DWI and complex brain network analysis predicts vascular cognitive impairment in spontaneous hypertensive rats undergoing executive function tests

The identification of biomarkers of vascular cognitive impairment is urgent for its early diagnosis. The aim of this study was to detect and monitor changes in brain structure and connectivity, and to correlate them with the decline in executive function. We examined the feasibility of early diagnostic magnetic resonance imaging (MRI) to predict cognitive impairment before onset in an animal model of chronic hypertension: Spontaneously Hypertensive Rats. Cognitive performance was tested in an operant conditioning paradigm that evaluated learning, memory, and behavioral flexibility skills. Behavioral tests were coupled with longitudinal diffusion weighted imaging acquired with 126 diffusion gradient directions and 0.3 mm(3) isometric resolution at 10, 14, 18, 22, 26, and 40 weeks after birth. Diffusion weighted imaging was analyzed in two different ways, by regional characterization of diffusion tensor imaging (DTI) indices, and by assessing changes in structural brain network organization based on Q-Ball tractography. Already at the first evaluated times, DTI scalar maps revealed significant differences in many regions, suggesting loss of integrity in white and gray matter of spontaneously hypertensive rats when compared to normotensive control rats. In addition, graph theory analysis of the structural brain network demonstrated a significant decrease of hierarchical modularity, global and local efficacy, with predictive value as shown by regional three-fold cross validation study. Moreover, these decreases were significantly correlated with the behavioral performance deficits observed at subsequent time points, suggesting that the diffusion weighted imaging and connectivity studies can unravel neuroimaging alterations even overt signs of cognitive impairment become apparent.

Establishing Zebrafish as a Novel Exercise Model: Swimming Economy, Swimming-enhanced Growth and Muscle Growth Marker Gene Expression

Zebrafish has been largely accepted as a vertebrate multidisciplinary model but its usefulness as a model for exercise physiology has been hampered by the scarce knowledge on its swimming economy, optimal swimming speeds and cost of transport. Therefore, we have performed individual and group-wise swimming experiments to quantify swimming economy and to demonstrate the exercise effects on growth in adult zebrafish.

Multigram synthesis and in vivo efficacy studies of a novel multitarget anti-Alzheimer's compound

We describe the multigram synthesis and in vivo efficacy studies of a donepezil‒huprine hybrid that has been found to display a promising in vitro multitarget profile of interest for the treatment of Alzheimer's disease (AD). Its synthesis features as the key step a novel multigram preparative chromatographic resolution of intermediate racemic huprine Y by chiral HPLC. Administration of this compound to transgenic CL4176 and CL2006 Caenorhabditis elegans strains expressing human Aβ42, here used as simplified animal models of AD, led to a significant protection from the toxicity induced by Aβ42. However, this protective effect was not accompanied, in CL2006 worms, by a reduction of amyloid deposits. Oral administration for 3 months to transgenic APPSL mice, a well-established animal model of AD, improved short-term memory, but did not alter brain levels of Aβ peptides nor cortical and hippocampal amyloid plaque load. Despite the clear protective and cognitive effects of AVCRI104P4, the lack of Aβ lowering effect in vivo might be related to its lower in vitro potency toward Aβ aggregation and formation as compared with its higher anticholinesterase activities. Further lead optimization in this series should thus focus on improving the anti-amyloid/anticholinesterase activity ratio.

The health effects of sea buckthorn berries and oil

Sea buckthorn (Hippophaë) berries are ingredients of the Chinese traditional medicine. In addition to China, they are nowadays cultivated for food in several European countries, Russia, Canada, the USA, and Japan. Sea buckthorn berries are a rich source of flavonoids, mainly flavonol glycosides and proanthocyanidins. Depending on the genetic background, growth conditions, and ripeness of the berries, vitamin C concentrations up to over 1 g/100 ml juice, have been reported. Sea buckthorn berries contain inositols and methyl inositols, components of messenger molecules in humans. Sea buckthorn seed oil is rich in essential aplha-linolenic and linoleic acids, whereas the most abundant fatty acids in the berry oil are palmitoleic, palmitic and oleic acids. Other potentially beneficial lipophilic compounds of sea buckthorn seeds and berries include carotenoids, phytosterols, tocopherols and tocotrienols. The effects of sea buckthorn fractions on inflammation, platelet aggregation, oxidation injuries, the liver, skin and mucosa, among others, have been reported. The aim of the thesis work was to investigate the health effects of sea buckthorn berries and oil in humans. The physiological effects of sea buckthorn berries, berry components, and oil have mostly been studied in vitro and in animal models, leaving a demand for more clinical trials. In the first randomized, placebo-controlled trial of this thesis healthy adults consumed 28 g/day of sea buckthorn berries for three months. The main objective was to investigate the effects on the common cold. In addition, effects on other infections, inflammation and circulating lipid markers associated with cardiovascular disease risk were studied. In the second randomized, placebocontrolled trial participants reporting dry eye symptoms consumed 2 g/day of sea buckthorn oil from the seeds and berries for three months. The effects on symptoms and clinical signs of dry eye were monitored. In addition, the effects on circulating markers of inflammation and liver functions were analyzed. Sea buckthorn berries did not affect the common cold or other infections in healthy adults. However, a decrease in serum C-reactive protein was detected, indicating effects on inflammation. Fasting concentrations of serum flavonols, typical to sea buckthorn berry, increased without affecting the circulating total, HDL, LDL cholesterol, or triacylglycerol concentrations. Tear film hyperosmolarity and activation of inflammation at the ocular surface are among the core mechanisms of dry eye. Combined sea buckthorn berry and seed oil attenuated the rise in tear film osmolarity taking place during the cold season. It also positively affected some of the dry eye symptoms. Based on the tear film fatty acid analysis, the effects were not mediated through direct incorporation of sea buckthorn oil fatty acids to tear film lipids. It is likely that the fatty acids, carotenoids, tocopherols and tocotrienols of sea buckthorn oil affected the inflammation of the ocular surface, lacrimal and/or meibomian glands. The effects on the differentiation of meibomian gland cells are also possible. Sea buckthorn oil did not affect the serum concentrations of inflammation markers or liver enzymes investigated. In conclusion, this thesis work suggests positive effects of sea buckthorn berries and oil on inflammation and dry eye, respectively, in humans.

Assessment of endocrine disrupting activities in the aquatic environment

Hormonstörande ämnen är syntetiska eller naturliga ämnen som stör organismers hormonsystem och bidrar till könsförvirring och sterilitet. Sådana ämnen kommer i ökande takt ut i vattenmiljön genom rester av läkemedel, bekämpningsmedel och industriprodukter. Eftersom det finns många likheter mellan hormonsystemen hos människan och övriga ryggradsdjur kan till exempel fiskar användas som modellsystem för att underöka detta problem. I tidigare undersökningar har man funnit hormonella störningar, bl.a. feminisering och maskulinisering hos fiskar som utsatts för avfallsvatten från kommunala reningsverk eller avfallsvatten från pappersindustrin. Målet med denna avhandling var att undersöka om renat avfallsvatten från kommunala reningsverk längs Finlands kust innehåller hormonstörande ämnen i sådana mängder att de kan försorsaka hormonstörande effekter på fisk. Målet i denna avhandling var också att utveckla cellulära testsystem baserade på fiskceller, eftersom behovet av tillförlitliga och kostnadseffektiva cellbaserade tester för att underlätta riskbedömningen av hormonstörande ämnen är mycket stort för tillfället. Som testsystem har storspiggen använts, som besitter flera användbara biomarkörer för att mäta hormonstörande ämnens påverkan. Resultaten tyder på att problemen med hormonstörande påverkan på fisk inte är lika utbredda i Finland som i många andra europeiska länder. Detta beror troligtvis på att finska reningsverk har effektiva reningstekniker som reducerar mängden hormonstörande ämnen, eller på att utspädningen av avloppen i recipienterna är större än i många andra länder. Dock kan problemen inte helt uteslutas eftersom vissa feminiserande (estrogena) effekter kunde observeras hos fisken i de undersökta recipienterna utanför kommunala reningsverk. I kontrollerade laboratorieförsök där storpsiggar exponerades för kommunalt avloppsvatten uppmättes även här effekter som tyder på förekomst av estrogener i avloppsvattnet. De cell-baserade testsystemen klarade av att förutspå hormonella effekter hos hel fisk och kan därför vara mycket användbara i fortsatta studier av hormonstörande ämnens verkningsmekanismer i preliminära toxicitetsbedömningar.

In vivo imaging of vascular adhesion protein 1 - preclinical studies with positron emission tomography

The golden standard in nuclear medicine imaging of inflammation is the use of radiolabeled leukocytes. Although their diagnostic accuracy is good, the preparation of the leukocytes is both laborious and potentially hazardous for laboratory personnel. Molecules involved in leukocyte migration could serve as targets for the development of inflammation imaging agents. An excellent target would be a molecule that is absent or expressed at low level in normal tissues, but is induced or up-regulated at the site of inflammation. Vascular adhesion protein-1 (VAP-1) is a very promising target for in vivo imaging, since it is translocated to the endothelial cell surface when inflammation occurs. VAP-1 functions as an endothelial adhesion molecule that participates in leukocyte recruitment to inflamed tissues. Besides being an adhesion molecule, VAP-1 also has enzymatic activity. In this thesis, the targeting of VAP-1 was studied by using Gallium-68 (68Ga) labeled peptides and an Iodine-124 (124I) labeled antibody. The peptides were designed based on molecular modelling and phage display library searches. The new imaging agents were preclinically tested in vitro, as well as in vivo in animal models. The most promising imaging agent appeared to be a peptide belonging to the VAP-1 leukocyte ligand, Siglec-9 peptide. The 68Ga-labeled Siglec-9 peptide was able to detect VAP-1 positive vasculature in rodent models of sterile skin inflammation and melanoma by positron emission tomography. In addition to peptides, the 124I-labeled antibody showed VAP-1 specific binding both in vitro and in vivo. However, the estimated human radiation dose was rather high, and thus further preclinical studies in disease models are needed to clarify the value of this imaging agent. Detection of VAP-1 on endothelium was demonstrated in these studies and this imaging approach could be used in the diagnosis of inflammatory conditions as well as melanoma. These studies provide a proof-of-concept for PET imaging of VAP-1 and further studies are warranted.

Use of preclinical and early clinical data for dose selection of a selective estrogen receptor modulator toremifene in treatment of breast cancer

In development of human medicines, it is important to predict early and accurately enough the disease and patient population to be treated as well as the effective and safe dose range of the studied medicine. This is pursued by using preclinical research models, clinical pharmacology and early clinical studies with small sample sizes. When successful, this enables effective development of medicines and reduces unnecessary exposure of healthy subjects and patients to ineffectice or harmfull doses of experimental compounds. Toremifene is a selective estrogen receptor modulator (SERM) used for treatment of breast cancer. Its development was initiated in 1980s when selection of treatment indications and doses were based on research in cell and animal models and on noncomparative clinical studies including small number of patients. Since the early development phase, the treatment indication, the patient population and the dose range were confirmed in large comparative clinical studies in patients. Based on the currently available large and long term clinical study data the aim of this study was to investigate how the early phase studies were able to predict the treatment indication, patient population and the dose range of the SERM. As a conclusion and based on the estrogen receptor mediated mechanism of action early studies were able to predict the treatment indication, target patient population and a dose range to be studied in confirmatory clinical studies. However, comparative clinical studies are needed to optimize dose selection of the SERM in treatment of breast cancer.

Cirurgia endoscópica transluminal por orifícios naturais: realidade atual

Natural orifice transluminal endoscopic surgery (NOTES) is an emerging experimental alternative to conventional surgery that eliminates abdominal incisions and incision-related complications by combining endoscopic and laparoscopic techniques to diagnose and treat abdominal pathology. Natural orifice transluminal endoscopic surgery refers to the method of accessing the abdominal cavity through a natural orifice under endoscopic visualization. Since its introduction in 2004, numerous reports have been published describing different surgical interventions. Recently, a group of expert laparoscopic surgeons and endoscopists outlined the limitations of this approach and issued recommendations for progress toward human trials. Transluminal surgery is a new method for accessing the abdomen under direct endoscopic visualization. Preliminary studies have demonstrated the feasibility of this technique in animal models; however, further research is warranted to validate its safety in humans.

Immunohistochemical detection of Tritrichomonas foetus in experimentally infected mice

The need to intensify knowledge of the pathogenesis of bovine genital trichomoniasis (BGT) led to the use of alternative animal models such as the mouse. Nevertheless, it is necessary to elucidate the dynamics of the infection in this animal species, evaluating different stages of the colonization and evolution of the pathological alterations. The immunohistochemistry (IHC) offers advantages over the routine histopathological staining techniques for the detection of the protozoan in tissues, cellular detritus and inside the macrophages. The goal of the present study was to demonstrate the presence of Tritrichomonas foetus in the reproductive tract of infected mice using an IHC technique. Female BALB/c mice were infected with a suspension of T. foetus by intravaginal route, in the estrum phase, detected by exfoliative vaginal cytology. After 10 weeks, the animals were sacrificed; uterus and vagina were fixed and histologically processed. Some slides were stained with HE. The rest of the slides were processed for IHC. An immunoadsorbed polyclonal serum against T. foetus was used. The avidine-biotine technique (HistoMouse, Zymed™) was employed. The histopathological studies showed a dilation of the uterine glands, presence of macrophages in the lumen of the organ and inner part of the endometrial glands. No T. foetus was identified using this method. The IHQ allowed additionally the identification of the protozoan in the endometrium, endometrial glands, uterine lumen and inside neutrophils and macrophages. The cytological studies stained with IHC showed either isolated T. foetus adhered to epithelial cells or inside macrophages. This technique proves to be a useful tool for the study of the pathogenesis of bovine genital trichomoniasis (BGT) in an experimental model.

Identification of Epigenetic Targets in Prostate Cancer for Therapeutic Development

Recurrent castration resistant prostate cancer remains a challenge for cancer therapies and novel treatment options in addition to current anti-androgen and mitosis inhibitors are needed. Aberrations in epigenetic enzymes and chromatin binding proteins have been linked to prostate cancer and they may form a novel class of drug targets in the future. In this thesis we systematically evaluated the epigenenome as a prostate cancer drug target. We functionally silenced 615 known and putative epigenetically active protein coding genes in prostate cancer cell lines using high throughput RNAi screening and evaluated the effects on cell proliferation, androgen receptor (AR) expression and histone patterns. Histone deacetylases (HDACs) were found to regulate AR expression. Furthermore, HDAC inhibitors reduced AR signaling and inhibited synergistically with androgen deprivation prostate cancer cell proliferation. In particular, TMPRSS2- EGR fusion gene positive prostate cancer cell lines were sensitive to combined HDAC and AR inhibition, which may partly be related to the dependency of a fusion gene induced epigenetic pathway. Histone demethylases (HDMs) were identified to regulate prostate cancer cell line proliferation. We discovered a novel histone JmjC-domain histone demethylase PHF8 to be highly expressed in high grade prostate cancers and mediate cell proliferation, migration and invasion in in vitro models. Additionally, we explored novel HDM inhibitor chemical structures using virtual screening methods. The structures best fitting to the active pocket of KDM4A were tested for enzyme inhibition and prostate cancer cell proliferation activity in vitro. In conclusion, our results show that prostate cancer may efficiently be targeted with combined AR and HDAC inhibition which is also currently being tested in clinical trials. HDMs were identified as another feasible novel drug target class. Future studies in representative animal models and development of specific inhibitors may reveal HDMs full potential in prostate cancer therapy

Evaluation of a model for induction of periodontal disease in dogs

There are several methods for inducing periodontal disease in animal models, being the bone defect one of the most reported. This study aimed to evaluate this model, through clinical, radiographic, tomographic and histological analyzes, thus providing standardized data for future regenerative works. Twelve dogs were subjected to the induction protocol. In a first surgical procedure, a mucoperiosteal flap was made on the buccal aspect of the right third and fourth premolars and a defect was produced exposing the furcation and mesial and distal roots, with dimensions: 5mm coronoapical, 5mm mesiodistal, and 3mm buccolingual. Periodontal ligament and cementum were curetted and the defect was filled with molding polyester, which was removed after 21 days on new surgical procedure. Clinical and radiographic examinations were performed after the two surgeries and before the collection of parts for dental tomography and histological analysis. All animals showed grade II furcation exposure in both teeth. Clinical attachment level increased after induction. Defect size did not change for coronoapical and buccolingual measurements, while mesiodistal size was significantly higher than at the time of defect production. Radiographic analysis showed decreased radiopacity and discontinuity of lamina dura in every tooth in the furcation area. The horizontal progression of the disease was evident in micro-computed tomography and defect content in the histological analysis. Therefore, it is concluded that this method promotes the induction of periodontal disease in dogs in a standardized way, thus being a good model for future work.

Assessment of the degree of contamination of rat germ cell preparations using specific cDNA probes

Recent reports showing a decrease in sperm count in men have brought new concerns about male infertility. Animal models have been widely used to provide some relevant information about the human male gamete, and extrapolations are made to men and to the clinical context. The present study assesses one of the methods used for separation of germ cells of the adult rat testis, namely centrifugal elutriation followed by density gradients (Percoll®). This method was chosen since it presents the best results for cell purity in separating germ cells from the rat testis. A comparison between continuous and discontinuous Percoll® gradients was performed in order to identify the best type of gradient to separate the cells. Maximal cell purity was obtained for spermatocytes (81 ± 8.2%, mean ± SEM) and spermatids (84 ± 2.6%) using centrifugal elutriation followed by continuous Percoll® gradients. A significant difference in purity was observed between elongating spermatids harvested from continuous Percoll® gradients and from discontinuous gradients. Molecular analysis was used to assess cell contamination by employing specific probes, namely transition protein 2 (TP2), mitochondrial cytochrome C oxidase II (COX II), and sulfated glycoprotein 1 (SGP1). Molecular analysis of the samples demonstrated that morphological criteria are efficient in characterizing the main composition of the cell suspension, but are not reliable for identifying minimal contamination from other cells. Reliable cell purity data should be established using molecular analysis

Response threshold to aversive stimuli in stimulated early protein-malnourished rats

Two animal models of pain were used to study the effects of short-term protein malnutrition and environmental stimulation on the response threshold to aversive stimuli. Eighty male Wistar rats were used. Half of the pups were submitted to malnutrition by feeding their mothers a 6% protein diet from 0 to 21 days of age while the mothers of the other half (controls) were well nourished, receiving 16% protein. From 22 to 70 days all rats were fed commercial lab chow. Half of the animals in the malnourished and control groups were maintained under stimulating conditions, including a 3-min daily handling from 0 to 70 days and an enriched living cage after weaning. The other half was reared in a standard living cage. At 70 days, independent groups of rats were exposed to the shock threshold or to the tail-flick test. The results showed lower body and brain weights in malnourished rats when compared with controls at weaning and testing. In the shock threshold test the malnourished animals were more sensitive to electric shock and environmental stimulation increased the shock threshold. No differences due to diet or environmental stimulation were found in the tail-flick procedure. These results demonstrate that protein malnutrition imposed only during the lactation period is efficient in inducing hyperreactivity to electric shock and that environmental stimulation attenuates the differences in shock threshold produced by protein malnutrition

Aluminum induces lipid peroxidation and aggregation of human blood platelets

Aluminum (Al3+) intoxication is thought to play a major role in the development of Alzheimer's disease and in certain pathologic manifestations arising from long-term hemodialysis. Although the metal does not present redox capacity, it can stimulate tissue lipid peroxidation in animal models. Furthermore, in vitro studies have revealed that the fluoroaluminate complex induces diacylglycerol formation, 43-kDa protein phosphorylation and aggregation. Based on these observations, we postulated that Al3+-induced blood platelet aggregation was mediated by lipid peroxidation. Using chemiluminescence (CL) of luminol as an index of total lipid peroxidation capacity, we established a correlation between lipid peroxidation capacity and platelet aggregation. Al3+ (20-100 µM) stimulated CL production by human blood platelets as well as their aggregation. Incubation of the platelets with the antioxidants nor-dihydroguaiaretic acid (NDGA) (100 µM) and n-propyl gallate (NPG) (100 µM), inhibitors of the lipoxygenase pathway, completely prevented CL and platelet aggregation. Acetyl salicylic acid (ASA) (100 µM), an inhibitor of the cyclooxygenase pathway, was a weaker inhibitor of both events. These findings suggest that Al3+ stimulates lipid peroxidation and the lipoxygenase pathway in human blood platelets thereby causing their aggregation

Monosodium glutamate (MSG)-obese rats develop glucose intolerance and insulin resistance to peripheral glucose uptake

Different levels of insulin sensitivity have been described in several animal models of obesity as well as in humans. Monosodium glutamate (MSG)-obese mice were considered not to be insulin resistant from data obtained in oral glucose tolerance tests. To reevaluate insulin resistance by the intravenous glucose tolerance test (IVGTT) and by the clamp technique, newborn male Wistar rats (N = 20) were injected 5 times, every other day, with 4 g/kg MSG (N = 10) or saline (control; N = 10) during the first 10 days of age. At 3 months, the IVGTT was performed by injecting glucose (0.75 g/kg) through the jugular vein into freely moving rats. During euglycemic clamping plasma insulin levels were increased by infusing 3 mU . kg-1 . min-1 of regular insulin until a steady-state plateau was achieved. The basal blood glucose concentration did not differ between the two experimental groups. After the glucose load, increased values of glycemia (P<0.001) in MSG-obese rats occurred at minute 4 and from minute 16 to minute 32. These results indicate impaired glucose tolerance. Basal plasma insulin levels were 39.9 ± 4 µU/ml in control and 66.4 ± 5.3 µU/ml in MSG-obese rats. The mean post-glucose area increase of insulin was 111% higher in MSG-obese than in control rats. When insulinemia was clamped at 102 or 133 µU/ml in control and MSG rats, respectively, the corresponding glucose infusion rate necessary to maintain euglycemia was 17.3 ± 0.8 mg . kg-1 . min-1 for control rats while 2.1 ± 0.3 mg . kg-1 . min-1 was sufficient for MSG-obese rats. The 2-h integrated area for total glucose metabolized, in mg . min . dl-1, was 13.7 ± 2.3 vs 3.3 ± 0.5 for control and MSG rats, respectively. These data demonstrate that MSG-obese rats develop insulin resistance to peripheral glucose uptake