Pilot Study of Endovascular Delivery of Mesenchymal Stromal Cells in the Aortic Wall in a Pig Model.

Abdominal aortic aneurysms (AAAs) have a high mortality. In small-animal models, multipotent mesenchymal stromal cells (MSCs) have shown benefits in attenuating aneurysm formation. However, an optimal cell delivery strategy is lacking. The NOGA system, which targets cell injections in a less-invasive way, has been used for myocardial cell delivery. Here, we assessed the safety and feasibility of the NOGA system for endovascular delivery of MSCs to the aortic wall in an AAA pig model. We induced AAA in 9 pigs by surgery or catheter induction. MSCs were delivered using the NOGA system 6 or 8 weeks after aneurysm induction. We euthanized the pigs and harvested the aorta for histologic analysis 1, 3, and 7 days after cell delivery. During AAA creation, 1 pig died; 8 pigs completed the study without acute adverse events or complications. The cell delivery procedure was safe and feasible. We successfully injected MSCs directly into the aortic wall in a targeted manner. Histologic and immunohistochemical analyses confirmed transmural injections in the aortic wall area of interest and the presence of MSCs. Our study showed the safety and feasibility of endovascular cell delivery to the aortic wall in a pig model.

Perforating the GORE® CARDIOFORM septal occluder and atrial septal defect occluder to gain access to the left atrium.

OBJECTIVE: Perforate the expanded polytetrafluoroethylene membrane of the GORE® CARDIOFORM Septal Occluder (GSO) and GORE® CARDIOFORM ASD Occluder (GCA) after implantation. BACKGROUND: Percutaneous transseptal access to the left atrium is necessary for many structural and electrophysiological procedures. The potential need to access the left atrium may influence decision-making for patent foramen ovale or atrial septal defect closure. METHODS: Sixteen canines underwent implantation of equal number GSO or GCA devices. A transseptal crossing procedure was performed through the device 85 (±1) days postoccluder implantation. The crossing procedure was performed utilizing commercially available equipment: radiofrequency/SureFlex sheath and standard needle/Mullin's sheath. Progressive dilation of the perforation was performed to allow passage of a 12 French Mullin's sheath into the left atrium. RESULTS: Left atrial access was achieved in all cases. Postmortem analysis demonstrated passage through both occluder discs in all radiofrequency/SureFlex sheath cases (4 GSO, 4 GCA) and half of the standard needle/Mullin's sheath cases (3 GSO, 1 GCA). The remaining standard needle/Mullin's sheath cases demonstrated perforation through the right atrial disc but passage around the septal aspect of the left atrial disc, thus not perforating the left atrial disc. No acute embolic complications from the procedure were observed. CONCLUSIONS: Left atrial access may be achieved through the GSO or GCA devices after implantation and endothelialization. The combination of a radiofrequency needle and steerable sheath provides benefit over a standard needle and Mullin's sheath in accomplishing passage through both occluder discs.

Osmotherapy With Hypertonic Saline Attenuates Global Cerebral Edema Following Experimental Cardiac Arrest via Perivascular Pool of Aquaporin-4.

OBJECTIVES: We tested the hypothesis that osmotherapy with hypertonic saline attenuates cerebral edema following experimental cardiac arrest and cardiopulmonary resuscitation by exerting its effect via the perivascular pool of aquaporin-4. We used mice with targeted disruption of the gene encoding α-syntrophin (α-Syn) that demonstrate diminished perivascular aquaporin-4 pool but retain the non-endfoot and ependymal pools. DESIGN: Laboratory animal study. SETTING: University animal research laboratory. INTERVENTIONS: Isoflurane-anesthetized adult male wild-type C57B/6 or α-Syn mice were subjected to cardiac arrest/cardiopulmonary resuscitation and treated with either a continuous IV infusion of 0.9% saline or various concentrations of hypertonic saline. Serum osmolality, regional brain water content, blood-brain barrier disruption, and aquaporin-4 protein expression were determined at 24 hours after cardiac arrest/cardiopulmonary resuscitation. MEASUREMENTS AND MAIN RESULTS: Hypertonic saline (7.5%) treatment significantly attenuated water content in the caudoputamen complex and cortex compared with 0.9% saline treatment in wild-type mice subjected to cardiac arrest/cardiopulmonary resuscitation. In contrast, in α-Syn mice subjected to cardiac arrest/cardiopulmonary resuscitation, 7.5% hypertonic saline treatment did not attenuate water content. Treatment with 7.5% hypertonic saline attenuated blood-brain barrier disruption at 24 hours following cardiac arrest/cardiopulmonary resuscitation in wild-type mice but not in α-Syn mice. Total aquaporin-4 protein expression was not different between 0.9% saline and hypertonic saline-treated wild-type mice. CONCLUSIONS: Following experimental cardiac arrest/cardiopulmonary resuscitation: 1) continuous hypertonic saline therapy maintained to achieve serum osmolality of ≈ 350 mOsm/L is beneficial for the treatment of cerebral edema; 2) perivascular pool of aquaporin-4 plays a critical role in water egress from brain; and 3) hypertonic saline attenuates blood-brain barrier disruption via perivascular aquaporin-4 pool.

Intra-arterial delivery is not superior to intravenous delivery of autologous bone marrow mononuclear cells in acute ischemic stroke.

BACKGROUND AND PURPOSE: Bone marrow-derived mononuclear cells (MNCs) are an investigational autologous cell-based therapy for acute ischemic stroke. Both intravenous (IV) and intra-arterial (IA) administration routes have been used in clinical trials. However, the route of administration to optimize the effect of MNCs is unknown. In this study, we compared the effect of IV versus IA route of administration of MNCs in the rat stroke model. METHODS: Long Evans rats were subjected to transient middle cerebral artery occlusion. At 24 hours after stroke, animals were randomly assigned to receive autologous bone marrow-derived MNCs using either the IV or IA delivery route. IV saline served as control. One million cells/kg (low dose) and 30 million cells/kg (high dose) were assessed. Neurological testing, cavity size, serum cytokines, neuroregenerative end points, and MNC biodistribution were evaluated. RESULTS: High-dose MNCs improved functional recovery, reduced lesion size and proinflammatory cytokines, and increased vessel density and neurogenesis markers compared with saline treatment (P<0.05). However, there were no significant differences between IV and IA MNC-treated groups, although IV MNCs reduced serum interleukin-1ß levels compared with IA MNCs (P<0.05). IA MNCs at high dose led to a greater number of cells in the brain at 1 and 6 hours after injection but not in the lungs and spleen. Low-dose MNCs (by IV or IA) did not improve any functional or structural end point compared with saline. CONCLUSIONS: At low and high doses of MNCs, we found that IV or IA achieves similar structural and functional outcomes after stroke.

Na(+)-K (+)-2Cl (-) cotransport inhibitor attenuates cerebral edema following experimental stroke via the perivascular pool of aquaporin-4.

INTRODUCTION: The Na(+)-K(+)-2Cl(-) cotransporter localized in the brain vascular endothelium has been shown to be important in the evolution of cerebral edema following experimental stroke. Previous in vivo studies have demonstrated that bumetanide, a selective Na(+)-K(+)-2Cl(-) cotransport inhibitor, attenuates ischemia-evoked cerebral edema. Recently, bumetanide has been shown to also inhibit water permeability via aquaporin-4 (AQP4) expressed in Xenopus laevis oocytes. We tested the hypothesis that the perivascular pool of AQP4 plays a significant role in the anti-edema effect of bumetanide by utilizing wild-type (WT) mice as well as mice with targeted disruption of alpha-syntrophin (alpha-Syn(-/-)) that lack the perivascular pool of AQP4. METHODS: Isoflurane-anesthetized adult male WT C57Bl6 and alpha-Syn(-/-) mice were subjected to 90 min middle cerebral artery occlusion (MCAO) followed by 24 or 48 h of reperfusion. Adequacy of MCAO and reperfusion was monitored with laser-Doppler flowmetry over the ipsilateral parietal cortex. Infarct volume (tetrazolium staining), cerebral edema (wet-to-dry ratios), and AQP4 protein expression (immunoblotting) were determined in different treatment groups in separate sets of experiments. RESULTS: Bumetanide significantly attenuated infarct volume and decreased ipsilateral hemispheric water content in WT mice compared to vehicle treatment. In alpha-Syn(-/-) mice, bumetanide treatment had no effect on infarct volume or ischemia-evoked cerebral edema. Bumetanide-treated WT mice had a significant attenuation of AQP4 protein expression at 48 h post-MCAO compared to vehicle-treated WT mice. CONCLUSIONS: These data suggest that bumetanide exerts its neuroprotective and anti-edema effects partly via blockade of the perivascular pool of AQP4 and may have therapeutic potential for ischemic stroke in the clinical setting.

Estradiol and G1 reduce infarct size and improve immunosuppression after experimental stroke.

Reduced risk and severity of stroke in adult females is thought to depend on normal endogenous levels of estrogen, a well-known neuroprotectant and immunomodulator. In male mice, experimental stroke induces immunosuppression of the peripheral immune system, characterized by a reduction in spleen size and cell numbers and decreased cytokine and chemokine expression. However, stroke-induced immunosuppression has not been evaluated in female mice. To test the hypothesis that estradiol (E2) deficiency exacerbates immunosuppression after focal stroke in females, we evaluated the effect of middle cerebral artery occlusion on infarct size and peripheral and CNS immune responses in ovariectomized mice with or without sustained, controlled levels of 17-beta-E2 administered by s.c. implant or the putative membrane estrogen receptor agonist, G1. Both E2- and G1-replacement decreased infarct volume and partially restored splenocyte numbers. Moreover, E2-replacement increased splenocyte proliferation in response to stimulation with anti-CD3/CD28 Abs and normalized aberrant mRNA expression for cytokines, chemokines, and chemokine receptors and percentage of CD4(+)CD25(+)FoxP3(+) T regulatory cells observed in E2-deficient animals. These beneficial changes in peripheral immunity after E2 replacement were accompanied by a profound reduction in expression of the chemokine, MIP-2, and a 40-fold increased expression of CCR7 in the lesioned brain hemisphere. These results demonstrate for the first time that E2 replacement in ovariectomized female mice improves stroke-induced peripheral immunosuppression.

Inhibition of aquaporin-1 and aquaporin-4 water permeability by a derivative of the loop diuretic bumetanide acting at an internal pore-occluding binding site.

Aquaporin (AQP) water channels, essential for fluid homeostasis, are expressed in perivascular brain end-feet regions of astroglia (AQP4) and in choroid plexus (AQP1). At a high concentration, the loop diuretic bumetanide has been shown to reduce rat brain edema after ischemic stroke by blocking Na(+)-K(+)-2Cl(-) cotransport. We hypothesized that an additional inhibition of AQP contributes to the protection. We show that osmotic water flux in AQP4-expressing Xenopus laevis oocytes is reduced by extracellular bumetanide (> or =100 microM). The efficacy of block by bumetanide is increased by injection intracellularly. Forty-five synthesized bumetanide derivatives were tested on oocytes expressing human AQP1 and rat AQP4. Of these, one of the most effective was the 4-aminopyridine carboxamide analog, AqB013, which inhibits AQP1 and AQP4 (IC(50) approximately 20 microM, applied extracellularly). The efficacy of block was enhanced by mutagenesis of intracellular AQP4 valine-189 to alanine (V189A, IC(50) approximately 8 microM), confirming the aquaporin as the molecular target of block. In silico docking of AqB013 supported an intracellular candidate binding site in rat AQP4 and suggested that the block involves occlusion of the AQP water pore at the cytoplasmic side. AqB013 at 2 microM had no effect, and 20 microM caused 20% block of human Na(+)-K(+)-2Cl(-) cotransporter activity, in contrast to >90% block of the transporter by bumetanide. AqB013 did not affect X. laevis oocyte Cl(-) currents and did not alter rhythmic electrical conduction in an ex vivo gastric muscle preparation. The identification of AQP-selective pharmacological agents opens opportunities for breakthrough strategies in the treatment of edema and other fluid imbalance disorders.

Experimental use of a castor bean (Ricinus communis L. ) oil derived polymer on lamellar, interlamellar and penetrating implahts in rabbit's cornea

Há muito que se estudam métodos e materiais reparadores de córnea, em busca de uma melhor reposição tecidual e, principalmente, manutenção ou recuperação da visão. A pesquisa de novos biomateriais tem permitido produzir próteses capazes de desempenhar a função requerida, sem reação importante. Conhecendo-se os bons resultados obtidos com a utilização da poliuretana vegetal derivada de óleo de mamona (Ricinus communis L ) em vários procedimentos, estudou-se sua implantação, em forma de membrana, na córnea, objetivando oferecer novo material para a reparação de lesões corneanas e a confecção de ceratopróteses biologicamente inertes. Utilizaram-se 28 coelhos, divididos em dois grupos (G 1 e G2) e estes em subgrupos para avaliações de implantes lamelares e interlamelares aos 2, 7, 15, 30, 60 e 120 dias, e implantes penetrantes, aos 2, 7,15, 30 e 60 dias de pós-operatório. Estudaram-se parâmetros como neovascularização, inflamação, transparência de córneas e implantes, bem como a aderência e viabilidade destes, através de exames oculares, e histopatológicos à microscopia óptica. Observou-se reação inflamatória branda em todos os períodos. Os implantes lamelares e penetrantes permitiram reparação da córnea e manutenção da integridade dos globos oculares, embora sofressem deiscência entre 5 e 28 dias de pós-operatório. Concluiu-se pela boa biocompatibilidade do material e pela possibilidade de empregar-se o polímero na reparação corneana e, possivelmente, em ceratopróteses.

Parathyroid hormone-related protein induction in focal stroke: a neuroprotective vascular peptide.

Parathyroid hormone-related protein (PTHrP) is a multifunctional peptide that enhances blood flow in non-central nervous system (CNS) vascular beds by causing vasodilation. PTHrP expression is induced in non-CNS organs in response to ischemia. Experiments were therefore undertaken to determine whether PTHrP can be induced in brain in response to ischemic injury and whether PTHrP can act locally as a vasodilator in the cerebral vasculature, an effect that could be neuroprotective in the setting of stroke. PTHrP expression was examined by Northern analysis and immunohistochemical staining in male Sprague-Dawley rats subjected to permanent middle cerebral artery occlusion (MCAO). Vasodilatory effects of superfused PTHrP(1-34) on pial arterioles were determined by intravital fluorescence microscopy. Effects of PTHrP(1-34) peptide administration on MCAO infarction size reduction were assessed. PTHrP expression was induced in the ischemic hemisphere as early as 4 h after MCAO and remained elevated for up to 24 h. Increased immunoreactive PTHrP at sites of ischemic tissue injury was located in the cerebral microvessels. Superfusion with PTHrP(1-34) peptide for up to 25 min increased pial arteriolar diameter by 30% in normal animals. In animals with permanent MCAO, PTHrP(1-34) peptide treatment significantly decreased cortical infarct size (-47%). In summary, PTHrP expression increases at sites of ischemic brain injury in the cerebrovasculature. This local increase in PTHrP could be an adaptive response that enhances blood flow to the ischemic brain, thus limiting cell injury.

Toracoscopia em eqüinos: técnica e emprego como método de avaliaçäo da cavidade pleural / Thoracoscopy in horses: technique and its use as pleural cavity evaluation method

Toracoscopia é o método de visibilizaçäo da cavidade torácica através de endoscópio rígido ou flexível, indicado como exame auxiliar para diagnóstico das diversas afecçöes do tórax. Pode ser realizada com o animal sob anestesia geral ou em posiçäo quadrupedal através de anestesia local associada ou näo a sedativos. Neste trabalho, foram utilizados seis eqüinos hígidos, que foram submetidos a toracoscopia, em posiçäo quadrupedal, nos dois hemitórax, com intervalo de 15 dias entre cada procedimento. Durante o exame, pôde-se observar as diversas estruturas da cavidade torácica, tais como: músculo diafragma (porçäo muscular e tendínea), hiato esofágico, mediastino, aorta, troncos dorsal e ventral do nervo vago, ducto torácico, veia àzigos, veias intercostais, músculos intercostais, tronco nervoso simpático, tronco vascular toracocervical, traquéia, pulmäo (lobo cranial e caudal), esôfago, pericárdio, veia cava caudal, nervo frênico e linfonodos aórticos. Discretas alteraçöes foram observadas ao exame físico, laboratorial e ultra-sonográfico no período pós-operatório, sem que trouxessem prejuízos ao desenvolvimento do experimento e recuperaçäo do animal. A toracoscopia mostrou ser exame de auxílio importante, pela facilidade, rapidez de execuçäo, ampla visibilizaçäo da cavidade pleural e trauma mínimo ao paciente.

Utilizaçäo de alfentanil, sufentanil e fentanil em cäes anestesiados com halotano / Use of alfentanil, sufentanil and fentanyl in dogs anesthetized with halothane

Os analgésicos opióides promovem analgesia intensa, possibilitando o emprego de concentraçöes reduzidas dos anestésicos inalatórios, minimizando assim a depressäo cardiovascular que ocorre durante a anestesia. Vários opióides podem ser empregados na anestesia, sendo que dentre os mais potentes, o fentanil, sufentanil e alfentanil säo os mais freqüentemente administrados no transoperatório. No presente estudo, comparou-se a açäo no sistema cardiovascular desses três agentes durante a anestesia inalatória em cäes, submetidos a procedimentos ortopédicos, bem como, avaliou-se a possibilidade de se manter a anestesia com baixas concentraçöes de halotano. Foram usados três grupos, de dez animais cada, que receberam, após estabilizaçäo da anestesia, doses equipotentes de um dos três agentes analgésicos - grupo I - 5µg/kg de fentanil IV, grupo II - 1µg/kg de sufentanil IV e grupo III - 25µg/kg de alfentanil IV. Os parametros cardiovasculares e respiratórios foram avaliados em diferentes tempos de observaçäo. Os resultados obtidos foram analisados através de análise estatística (ANOVA seguida de Dunnett e Bonferroni). Os três agentes promoveram bradicardia importante durante o decorrer do estudo, sendo que apenas o alfentanil promoveu hipotensäo significativa após sua administraçäo. Apenas um animal tratado com fentanil apresentou aumento da pressäo arterial e freqüência cardíaca durante a anestesia. Na recuperaçäo da anestesia, um número maior de animais tratados com alfentanil apresentou sinais de excitaçäo. Através dos resultados obtidos, pode-se concluir que a administraçäo de analgésicos opióides possibilita de fato o emprego de menores concentraçöes de halotano, promovendo anestesia estável com a manutençäo da pressäo arterial dentro dos valores normais para a espécie. Dos agentes empregados, o alfentanil é o que promove efeitos cardiovasculares mais pronunciados.

Implantação intra-orbital, após a enucleação transpalpebral, de resina acrílica ou pericárdio em coelhos / Intraorbital implantation of acrylic resin or pericardium after transpalpebral enucleation in rabbits

Dezoito coelhos, Norfolk, fêmeas, com 45 dias de idade foram divididos em três grupos de seis animais e submetidos a enucleação transpalpebral. Os animais do grupo I receberam na cavidade orbitaria acrílico auto-polimerizável, os do grupo II pericárdio eqüino conservado em glicerina e os do grupo III foram mantidos como controle. Para avaliação macroscópica e histopatológica das cavidades orbitarias, três animais de cada grupo foram sacrificados com 30 e 60 dias após a implantação. Apesar da resina ter sido aplicada na fase pastosa, na qual a alta temperatura que ocorre durante a polimerizaçâo pode ser lesiva aos tecidos, foi o produto que apresentou os melhores resultados.

Eighteen Norfolk female rabbits, 45 days old, were divided into three groups of six animals and submitted to transpalpebral enucleation. Animals of group I received intraorbital auto-polimerized acrylic resin, the ones of group II received eqüine pericardium preserved in glycerin solution and animals of group III were control. Three animals of each group were euthanised 30 and 60 days after implantation for macroscopic and histopathologic evaluation of the orbital cavities. Although the resin has been applied during the doughy phase in which the high temperature produced by polimerization could be dangerous to the tissue, it was the product that showed the best result.