Maternal protein restriction increases respiratory and sympathetic activities and sensitizes peripheral chemoreflex in male rat offspring

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Força muscular e massa corpórea de ratos Wistar

The use of rats in studies that seek to monitor the effects of physical exercise is fairly common, even in works that make use of overload. However, the correct determination of the training load is critical to success of studies. Thus, with the objective of verifying the correlation of total body mass of the rats with their respective strength, and the correlation of the 1RM test with test repetitions, 40 rats, 90 days old and weighing average 0,49 pounds underwent a program of adaptation to the water and then were subjected to tests to determine the maximal and submaximal loads. After measuring the total body mass and maximal and submaximal strenght, it was possible to observe directly proportional relationship between maximum strenght and total body mass of 0.97 (P ≤ 0.01), being the maximum muscle strength equivalent to 109% of the total body mass. Between muscle strength and mass submaximal muscle strength was also found direct correlation between the variables, allowing to conclude that the submaximal strength test and total body mass are effective tools in determining the training load of rats.

Inhomogeneity in optical properties of rat brain: a study for LLLT dosimetry.

Over the last few years, low-level light therapy (LLLT) has shown an incredible suitability for a wide range of applications for central nervous system (CNS) related diseases. In this therapeutic modality light dosimetry is extremely critical so the study of light propagation through the CNS organs is of great importance. To better understand how light intensity is delivered to the most relevant neural sites we evaluated optical transmission through slices of rat brain point by point. We experimented red (λ = 660 nm) and near infrared (λ = 808 nm) diode laser light analyzing the light penetration and distribution in the whole brain. A fresh Wistar rat (Rattus novergicus) brain was cut in sagittal slices and illuminated with a broad light beam. A high-resolution digital camera was employed to acquire data of transmitted light. Spatial profiles of the light transmitted through the sample were obtained from the images. Peaks and valleys in the profiles show sites where light was less or more attenuated. The peak intensities provide information about total attenuation and the peak widths are correlated to the scattering coefficient at that individual portion of the sample. The outcomes of this study provide remarkable information for LLLT dose-dependent studies involving CNS and highlight the importance of LLLT dosimetry in CNS organs for large range of applications in animal and human diseases.

Successful transnasal delivery of stem cells in a rat model of perinatal hypoxic-ischemic brain injury

OBJECTIVE: New routes for cell transplantation into the brain need to be explored as intracerebral or intrathecal applications have a high risk to cause damage to the central nervous system. It has been hypothesized that transnasally administrated cells bypass the blood-brain barrier and migrate along the olfactory neural route into the brain and cerebrospinal fluid. Our goal is to confirm this hypothesis by transnasally administrating Wharton’s Jelly mesenchymal stem cells (WJ-MSC) and neural progenitor cells (NPC) to perinatal rats in a model of hypoxic-ischemic brain injury. STUDY DESIGN: Four-day-old Wistar rat pups, previously brain-damaged by combined hypoxic-ischemic and inflammatory insult, either received WJ-MSC or green fluorescent protein-expressing NPC: The heads of the rat pups were immobilized and 3 ml drops containing the cells (50’000 cells/ml) were placed on one nostril allowing it to be snorted. This procedure was repeated twice, alternating right to left nostril with an interval of one minute between administrations. The rat pups received a total of 600’000 cells. Animals were sacrificed 24h, 48h or 7 days after the application of the cells. Fixed brains were collected, embedded in paraffin and sectioned. RESULTS: Transplanted cells were found in the layers of the olfactory bulb (OB), the cerebral cortex, thalamus and the hippocampus. The amount of cells was highest in the OB. Animals treated with transnasally delivered stem cells showed significantly decreased gliosis compared to untreated animals. CONCLUSION: Our data show that transnasal delivery of WJ-MSC and NPC to the newborn brain after perinatal brain damage is successful. The cells not only migrate the brain, but also decrease scar formation and improve neurogenesis. Therefore, the non-invasive intranasal delivery of stem cells to the brain may be the preferred method for stem cell treatment of perinatal brain damage and should be preferred in future clinical trials.

Abolition of valproate-derived choleresis in the Mrp2 transporter-deficient rat

Valproic acid (VPA) is a major therapeutic agent in the treatment of epilepsy and other neurological disorders. It is metabolized in humans and rats primarily along two pathways: direct glucuronidation to yield the acyl glucuronide (VPA-G) and beta-oxidation. We have shown much earlier in the Sprague-Dawley rat that i.v. administration of sodium valproate (NaVPA) caused a marked choleresis ( mean of 3.3 times basal bile flow after doses of 150 mg/kg), ascribed to the passive osmotic flow of bile water following excretion of VPA-G across the canalicular membrane. Active biliary pumping of anionic drug conjugates across the canalicular membrane is now believed to be attributable to transporter proteins, in particular Mrp2, which is deficient in the TR- ( a mutant Wistar) rat. In the present study, normal Wistar and Mrp2-deficient TR- rats were dosed i.v. with NaVPA at 150 mg/kg. In the Wistar rats, there was a peak choleretic effect of about 3.2 times basal bile flow, occurring at about 30 to 45 min postdose ( as seen previously with Sprague-Dawley rats). In TR- rats given the same i.v. dose, there was no evidence of postdose choleresis. The choleresis was correlated with the excretion of VPA-G into bile. In Wistar rats, 62.8 +/- 7.7% of the NaVPA dose was excreted in bile as VPA-G, whereas in TR- rats, only 2.0 +/- 0.6% of the same dose was excreted as VPA-G in bile ( with partial compensatory excretion of VPA-G in urine). This study underlines the functional ( bile flow) consequences of biliary transport of xenobiotic conjugated metabolites.

Cryosections of pre-irradiated adult rat spinal cord tissue support axonal regeneration in vitro

Neonatal X-irradiation of central nervous system (CNS) tissue markedly reduces the glial population in the irradiated area. Previous in vivo studies have demonstrated regenerative success of adult dorsal root ganglion (DRG) neurons into the neonatally-irradiated spinal cord. The present study was undertaken to determine whether these results could be replicated in an in vitro environment. The lumbosacral spinal cord of anaesthetised Wistar rat pups, aged between 1 and 5 days, was subjected to a single dose (40 Gray) of X-irradiation. A sham-irradiated group acted as controls. Rats were allowed to reach adulthood before being killed. Their lumbosacral spinal cords were dissected out and processed for sectioning in a cryostat. Cryosections (10 mum-thick) of the spinal cord tissue were picked up on sterile glass coverslips and used as substrates for culturing dissociated adult DRG neurons. After an appropriate incubation period, cultures were fixed in 2% paraformaldehyde and immunolabelled to visualise both the spinal cord substrate using anti-glial fibrillary acidic protein (GFAP) and the growing DRG neurons using anti-growth associated protein (GAP-43). Successful growth of DRG neurites was observed on irradiated, but not on non-irradiated, sections of spinal cord. Thus, neonatal X-irradiation of spinal cord tissue appears to alter its environment such that it can later support, rather than inhibit, axonal regeneration. It is suggested that this alteration may be due, at least in part, to depletion in the number of and/or a change in the characteristics of the glial cells. (C) 2000 ISDN. Published by Elsevier Science Ltd. All rights reserved.

A truncation in the Aryl Hydrocarbon Receptor of the CRL:WI(Han) rat does not affect the developmental toxicity of TCDD

The Aryl Hydrocarbon Receptor (AhR) is required for the toxicity of TCDD, and so the AhR of CRL:WI and CRL:WI(Han) rats was characterised. Western blot showed AhR proteins of ~110 and ~97 kDa in individual rats from both strains. The AhR cDNA from a CRL:WI(Han) rat with the ~110kDa protein revealed a sequence that was identical to that of the CRL:WI and SD rat. However, cloning of the AhR from a rat with the ~97kDa protein revealed a point mutation, and five variants encoding two C-terminally truncated variants of the AhR protein, arising from a point mutation in the intron/exon junction and consequent differential splicing. These C-terminally truncated variants were expressed and shown to give rise to a protein of ~97kDa; the recombinant AhR bound TCDD with an affinity that was not statistically different from the full-length protein. A single-nucleotide polymorphism (SNP) assay was developed, and showed that both alleles were represented in a Hardy-Weinberg equilibrium in samples of CRL:WI and CRL:WI(Han) populations; both alleles are abundant. Rats from two studies of TCDD developmental toxicity were genotyped, and the association with toxicity investigated using statistical analysis. There was no plausible evidence that the AhR allele had a significant effect on the toxic endpoints examined. These data show that the two AhR alleles are common in two strains of Wistar rat, and that the AhR alleles had no effect on TCDD-induced developmental toxicity in two independent studies.

POP-inhibiittorin KYP-2047 vaikutukset angiogeneesiin in vitro

Angiogeneesi on tärkeä ilmiö elimistön fysiologiassa, mutta myös lukuisissa patologisissa tiloissa. Angiogeneesi on monivaiheinen prosessi, joka sisältää angiogeneesiä indusoivia ja sitä inhiboivia tekijöitä tasapainossa keskenään. Useat tutkimukset puoltavat sitä, että tymosiini ȕ4 (Tȕ4) ja tetrapeptidi Ac-SDKP (N-asetyyliseryyli- aspartyyli-lysyyli-proliini) indusoivat angiogeneesiä in vitro ja in vivo. Tutkimukset viittaavat myös siihen, että prolyylioligopeptidaasi (POP) hydrolysoi peptidifragmentin Ac- SDKP Tȕ4:n (43 ah) proliinin jälkeen. POP on laajalti esiintyvä seriiniproteaasi, joka pystyy pilkkomaan vain alle 30 aminohapon oligopeptidejä. Tȕ4:n tulee siksi pilkkoutua ensin jonkin, vielä tuntemattoman peptidaasin johdosta. POP:ia on löydetty eniten aivoista, minkä vuoksi sitä on tutkittu varsinkin muistin ja oppimisen häiriötiloissa sekä neurodegeneratiivisten sairausten yhteydessä. POP:in todellinen fysiologinen merkitys on kuitenkin vielä selvittämättä. Tämän pro gradun kirjallisuusosiossa selvitetään angiogeneesiin liittyvien tekijöiden yhteyksiä sekä kuvataan angiogeenisten Tȕ4:n, Ac-SDKP:n ja POP:in ominaisuuksia, esiintymistä ja toimintaa. Kokeellisen osion tarkoituksena oli osoittaa, osallistuvatko POP ja Tȕ4 tetrapeptidin Ac-SDKP muodostumiseen ja kapillaarimuodostumiseen ja edelleen, voidaanko POPaktiivisuutta, tetrapeptidi- ja kapillaarimuodostumista estää spesifisellä POP-inhibiittorilla, KYP-2047:llä. Kokeellinen osa oli kaksiosainen. Ensimmäisessä osassa tutkittiin POPaktiivisuutta ja suoritettiin Ac-SDKP –pitoisuusmittauksia ajanjaksolla 0-180 min Wistarkannan rotista tehdyillä homogenaateilla. Tutkimusryhminä olivat 0,1 ja 0,5 μM KYP-2047 (+2 μM Tȕ4), 1:20 (0,625 μM) humaaniperäinen rekombinantti-POP (+ 2 μM Tȕ4), 2 μM Tȕ4 (pos. kontrolli) ja raakahomogenaatti (neg. kontrolli). Toisessa osassa tutkittiin kapillaarimuodostumista ajanjaksolla 0-180 min humaaniperäisillä napanuoralaskimon primaariendoteelisoluilla MatrigelTM Matrix -päällystetyllä 48- kuoppalevyllä, jolle oli siirrostettu 50 000 solua/kuoppa. Naudan seerumilla ja antibiooteilla käsitellyt tutkimusryhmät olivat 5 ja 10 μM KYP-2047 (+4 μM Tȕ4), 1:20 (0,625 μM) humaaniperäinen rekombinantti-POP (+4 μM Tȕ4), 4 μM Tȕ4 (pos. kontrolli) ja DMEM (neg. kontrolli). Kuoppia inkuboitiin ja kapillaarimuodostuminen kuvattiin valomikroskoopilla digitaalikameralla. Kutakin tutkimusryhmää pipetoitiin kolmeen rinnakkaiseen kuoppaan ja kokeet toistettiin neljästi. Sulkeutuneiden kapillaarien lukumäärä laskettiin manuaalisesti ja tuloksista tehtiin tilastollinen analyysi. 7ȕ4:n ja POP:in havaittiin molempien osallistuvan tetrapeptidin AC-SDKP muodostumiseen munuaishomogenaateissa. Primaariendoteelisolut muodostivat selkeitä kapillaareja Matrigelilla, erityisesti POP- ja Tȕ4–ryhmissä. KYP-2047 inhiboi tehokkaasti POP:ia kaikissa kokeissa osoittautuen hyväksi antiangiogeeniseksi yhdisteeksi. Angiogeneesin mekanismien ja POP:in, Tȕ4:n ja Ac-SDKP:n yhteyksien selvittäminen vaatii luonnollisesti vielä lisätutkimuksia.

Dextran and protamine-based solid lipid nanoparticles as potential vectors for the treatment of X-linked juvenile retinoschisis

This is a copy of an article published in the Human gene therapy © 2012 copyright Mary Ann Liebert, Inc.; Human gene therapy is available online at: http://online.liebertpub.com.

Evaluation and application of micro-sampling system for inductively coupled plasma mass spectrometry

Two Meinhard microconcentric nebulizers, model AR30-07-FM02 and AR 30-07-FM005, were employed as a self-installed micro-sampling system for inductively coupled plasma-mass spectrometry (ICP-MS). The FM02 nebulizer at 22 muL/min of solution uptake rate gave the relative standard deviations of 7.6%, 3.0%, 2.7%, 1.8% for determinations (n = 10) of 20 mug/L Be, Co, In and Bi, respectively, and the detection limits (3s) of 0.14, 0.10, 0.02 and 0.01 mug/L for Be, Co In and Bi, respectively. The mass intensity of In-115 obtained by this micro-sampling system was 60% of that by conventional pneumatic nebulizer system at 1.3 mL/min. The analytical results for La, Ce, Pr and Nd in 20 muL Wistar rat amniotic fluid obtained by the present micro-sampling system were precisely in good agreement with those obtained using conventional pneumatic nebulization system.

Multigenerational Undernutrition Increases Susceptibility to Obesity and Diabetes that Is Not Reversed after Dietary Recuperation

People in developing countries have faced multigenerational undernutrition and are currently undergoing major lifestyle changes, contributing to an epidemic of metabolic diseases, though the underlying mechanisms remain unclear. Using a Wistar rat model of undernutrition over 50 generations, we show that Undernourished rats exhibit low birth-weight, high visceral adiposity (DXA/MRI), and insulin resistance (hyperinsulinemic-euglycemic clamps), compared to age-/gender-matched control rats. Undernourished rats also have higher circulating insulin, homocysteine, endotoxin and leptin levels, lower adiponectin, vitamin B12 and folate levels, and an 8-fold increased susceptibility to Streptozotocin-induced diabetes compared to control rats. Importantly, these metabolic abnormalities are not reversed after two generations of unrestricted access to commercial chow (nutrient recuperation). Altered epigenetic signatures in insulin-2 gene promoter region of Undernourished rats are not reversed by nutrient recuperation, and may contribute to the persistent detrimental metabolic profiles in similar multigenerational undernourished human populations.

Estudo da vascularização dos nervos periféricos e da sua influência na regeneração nervosa : estudo experimental e no cadáver humano

RESUMO: Na clínica, a recuperação funcional que se segue a uma lesão nervosa raramente é atingida na sua totalidade. A reinervação, quer motora, quer sensitiva, ocorre geralmente com maior ou menor deficit. Interessa, então, identificar os factores que podem interferir na regeneração nervosa. O neurónio é a unidade anatómica fundamental do sistema nervoso periférico e é muito vulnerável à isquemia pela grande distância que existe entre o corpo neuronal e a extensão do axónio, que pode ser de apenas alguns milímetros ou até atingir um metro. É, por isso, fundamental o estudo da vascularização do nervo periférico e da sua influência na regeneração nervosa. O resultado deste estudo pode levar ao desenvolvimento de técnicas cirúrgicas que criem as condições que garantam, por sua vez, a revascularização precoce do nervo periférico em caso de lesão, ou mesmo em caso de doenças, nas quais a vascularização do nervo está alterada como, por exemplo, na neuropatia diabética. O estudo da vascularização do nervo periférico realizou-se através da investigação da vascularização do nervo mediano do cadáver humano, pela investigação da vascularização do nervo isquiático do rato Wistar e do Plexo Braquial (PB) do mesmo. A vascularização do PB do rato não é muito diferente daquela que é reportada na espécie humana, existindo uma homologia entre o rato e o Homem no que diz respeito à morfologia e à vascularização do PB. Através da comparação angiomorfológica entre o nervo isquiático do rato e o nervo mediano humano, concluíu-se que a microvascularização do nervo isquiático do rato e do mediano humano são muito semelhantes, o que suporta a utilização do rato como modelo experimental de lesões do nervo mediano humano. Para a avaliação da influência da vascularização na regeneração nervosa foi feita a análise da eficácia de enxerto de tubo de membrana amniótica humana imunologicamente inerte, de enxerto de veia jugular externa autóloga e de auto-enxerto de nervo, na reparação de um defeito de 10 milímetros no nervo isquiático do rato, na presença de um fornecimento vascular axial, comparando-se com os mesmos procedimentos em estudos realizados anteriormente, sem suprimento vascular. Os ratos foram avaliados funcionalmente através do estudo das pegadas, da electroneurografia e da força de flexão ao nível do tornozelo, e estruturalmente, através das avaliações histológicas e morfométricas, da taxa de recuperação do peso dos músculos gastrocnémio e solhear e da marcação axonal retrógrada com True Blue às 4, 8 e 12 semanas. Os nervos reconstruídos apresentaram uma arquitectura normal, incluindo a arquitectura vascular. A membrana amniótica foi bem tolerada, persistindo imunologicamente em torno do nervo até à 12.ª semana. Concluiu-se também que, na presença de um suprimento vascular axial local, a membrana amniótica humana e as veias autólogas são, pelo menos, tão eficazes como os auto-enxertos nervosos na reconstrução de defeitos nervosos de 10 milímetros.--------------------------------------ABSTRACT: At the clinic, the functional recovery that follows a nerve lesion is rarely achieved in full. The neuron is very vulnerable to ischemia that’s why it is essential to study the vascularization of the peripheral nerve and its influence on the nerve’s regeneration. The outcome of this study may lead to the development of surgical techniques that create the conditions which are necessary to ensure an early revascularization in case of a peripheral nerve injury. This study investigated the vascularization of the median nerve of the human cadaver and the vascularization of the sciatic nerve of the Wistar rat and his Brachial Plexus (BP) through animal experimentation. The mouse's BP vascularization is not so different from the one that is reported in the human species. An angiomorphological comparison between the mouse sciatic nerve and the human median nerve concluded that the microvascularizations are very similar, which supports the use of the mouse as an experimental model for the study of median nerve’s lesions. To evaluate the influence of vascularization in the nerve’s regeneration, it was made an assessment of the effectiveness of the human amniotic immuno-inert membrane grafts, of the autologous external jugular vein grafts and of the nerve auto-graft in the repair of a defect of 10 mm on the sciatic nerve of the rat, in the presence of an axial vascular supply, comparing these with the same procedures that were adopted in the previous studies, without vascular supply. The rats were functionally assessed and structurally evaluated (through histological and morphometric evaluations) at the 4.th, 8.th and 12.th weeks. The nerves reconstructed presented a normal architecture, including vascular architecture. The amniotic membrane was well-tolerated immunologically, persisting around the nerve until the 12.th week. As a result, it was also concluded that in the presence of a local axial vascular supply, the human amniotic membrane and the autologous veins are, at least, as effective as the nerve auto-grafts in the reconstruction of the nerve’s defects of 10 mm.

A fisiologia da pele de rato é modificada pela idade

Os modelos animais têm sido úteis para estudar mecanismos específicos que afetam a pele humana. É o caso do envelhecimento e das alterações micromecânicas que determinam o enrugamento (a formação de rugas) nos ratinhos irradiados com UV. Estes modelos permitiram perceber que o envelhecimento envolve muitas respostas mecânicas peculiares que não podem ser explicadas pela deformação homogénea da pele. No entanto, a diferença entre o tempo de vida destas espécies afeta também os processos e este é um aspeto principal a ter em conta. Este projeto é direcionado à comparação das propriedades cutâneas de dois grupos de ratos Wistar com diferentes idades – ratos jovens-adultos (n=7, 20-24 semanas) e adultos-velhos (n=5, 48-72 semanas). Medições não-invasivas de perda transepidérmica de água (PTEA), hidratação superficial (MoistureMeter) e biomecânica (Reviscometer e Cutometer por MPA80) foram realizadas nas zonas dorsais (pescoço, dorso-posterior) do animal durante 5 dias, permitindo comparações estatísticas entre grupos. Os resultados não revelaram diferenças significativas para a PTEA, firmeza ou viscoelasticidade entre os dois grupos. Contudo, foi notada uma diminuição significativa, próxima de 40%, da hidratação da pele no grupo dos ratos adultos-velhos. Estes resultados, apesar de preliminares, se fizermos a translação para os seres humanos poderiam ser úteis para avaliar mudanças relacionadas à idade em processos como a recuperação cutânea, ou eficácia / segurança de pensos, onde a hidratação é um factor determinante. Asua potencial utilização para a predição da eficácia e/ou segurança dos produtos de aplicação tópica para idosos em comparação aos adultos merecem ser exploradas.

Hydralazine reduces leukocyte migration through different mechanisms in spontaneously hypertensive and normotensive rats

In addition to reducing blood pressure, hydralazine can reduce the production of inflammatory cytokines and reduce the expression of leukocyte adhesion molecules. Differences in leukocyte behavior and leukocyte adhesion molecule expression in spontaneously hypertensive rats (SHR) compared to normotensive rats have been reported. However, whether hydralazine can reduce leukocyte migration in vivo in hypertension and in normotension remains unknown. To address this question, male SHR and Wistar rats were treated for 15 days with hydralazine at a dose of similar to 3.5 mg/kg or similar to 14 mg/kg in their drinking water. The numbers of rollers and adherent and migrated cells were determined by direct vital microscopy, and blood pressure was assessed by tail plethysmography. In addition, following treatment with the higher dose, immunohistochemistry was used to measure the expression of intercellular adhesion molecule-1 (ICAM-1), P-selectin, and platelet-endothelial cell adhesion molecule-1 (PECAM-1) in endothelial cells, while flow cytometry was used to evaluate the expression of leukocyte CD18 and L-selectin. Hydralazine reduced leukocyte adherence and migration in SHR either at the higher, that reduced blood pressure levels, or lower dose, which did not reduce it. Reduced ICAM-1 expression might be involved in the reduced migration observed in SHR. In Wistar rats, only at the higher dose hydralazine reduced blood pressure levels and leukocyte migration. Reduced P-selectin expression might be involved. We therefore conclude that hydralazine reduces leukocyte migration by different mechanisms in SHR and Wistar rats, specifically by reducing ICAM-1 expression in the former and P-selectin expression in the latter. (c) 2008 Elsevier B.V. All rights reserved.

Simvastatin improves the healing of infected skin wounds of rats

This study explores the potential of the simvastatin to ameliorate inflammation and infection in open infected skin wounds of rats. Methods: Fourteen Wistar rats weighing 285±12g were used. The study was done in a group whose open infected skin wounds were treated with topical application of sinvastatina microemulsion (SIM, n=7) and a second group with wounds treated with saline 0.9 % (SAL, n=7). A bacteriological exam of the wounds fluid for gram positive and gram negative bacteria, the tecidual expression of TNFá and IL-1â by imunohistochemical technique, and histological analysis by HE stain were performed. Results: The expression of TNFa could be clearly demonstrated in lower degree in skin wounds treated with simvastatin (668.6 ± 74.7 ìm2) than in saline (2120.0 ± 327.1 ìm2). In comparison, wound tissue from SIM group displayed leukocyte infiltration significantly lower than that observed in SAL group (p<0.05). Culture results of the samples taken from wound fluid on fourth post treatment day revealed wound infection in only one rat of group simvastatin (SIM), where Proteus mirabilis, Escherchia coli and Enterobacter sp were isolated. In the rats whose wounds were treated with saline (SAL), polymicrobial infection with more than 100,000 CFU/g was detected in all the wounds. Conclusion: In addition to its antiinflammatory properties, the protective effects of simvastatin in infected open skin wounds is able to reduce infection and probably has antibacterial action. The potential to treat these wounds with statins to ameliorate inflammation and infection is promising