20.2 ± 2.2 g (p = 0.0004), while in the SIH group it increased from 23.3 ± 3.81 to 24.1 ± 2.96 g (p = 0.23). BATww was also lower in IH than in SIH group (p = 0.00003). TPC of BAT, however, was similar in IH (204.4 ± 44.3 µg/100 µL) and SIH groups (213.2 ± 78.7 µg/100 µL; p = 0.74) and correlated neither with body weight nor with BATww. TPC appeared to be unaffected by exposure to IH also in multivariate analysis, adjusting for body weight and BATww. The correlation between body weight and BATww is significant (rho= 0.63) for the whole sample. When IH and SIH groups are tested separately, the correlations are no longer significant (rho = 0.48 and 0.05, respectively). CONCLUSION: IH during 35 days in a mice model of sleep apnea causes weight loss, BATww reduction, and no change in TPC of BATww. The mechanisms of weight loss under IH demands further investigation.