O autismo é um transtorno complexo do desenvolvimento caracterizado por inúmeros prejuízos comportamentais, tais como perdas na comunicação, socialização e indução da inflexibilidade cognitiva. A reprodução de modelos experimentais de autismo é bastante importante no estudo desse transtorno. A exposição pré-natal ao VPA reproduz sintomas similares àqueles encontrados na condição humana de autismo. Apesar de o VPA ser considerado um provável teratógeno em humanos, pouco se sabe sobre seus mecanismos de ação, ou até mesmo, como ele é capaz de induzir o autismo. O VPA parece influenciar o metabolismo do zinco durante o período pré-natal, podendo comprometer o desenvolvimento fetal normal. Esse estudo foi dividido em duas partes: (1) reproduzir um modelo de autismo induzido por VPA pré-natal (400 mg/kg no GD 12,5) e (2) avaliar se a administração de zinco pré-natal (2 mg/kg 1h após) previne ou ameniza os prejuízos comportamentais e a expressão de TH-estriatal associados ao autismo causados pela exposição pré-natal ao VPA em ratos. Foram avaliados parâmetros reprodutivos, anormalidades comportamentais na comunicação (vocalização ultrassônica), comportamentos repetitivos e na cognição (labirinto em T) e interação social (comportamento de brincar), além de verificar o sistema dopaminérgico por meio da expressão proteica de TH-estriatal na prole masculina e feminina de ratas. O VPA causou danos reprodutivos, prejuízos na vocalização ultrassônica, comportamento repetitivo/restrito, inflexibilidade cognitiva, comprometimento na socialização com diminuição da brincadeira e redução nos níveis de TH- estriatal na prole masculina. A administração de zinco não foi capaz de impedir os danos reprodutivos causados pelo VPA, porém amenizou a inflexibilidade cognitiva e atenuou a brincadeira social, sem reestabelecer os níveis de TH-estriatal. A prole feminina foi menos afetada que a masculina, apresentando apenas a vocalização ultrassônica alterada. O zinco reestabeleceu esse dano. A redução da TH-estriatal na prole masculina sugere que, possivelmente o fenótipo tipo-autista investigado no presente estudo, esteja relacionado a modificações funcionais no sistema dopaminérgico. / Autism is a complex developmental disorder characterized by numerous behavioral impairments, such as communication, socialization and induction of cognitive inflexibility. Reproduction of experimental models of autism is an important tool in the study of this disorder. Prenatal exposure to VPA reproduces symptoms similar to those found in the human condition of autism. The VPA is known as a potential teratogen in humans and its mechanisms of action is not well understood. VPA appears to influence the zinc metabolism during the prenatal period, compromising normal fetal development. This study was done into two parts: (1st) obtain an autism model induced by prenatal VPA (400 mg/kg 12.5 GD) and (2nd) evaluate whether prenatal administration of zinc (2 mg/kg after 1h) prevents or reverses the behavioral impairments and protein striatal TH-expression associated with autism caused by prenatal exposure to VPA in rats. It was evaluated the reproductive parameters, abnormal behavior in the communication (ultrasonic vocalization test), repetitive behavior and cognitive capacity (T-maze test) and social interaction (play behavior test), and the evaluation of the dopaminergic system by quantification of TH-striatal expression in male and female offspring of rats. The VPA was able to cause reproductive damage, impairment in ultrasonic vocalization, repetitive/restricted behavior and cognitive inflexibility, impairment in socialization with decreased play behavior and reduction in striatal TH levels in male offspring. The zinc administration has not been able to prevent reproductive damage caused by VPA, but ameliorates the cognitive inflexibility and attenuated the social play behavior, without restoring TH-striatal levels. The offspring females were less affected as males, with alterations into ultrasonic vocalization only and zinc reestablished this damage. The reduction in striatal TH-male offspring suggests that the autistic-like phenotype, investigated in this study is, may be is related to functional modifications in the dopaminergic system.

O transtorno do espectro autista atinge uma em cada 150 crianças. Sua etiologia ainda é desconhecida, apesar de fortes evidências de fatores genéticos e recentes achados de interferências ambientais, particularmente a ativação imune materna durante a gestação. Em nossos estudos prévios expusemos ratas Wistar no início da gestação ao lipopolissacarídeo (LPS), que mimetiza uma infecção bacteriana (100 µg/kg, intraperitoneal [i.p.], no dia gestacional [GD] 9,5) e observamos prejuízos no comportamento de brincar da prole masculina. Sabendo que esse teste é classicamente usado para avaliação de modelos animais de autismo e da ligação do autismo com fatores ambientais, propusemos que nosso modelo seria um modelo animal de autismo. Para avaliar essa possibilidade, foi objetivo do presente trabalho estudar se nosso modelo de LPS pré-natal (100 µg/kg, i.p., no GD 9,5) causaria os outros sintomas típicos de autistas: anormalidades na comunicação (avaliado pelo teste da vocalização ultrassônica), comportamentos repetitivos e inflexibilidade cognitiva (teste de alternação espontânea no labirinto em T), ausência de demonstração de medo em situações potencialmente perigosas (estímulo olfativo aversivo do odor de gato) e hiperatividade (atividade geral em campo aberto). Visto que alguns autistas apresentam níveis elevados de citocinas pró-inflamatórias e cortisol, bem como neuroinflamação, foram quantificados os níveis de IL-1β, TNF-α e corticosterona séricos, e estudado os astrócitos e micróglia do estriado e bulbo olfatório. Outro objetivo deste trabalho foi estudar os mecanismos centrais associados ao LPS pré-natal, especialmente o sistema dopaminérgico (expressão gênica e proteica de tirosina hidroxilase e receptores dopaminérgicos D1a e D2) do estriado e bulbo olfatório, baseado nos nossos achados prévios de redução de dopamina nessas regiões cerebrais. Também foram avaliados os níveis séricos de corticosterona, o desempenho reprodutivo e o tecido placentário nas mães expostas ao LPS durante a gestação. Por fim, sabendo que o autismo é mais prevalente em homens do que em mulheres, foram avaliadas também as fêmeas expostas pré-natalmente ao LPS: comportamento de brincar, interação social adulta e níveis séricos de corticosterona. A exposição pré-natal ao LPS prejudicou a comunicação, a cognição, a demonstração de medo em situações potencialmente perigosas, além de induzir comportamentos repetitivos/restritos e elevação dos níveis de IL-1β na prole masculina de ratas; as fêmeas não apresentaram os prejuízos observados nos machos. Esses achados sugerem fortemente que nosso modelo de LPS pré-natal induziu comportamentos do tipo autismo em ratos, corroborando com a hipótese dos fatores ambientais no autismo. Acrescenta de maneira inédita um modelo que mimetiza uma infecção bacteriana no início da gestação como indutor do autismo. Mais ainda, a redução da expressão proteica de tirosina hidroxilase estriatal causada pelo LPS pré-natal inclui a hipoatividade do sistema dopaminérgico estriatal como um possível aspecto para explicar o autismo. A elevação nos níveis de corticosterona, prejuízo no desempenho reprodutivo e danos no tecido placentário das ratas ajudaram no entendimento da gênese dos prejuízos na prole. O estabelecimento do modelo experimental de autismo com o LPS pré-natal significa um passo importante para o entendimento dessa desordem e pode auxiliar também na busca por eventuais tratamentos, baseados nas alterações neuroimunes encontradas. / One child in about 150 children has autism spectrum disorder. Despite strong evidence of genetic factors and recent findings of environmental interferences, particularly maternal immune activation during pregnancy, autism etiology is still unknown. In our previous study, we exposed Wistar rats in the beginning of the gestation to lipopolysaccharide (LPS, 100 µg/kg, intraperitoneally [i.p.], on gestational day [GD] 9.5), which mimics a bacterial infection and observed impairments in the play behavior of male offspring. Knowing that play behavior is classically used to evaluate autism animal models and the link between autism and environmental factors, we proposed that our model would be an autism animal model. To evaluate this possibility, the aim of this study was to know whether our prenatal LPS model (100 µg/kg, i.p., on GD 9.5) causes other autism typical symptoms: communication abnormalities (evaluated by the ultrasonic vocalization test), repetitive behavior and cognitive inflexibility (T-maze spontaneous alternation test), absence of fear demonstration in potentially dangerous situations (aversive exposure to cat odor test) and hyperactivity (open field general activity). Because some autistic patients presents high levels of proinflammatory cytokines and cortisol as well as neuroinflammation, we quantified IL-1β, TNF-α and corticosterone serum levels, and studied astrocytes and microglia of striatum and olfactory bulb. Another objective was to study the central mechanisms associated with prenatal LPS, particularly the dopaminergic system (gene and protein expression of tyrosine hydroxylase and dopamine D1a and D2 receptors) of striatum and olfactory bulb, based on our previous findings of reduced dopamine in these brain regions. We also evaluated the corticosterone serum levels, the reproductive performance and the placental tissue of mothers exposed to LPS during pregnancy. Finally, because autism is more prevalent in men than in women, we also evaluated females prenatally exposed to LPS: play behavior, social interaction and adult corticosterone serum levels. Prenatal LPS exposure impaired communication, cognition, fear demonstration of potentially dangerous situations, induced repetitive/restricted behaviors and elevated IL-1β levels of male offspring; females did not present the impairments observed in males. These findings strongly suggest that our model of prenatal LPS induced autism like behaviors in rats, corroborating the hypothesis of environmental factors in autism. It adds an unprecedented way a model that mimics a bacterial infection in early pregnancy as an inducer of autism. Moreover, the striatal tyrosine hydroxylase protein expression reduction caused by prenatal LPS includes hypoactivity of the striatal dopaminergic system as a possible aspect to explain autism. The increase in the corticosterone levels, impairment of reproductive performance and placental tissue injuries of dams helped in understanding the genesis of the offspring impairments. The importance of establishing an experimental model of autism with prenatal LPS is an extra step to better understand this disorder and may also help in the search for possible treatments, based on the observed neuroimmune changes.

Human herpesvirus 6A (HHV-6A) has yet to be definitively linked to a specific disease. This is due in part to the ubiquitous nature of the virus. Humanized Rag2-/-γc-/- (Rag-hu) mice were tested to determine if these were a suitable animal model to study the virus. Both cell-free and cell-associated virus was used for infection and both were found to be efficient at infecting the mice. Viral DNA was found in the plasma and cellular blood fractions, bone marrow, lymph node, and thymus, indicating successful infection and propagation of the virus in vivo. The CD3+CD4- population was depleted, while the CD3-CD4+ was increased in infected animals. The CD3-CD4+CD8- and CD3+CD4+CD8- populations were depleted and the CD3+CD4+CD8+ population increased when analysis was gated upon CD4+ cells. The CD3-CD4+CD8+ population expanded and the CD3-CD4+CD8- population was reduced when analysis was gated on the CD3- population. Additional flow cytometry analysis revealed increases in CD4+CD8+ double positive cells in the peripheral blood of cell-free infected mice, which could indicate improper T cell selection and a premature departure of these cells from the thymus, possibly contributing to autoimmunity. Previous research has shown that HIV and HHV-6A may have a synergistic effect on one another and that HHV-6A may act as a cofactor in the progression to AIDS. After determining the Rag-hu mouse model was suitable for studying HHV-6A infection, a coinfection of HHV-6A and HIV-1 was performed. Coinfected mice had fewer thymocytes when compared with the HIV-1 only, mock-infected, and to a lesser extent HHV-6A only groups which could indicate increased cell death in the coinfected group as well as possible disruptions in migration of cells, either causing cells to be sequestered in the bone marrow and unable to migrate to the thymus, or causing premature egress of the cells in the thymus due in part to premature upregulation of CCR7, both of which would explain the smaller cellular populations found in the coinfected mouse thymi. Additional studies were performed to determine if a preferential targeting existed between HHV-6A and HIV-1 as these viruses are found simultaneously coinfecting the same cell. Preferential targeting was not observed by cell-associated migration assay, but increased migration of HHV-6A-infected cells was observed in a CCL21 dependent manner. These studies have provided useful information about HHV-6A and its relevance to HIV/AIDS as well as a possible mechanism of the involvement of HHV-6A in multiple sclerosis (MS) and other autoimmune diseases.

The current study analyzed the effects of environmental enrichment versus isolation housing on the behavioral sensitization to nicotine in the neonatal quinpirole (NQ; dopamine D2-like agonist) model of dopamine D2 receptor supersensitivity, a rodent model of schizophrenia. NQ treatment in rats increases dopamine D2 receptor sensitivity throughout the animal's lifetime, consistent with schizophrenia. Animals were administered NQ (1 mg/kg) or saline (NS) from postnatal day (P)1 to P21, weaned, and immediately placed into enriched housing or isolated in wire cages throughout the experiment. Rats were behaviorally sensitized to nicotine (0.5 mg/kg base) or saline every consecutive day from P38 to P45, and brain tissue was harvested at P46. Results revealed that neither housing condition reduced nicotine sensitization in NQ rats, whereas enrichment reduced sensitization to nicotine in NS-treated animals. The nucleus accumbens (NAcc) was analyzed for glial cell line-derived neurotrophic factor (GDNF), a neurotrophin important in dopamine plasticity. Results were complex, and revealed that NAcc GDNF was increased in animals given nicotine, regardless of housing condition. Further, enrichment increased GDNF in NQ rats regardless of adolescent drug treatment and in NS-treated rats given nicotine, but did not increase GDNF in NS-treated controls compared to the isolated housing condition. This study demonstrates that environmental experience has a prominent impact on the behavioral and the neural plasticity NAcc response to nicotine in adolescence.

The purpose of lactation is for nutrient provision and also importantly for protection from various environmental stressors. Milk polar lipids reduce cholesterol, protect against bacterial infection, reduce inflammation and help maintain gut integrity. Dynamic interactions within dietary fat, lipid metabolism, gut permeability and inflammatory cytokines remain unclear in the context of obesity and systemic inflammation. A rat model and three mouse models were developed to test the hypotheses that dietary milk polar lipids may affect lipid metabolism and intestinal integrity and may protect against systemic inflammation in the context of stressful diet, systemic inflammation, and obesity. The milk polar lipids isolates had complex effects on lipid metabolism and associated gene expression in the rat model. There were complex dynamics in lipid metabolism, gut permeability and systemic inflammation at different time points in all mouse models. The milk phospholipids increased gut permeability in genetic and diet-induced obesity and during the lipopolysaccharide (LPS) -induced inflammation. The phospholipids increased the plasma LPS level in genetic obesity and during the LPS stress. The phospholipids reduced liver mass and liver lipids in genetic obesity and during the LPS-induced inflammation. The phospholipids increased the body fat in the diet-induced obesity model. The milk gangliosides did not significantly affect gut permeability, systemic inflammation, and lipid metabolism in all three mouse models. Current estimate by the Centers for Disease Control is that about 1/3 Americans are obese (body mass index, BMI ≥ 30) and 1/3 Americans are overweight (25 ≤ BMI < 30). More than 25% of Americans today have a fatty liver which could lead to further health problems. The data from this dissertation shed light on the complicated interrelationships between gut permeability, systemic inflammation, and lipid metabolism in obesity. The results contribute to our understanding of the bioactivities of milk polar lipids and provide scientific evidence for the role of milk polar lipids rich materials in affecting biological functions. The study of the influence of milk polar lipids on gut barrier integrity adds new information on understanding the mechanisms of gut leakiness and recovery. The investigation of the impact of milk polar lipids on lipid metabolism reveals new perspectives for the development of diet-induced obesity.

<p>Predisposition to autoimmune diseases such as, rheumatoid arthritis, diabetes, and multiple sclerosis, is caused by the effect of multiple genes and a strong influence from the environment. </p><p>In this study, I have investigated genetic factors that confer susceptibility to rheumatoid arthritis in a rat model. This work has led to the identification of several chromosomal regions, containing uncharacterized genes that directly or indirectly are associated to the arthritis development in these rats. We have observed that timing, gender, and genetic interactions are features that play a part in the effect that these genetic factors exert. </p><p>Unarguably, animal models for human disorders display differences to the human form of disease. An important fact is however that the same chromosomal regions are identified in both rodent and human studies, which suggests that there are genetic factors that we have in common, which are involved directly or indirectly with an autoimmune response. </p><p>Focusing the interest on these similarities, and on the possibility to apply a wide set of genetic tools, make animal models an invaluable, and probably necessary, instrument to dissect the genetic component of complex disorders. To fully comprehend the genetic basis for a complex disorder like this, will require understanding of how multiple genes interact with each other to cause disease. </p><p>We have been able to demonstrate that chronic arthritis, in a rat model for rheumatoid arthritis, is regulated by several genes and that these act during different temporal phases of the disease. These findings will hopefully contribute to our understanding of the etiology and progression of rheumatoid arthritis.</p>

<p>In this thesis, occurrence of heterotopic ossification (HO) following total hip arthroplasty (THA) was studied. Preventive effects and complications with non-steroidal anti-inflammatory drugs (NSAIDs) were analyzed. Experimental investigations on bone formation were employed to gain insight to the mechanism of NSAIDs action on bone.</p><p>(I). Fifty-six patients with bilateral THAs were analyzed. We found a strong correlation between HO on the two sides. Incidence and grade of HO were higher in men than in women.</p><p>(II). Sixty-nine patients with bilateral THAs who had been treated with NSAIDs after one or both THAs were analyzed for HO. Widespread HO occurred in untreated THAs, but in none of the treated THAs.</p><p>(III). A consecutive series of THAs were analyzed for HO. No widespread HO occurred in patients treated with NSAIDs for 21 days. In contrast, widespread HO occurred in 23% of patients not treated.</p><p>(IV). A randomized, double-blind, prospective study on 144 patients was performed to determine the efficacy and minimum treatment time with Ibuprofen for prophylaxis of HO after THA. Treatment with Ibuprofen was effective for preventing HO and a treatment time of 8 days was sufficient.</p><p>(V). A ten-year follow-up examination was performed on the patients from study IV. Thirteen patients had been revised. All but one belonged to groups treated with Ibuprofen. However, the prosthetic survival time was not statistically different for patients treated with NSAIDs compared to the control group. Eighty-four more patients underwent radiographic examination10 years after THA. Nine loose prostheses were found. These were equally distributed between NSAIDs-treated and non-treated THAs. When combining complications (revisions and radiographic loosening) no significant effects could be verified.</p><p>(VI). Experimental induction of heterotopic new bone with demineralized allogeneic bone matrix (DABM) and with bone autografts, was used in rats to study effects of NSAIDs on new bone formation. Indomethacin inhibited net bone formation in DABMs and in orthotopic fractured bone. In contrast, a net mineral loss occurred in autografts, but neither mineral content nor ⁴⁵Ca incorporation was affected by Indomethacin treatment. The amount of bone formed per mg implanted DABM was linearly correlated to implant size.</p>

<p>The genetic basis for behavioural traits is largely unknown. The overall aim of this thesis was to find genes with importance for behavioural traits related to fear and anxiety. Microarray analysis was used to screen expression profiles of brain regions important for emotional behaviour in dogs, wolves, foxes and mice. In a first experiment, dogs and their wild ancestors the wolves were compared. Our results suggested that directed selection for behaviour might have resulted in expression changes in few genes acting on several brain functions, possibly affecting behaviour. However, the observed expressional differences were confounded with environmental effects. This was addressed in a second study on domesticated silver foxes. By correlating behaviour and brain gene expression in foxes selected for tameness to non-selected foxes raised in the same environment, we found large behavioural differences but only few genes with differential expression in the brain. Fifteen of the 40 genes showing evidence of expression difference were related to haem or haemoglobins. Further studies showed an additive genetic effect on brain gene expression, similar to the additive genetic inheritance of behaviour, indicating an involvement in domestication. Transcriptional profiling was also used for finding genes involved with the sleep disorder narcolepsy. Narcoleptic Doberman pinschers homozygous for the canarc-1 mutation were compared to their unaffected heterozygots revealing reduced expression of three genes, TAC1, PENK and SOCS2, with relevance to the narcoleptic phenotype. Finally gene expression was investigated in relation to anxiety-related traits in a mouse model. Surprisingly, as in the fox study, genes coding for haemoglobins indicated differential expression in the brain between animals with different anxiety levels. Our combined results suggest that genes like haemoglobins, best known for their function in oxygen transport in blood, may also participate in brain functions related to decreased anxiety in domestic animals. </p>

Predisposition to autoimmune diseases such as, rheumatoid arthritis, diabetes, and multiple sclerosis, is caused by the effect of multiple genes and a strong influence from the environment. In this study, I have investigated genetic factors that confer susceptibility to rheumatoid arthritis in a rat model. This work has led to the identification of several chromosomal regions, containing uncharacterized genes that directly or indirectly are associated to the arthritis development in these rats. We have observed that timing, gender, and genetic interactions are features that play a part in the effect that these genetic factors exert. Unarguably, animal models for human disorders display differences to the human form of disease. An important fact is however that the same chromosomal regions are identified in both rodent and human studies, which suggests that there are genetic factors that we have in common, which are involved directly or indirectly with an autoimmune response. Focusing the interest on these similarities, and on the possibility to apply a wide set of genetic tools, make animal models an invaluable, and probably necessary, instrument to dissect the genetic component of complex disorders. To fully comprehend the genetic basis for a complex disorder like this, will require understanding of how multiple genes interact with each other to cause disease. We have been able to demonstrate that chronic arthritis, in a rat model for rheumatoid arthritis, is regulated by several genes and that these act during different temporal phases of the disease. These findings will hopefully contribute to our understanding of the etiology and progression of rheumatoid arthritis.

In this thesis, occurrence of heterotopic ossification (HO) following total hip arthroplasty (THA) was studied. Preventive effects and complications with non-steroidal anti-inflammatory drugs (NSAIDs) were analyzed. Experimental investigations on bone formation were employed to gain insight to the mechanism of NSAIDs action on bone. (I). Fifty-six patients with bilateral THAs were analyzed. We found a strong correlation between HO on the two sides. Incidence and grade of HO were higher in men than in women. (II). Sixty-nine patients with bilateral THAs who had been treated with NSAIDs after one or both THAs were analyzed for HO. Widespread HO occurred in untreated THAs, but in none of the treated THAs. (III). A consecutive series of THAs were analyzed for HO. No widespread HO occurred in patients treated with NSAIDs for 21 days. In contrast, widespread HO occurred in 23% of patients not treated. (IV). A randomized, double-blind, prospective study on 144 patients was performed to determine the efficacy and minimum treatment time with Ibuprofen for prophylaxis of HO after THA. Treatment with Ibuprofen was effective for preventing HO and a treatment time of 8 days was sufficient. (V). A ten-year follow-up examination was performed on the patients from study IV. Thirteen patients had been revised. All but one belonged to groups treated with Ibuprofen. However, the prosthetic survival time was not statistically different for patients treated with NSAIDs compared to the control group. Eighty-four more patients underwent radiographic examination10 years after THA. Nine loose prostheses were found. These were equally distributed between NSAIDs-treated and non-treated THAs. When combining complications (revisions and radiographic loosening) no significant effects could be verified. (VI). Experimental induction of heterotopic new bone with demineralized allogeneic bone matrix (DABM) and with bone autografts, was used in rats to study effects of NSAIDs on new bone formation. Indomethacin inhibited net bone formation in DABMs and in orthotopic fractured bone. In contrast, a net mineral loss occurred in autografts, but neither mineral content nor 45Ca incorporation was affected by Indomethacin treatment. The amount of bone formed per mg implanted DABM was linearly correlated to implant size.