Thesis (M.S.)--University of Tennessee Health Science Center, 2008. / Title from title page screen (viewed on February 9, 2009). Research advisor: Denis J DiAngelo, Ph.D. Document formatted into pages (x, 62 p. : ill.). Vita. Abstract. Includes bibliographical references (p. 35-36).

Regional anestesi är en uppskattad metod inom operationssjukvård för sina fördelar; patienten är vaken och kan kommunicera, operationsområdet är väl smärtlindrat och förekomst av illamående och kräkningar är minimal. Patienten kan även komma hem samma dag, vilket är både kostnadseffektivt för Hälso- och sjukvården och till fördel för patienten. Dock läggs det ett stort ansvar på patienten och egenvården blir omfattande i samband med dagkirurgiska ingrepp som innebär att patienten lämnar sjukhuset med en pågående nervblockad. Efter att en operationsmottagning i södra Sverige har börjat lägga blockader på patienter inför operation i övre och nedre extremiteter, har det blivit viktigt att utvärdera den nya metoden för postoperativ smärtlindring. Syftet med denna uppsats är att beskriva patienters erfarenheter av att lämna sjukhuset och att vara postoperativt smärtlindrad med regional anestesi. Studien genomfördes i form av ett frågeformulär som utformats av operationsmottagningen. Urvalet utgjordes av patienter (n=30) som opererats för handledsfrakturer, ligament i knäled och operationer i axel/överarm. En sjuksköterska genomförde datainsamlingen via telefonintervjuer. Analysen var kvantitativ och dataprogrammet IBM SPSS Statistic Software (SPSS) tillämpades. Resultatet visar att patienterna var nöjda och påvisar vidare att patienterna inte upplevde några större problem med illamående/kräkning, förband eller information. Emellertid framkom, att smärtlindringen var otillfredsstillande såväl vid hemgång som på första postoperativa dagen. Diskussionen fokuserar därför på bristande smärtlindring och hur smärtlindringen kan förbättras, samt tänkbara orsaker till varför patienterna förefaller acceptera postoperativ smärta. Ett förbättringsarbete i behandlingsstrateginer för postoperativ smärtlindring efter regional anestesi rekommenderas därför i yttersta syfte att stödja patientens egenvård. Urvalet av patienter (n=30) anses inte kunna ge en generaliserbar bild men är statiskt signifikant beträffande postoperativ smärta, vilket understryker vikten av att identifiera förbättringspotentialer.

Orientador: Lourenço Sbragia Neto / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-08T14:55:03Z (GMT). No. of bitstreams: 1 Franca_WillyMarcusGomes_D.pdf: 3673624 bytes, checksum: 691804a882449e544abe2d0c2ae420af (MD5) Previous issue date: 2006 / Resumo: Na gastrosquise (G), o grau de lesão das alças intestinais exteriorizadas está diretamente relacionado ao tempo de contato com o líquido amniótico (LA) e exposição ao mecônio, que causam alterações morfológicas e histológicas do intestino, além da formação de uma fina camada de fibrina (fibrous peel) sobre a serosa, formando aderências entre as alças intestinais. Estas lesões levam ao hipoperistaltismo intestinal e deficiência na absorção dos nutrientes, contribuindo para o aumento da morbidade e alto custo médico-hospitalar. O hipoperistatismo na G é atribuído à desorganização e à imaturidade dos plexos mioentéricos. Estas características podem servir como marcadores do grau de lesão intestinal, e que podem ser identificados pela presença de neurofilamentos dos plexos nervosos intestinais e sugerir a antecipação do parto. Nesse estudo identificamos as alterações morfológicas e histológicas intestinais e dos plexos mioentéricos em dois diferentes tempos de contato com o LA. A G experimental em fetos de ratas Spreague-Dowley foi realizada em duas idades gestacionais, 18,5o dia (E18,5) e 19,5o dia (E19,5), que foram divididos em 3 subgrupos: controle (C), sham (S) e gastrosquise (G). Medimos o peso corporal fetal (PC), peso (PI) e comprimento intestinais (CI). As camadas da parede intestinal e os plexos mioentéricos foram avaliados pela coloração de H&E e imunofluorescência (?-Internexina), respectivamente. O PC não apresentou diferença significativa entre C/S/G, nos 2 grupos. O PI e CI foram respectivamente maior e menor nos fetos G (p<0,001) nos 2 grupos. Os diâmetros intestinais e as camadas da parede apresentaram diferença significativa entre C/S/G, em ambos os grupos (p<0,001), mas o tempo de contato com LA e exposição ao mecônio comprometeram a serosa e D-II (diâmetro II) (p<0,001), e CI (p=0,001). A ?-internexina apresentou imunorreatividade mais intensa nos fetos G de E18,5. Concluímos que no modelo de G em fetos de ratos, as alterações da parede intestinal, principalmente da camada serosa e do comprimento intestinal, bem como a imaturidade dos plexos mioentéricos, apresentaram-se mais intensas nos fetos cujo contato com o LA e exposição ao mecônio foram mais prolongados (G/E18,5). Estudos experimentais adicionais devem ser desenvolvidos e direcionados para compreensão da motilidade intestinal na G com o objetivo de minimizar os danos neuromusculares nas alças intestinais expostas ao LA e permitir avaliar as supostas vantagens da antecipação do parto para fetos humanos com G / Abstract: BACKGROUND: The amniotic fluid (AF) and its components such as fetal urine and meconium may lead to intestinal alterations in gastroschisis (G), which cause immaturity of the myenteric plexus and consequent intestinal hypomotility and malabsorption. In this study we identified morphological and histological alterations of the intestine and of the myenteric plexus with two different times of exposure to the AF. METHODS: The experimental G was achieved at two different gestational ages, on day 18.5 (E18.5) and on day 19.5 (E19.5) of gestation in fetal rats which were divided into 3 subgroups: control (C), sham (S) e gastroschisis (G). We measured fetal body weight (BW), intestinal weight (IW) and intestinal length (IL). The layers of intestinal wall and myenteric plexus were evaluated by hematoxylin and eosin staining (H&E staining) and immunofluorescence (?-Internexin), respectively. RESULTS: BW did not show significant differences among C/S/G, in both groups. IW and IL were respectively larger and shorter in the G fetuses (p<0.001) in both groups. Intestinal diameters and wall layers presented significant differences among C/S/G in both groups (p<0.001), but time of exposure to AF compromised the serous membrane, D-II (diameter II) (p<0.001) and IL (p=0.001). The ?-Internexin presented more intensive immunoreactivity in G/E18.5 fetuses. CONCLUSION: In gastroschisis, the longer the time of exposure to AF, the more severe will be bowel impairment, especially concerning IL and the serous layer; and the more immature will be the myenteric plexus / Doutorado / Cirurgia / Doutor em Cirurgia

The choroid plexuses are largely responsible for cerebrospinal fluid (CSF) secretion and therefore play a fundamental role in brain homeostasis. The membrane proteins involved in CSF secretion are not fully known. Several electroneutral transporters have been identified by molecular methods in choroid plexus epithelial cells but there is a lack of functional data to support their expression making it impossible to elucidate their role in CSF secretion fully. The activity of many of these transporters can be observed in cell volume regulation. Thus, the main aim of the present study was to determine the ability of mammalian choroid plexus epithelial cells to regulate their volume in response to anisosmotic challenge and to investigate the transporters involved.Experiments were performed on cells isolated from the mouse fourth ventricle choroid plexus. Cells were isolated using a combination of manual perturbation, the enzyme dispase and a Ca2+ free incubation to disrupt tight junctions. Cell volume was measured using a video-imaging method. Cells used in this study were all of a similar morphology and had a mean volume of 0.71 pL.Cells exhibited a HCO3- dependent regulatory volume increase (RVI) in response to hypertonic challenge. Strong evidence is presented that the Na+/H+ exchanger (NHE1) and the Cl-/HCO3- exchanger (AE2) contribute to the RVI but the Na+K+2Cl- cotransporter (NKCC1) and the epithelial Na+ channel (ENaC) do not. Choroid plexus cells exhibit a HCO3- dependent regulatory volume decrease (RVD) in response to hypotonic challenge. The RVD was unaffected by DIOA (an inhibitor of KCC activity), the K+ channel inhibitors TEA+, Ba2+ or 4AP or the Cl- channel inhibitors DIDS or NPPB. However removal of extracellular Ca2+ completely abolished cell swelling in response to hypotonic challenge. This sensitivity of volume change to Ca2+ was specific to cell swelling as cell shrinkage in hypertonic artificial CSF was unaffected by removal of extracellular Ca2+.Thus functional evidence is presented to further elucidate the role of several proteins in the choroid plexus cell volume regulatory response to anisosmotic challenge.

L’activateur tissulaire du plasminogène (tPA) est une protéase initialement découverte dans le sang pour son rôle fibrinolytique. C’est pour cette fonction que le tPA recombinant est utilisé pour traiter la phase aigüe de l’accident vasculaire cérébral (AVC) ischémique, même s’il présente quelques limites. Le tPA exogène peut passer du compartiment vasculaire au parenchyme cérébral où il peut influencer des processus physiologiques, et participer au devenir neuronal, notamment aggraver la mort neuronale lors d’un AVC ischémique. Le laboratoire a montré que le tPA peut traverser la barrière-hémato-encéphalique (BHE), par transcytose au travers des cellules endothéliales de la BHE et cela sous le contrôle des récepteurs LRP1 (Low density lipoprotein receptor-related protein 1). D’autres barrières existent au sein du système nerveux central notamment la barrière sang-liquide cérébrospinal (BSLCS), formée par les plexus choroïdes (PCs). Les PCs sont une route de migration pour les cellules inflammatoires et le LCS peut véhiculer des solutés, via les espaces péri-artériels, vers le parenchyme cérébral. Ainsi, dans notre première étude, nous avons testé l’hypothèse d’un passage du tPA vasculaire par les PCs. Pour cela, nous avons produit un tPA traçable in vivo et in vitro. Nous avons commencé par étudier la distribution du tPA suite à une injection intraveineuse (IV) avec comme focus les PCs et le LCS. Nos résultats montrent que le tPA exogène, suite à une injection IV, est retrouvé de manière séquentielle dans les PCs puis dans le LCS. Le tPA est donc capable de traverser les PCs. Nous avons alors développé un modèle de culture primaire de cellules épithéliales de PCs (CPECs) de souris pour disséquer le(s) mécanisme(s) sous-jacents à l’internalisation du tPA par les CPECs. Ce modèle nous a permis de montrer que l’internalisation du tPA par les CPECs est un phénomène actif, médié par un membre de la famille des récepteurs LRP, mais qui n’est ni LRP1, ni LRP2. Nous avons également mis en évidence la nécessité du domaine Finger du tPA pour son internalisation par les CPECs. Une étude préliminaire dans un modèle d’AVC suggère que l’ischémie modifie la cinétique de passage du tPA, puisqu’il y a plus de tPA dans les PCs des souris ischémiées que les souris non ischémiées.Dans une deuxième étude nous nous sommes intéressés à l’effet du tPA endogène sur les PCs. Nous montrons que l’absence de tPA endogène n’influence ni la morphologie des PCs, ni la diffusion du LCS. De plus, nous montrons que cette absence de tPA n’influence pas le nombre de macrophages et de lymphocytes T dans les PCs en conditions basales. / Tissue-type plasminogen activator (tPA) is a protease initially discovered in the blood for its fibrinolytic role. Accordingly, recombinant tPA has become the gold standard to treat the acute phase of ischemic stroke, despite some limitations. Exogenous tPA can switch from the vascular compartment to the brain parenchyma, where it can influence physiological processes, and participate in neuronal fate, including a worsening of neuronal death during ischemic stroke. In the team, it has been shown that tPA can cross the blood-brain barrier (BBB), by a transcytosis through BBB endothelial cells, under the control of LRP1 receptors (Low density lipoprotein receptor-related protein 1). The central nervous system has other barriers, including the blood-cerebrospinal fluid barrier (BCSFB), that relies on choroid plexuses (CPs). CPs are a migration route for inflammatory cells and a major source of CSF, which carries solutes to the cerebral parenchyma, via peri-arterial spaces. Thus, in a first study, we tested the hypothesis of a passage of vascular tPA through CPs. We thus produced a fluorescent tPA that can be tracked in vivo and in vitro. We first studied the distribution of tPA following intravenous (IV) injection, focusing on CPs and CSF. We show that after an IV injection, exogenous tPA is sequentially found in the CPs and then in the CSF. tPA is therefore able to cross the CPs. We then developed a model of primary culture of mouse choroid plexus epithelial cells (CPECs) to dissect the mechanism (s) underlying the internalization of tPA. This model allowed us to demonstrate that the internalization of tPA by CPECs is an active phenomenon, mediated by a member of the family of LRP receptors, but which is neither LRP1 nor LRP2. We also highlight the requirement for the Finger domain of tPA for its internalization by CPECs. A preliminary study in a murine stroke model suggests that ischemia alters the tPA passage kinetics, since there is more tPA in ischemic CPs than non-ischemic CPs.In a second study, we investigated the effect of endogenous tPA on CPs. We show that the absence of endogenous tPA influences neither CPs morphology nor CSF diffusion . Moreover we show that the absence of tPA does not influence the number of macrophages and T cells in the stroma of PCs under basal conditions.

Indiana University-Purdue University Indianapolis (IUPUI) / Hydrocephalus is a neurological disorder characterised by the pathological accumulation of cerebrospinal fluid (CSF) within the brain ventricles. Surgical interventions, including shunt placement, remain the gold standard treatment option for this life-threatening condition, despite these often requiring further revision surgeries. Unfortunately, there is currently no effective, pharmaceutical therapeutic agent available for the treatment of hydrocephalus. CSF is primarily produced by the choroid plexus (CP), a specialized, branched structure found in the ventricles of the brain. The CP comprises a high resistance epithelial monolayer surrounding a fenestrated capillary network, forming the blood-CSF barrier (BCSFB). The choroid plexus epithelium (CPe) critically modulates CSF production by regulating ion and water transport from the blood into the intraventricular space. This process is thought to be controlled by a host of intracellular mediators, as well as transporter proteins present on either the apical or basolateral membrane of the CPe. Though many of these proteins have been identified in the native tissue, exactly how they interact and modulate signal cascades to mediate CSF secretion remains less clear. Transient potential receptor vanilloid 4 (TRPV4) is a non-selective cation channel that can be activated by a range of stimuli and is expressed in the CP. TRPV4 has been implicated in the regulation of CSF production through stimulating ion flux across the CPe. In a continuous CP cell line, activation of TRPV4, through the addition of a TRPV4 specific agonist GSK1016790A, stimulated a change in net transepithelial ion flux and increase in conductance. In order to develop a pharmaceutical therapeutic for the treatment of hydrocephalus, we must first understand the mechanism of CSF secretion in health and disease. Therefore, a representative in vitro model is critical to elucidate the signaling pathways orchestrating CSF production in the CP. This research aims to characterize an in vitro culture model that can be utilized to study both the BCSFB and CSF production, to investigate and identify additional transporters, ion channels and intracellular mediators involved in TRPV4-mediated signaling in the CPe, primarily through a technique called Ussing-style electrophysiology which considers electrogenic ion flux across a monolayer. These studies implicated several potential modulators, specifically phospholipase C (PLC), phosphoinositide 3-kinase (PI3K), protein kinase C (PKC), intermediate conductance K+ channel (IK), transmembrane member 16A (TMEM16A), cystic fibrosis transmembrane conductance regulator (CFTR) and protein kinase A (PKA), in TRPV4-mediated ion flux.

Indiana University-Purdue University Indianapolis (IUPUI) / Hydrocephalus is a medical condition characterized by a buildup of cerebrospinal fluid which causes hydrostatic pressure to increase resulting neuronal destruction and can ultimately cause death. Hydrocephalus is seen in both the pediatric population and adults. Treatment of hydrocephalus usually involves surgical placement of a relocation system to drain the fluid into the abdominal cavity. Hydrocephalus may be caused by mechanical obstruction of the outflow of CSF from the ventricles or by faulty reabsorption. It can be also caused by CSF overproduction by the choroid plexus found in the lateral, third, and fourth ventricles of the brain. The choroid plexus is composed of a high resistance monolayer epithelium which surrounds a network of capillaries. Its primary function is to regulate transport of ions and water that control the production and movement of CSF. Therefore it is important to understand the mechanism of CSF production by the choroid plexus. Recently, a stable porcine choroid plexus (PCP-R) epithelial cell line with a high transepithelial resistance (TER) was developed that provides an important model to study regulation of CSF production. Ussing style electrophysiology was used to measure short circuit current (SCC) to characterize stimulated transepithelial ion transport in confluent PCP-R cells. GSK1016790, a TRPV4 agonist, was used to understand the role of TRPV4 in CSF production by the choroid plexus using PCP-R cell model. TRPV4 activation produces a sustained ion transport response that is consistent with an increase in cation secretion and/or anion absorption which is accompanied by a reversible decrease in TER. The effect of the agonist on both SCC and TER was blocked by HC067047, a TRPV4 antagonist, showing that the sustained ion transport and TER change is TRPV4 specific. TRPV4 mediated ion flux was inhibited by CFTR inhibitor II GlyH-101, a cell permeable inhibitor of the cAMP activated chloride channel CFTR, when added on either side of the membrane and was not accompanied by a TER reversal which showed that CFTR is activated by TRPV4 mediated ion flux. TMEM16A, a calcium activated chloride channel, was speculated to be located in that basal membrane as T16Ainh-AO1, a membrane permeable TMEM16A inhibitor, reversed the TRPV4 mediated ion flux when added on either side of the membrane. Slight reversal in TER was observed when T16Ainh-AO1 was added on the apical side. Apamin, a differential inhibitor of calcium activated small conductance potassium channel 1, 2 and 3 (SK1, SK2 and SK3) had no effect on the TRPV4 mediated ion flux. Whereas, fluoxetine, a membrane permeable inhibitor of SK1, SK2 and SK3 channel, inhibited the TRPV4 mediated ion flux and TER change. Bumetanide, an inhibitor of the sodium-potassium-chloride cotransporter reversed TRPV4 mediated ion flux when added on the apical membrane but not on the basal membrane indicating a possible K+ secretion via SK1 and/or SK4/IK channels and Cl- absorption through CFTR and TMEM16A channels. Acetazolamide, a carbonic anhydrase inhibitor and a compound used to treat hydrocephalus had no effect on the TRPV4 mediated ion flux. cAMP is an intracellular mediator involved in neuromodulator effects, inflammatory responses and other regulatory mechanisms and is constitutively activated by forskolin. In PCP-R cells, forskolin stimulated an increase in transepithelial ion flux that is consistent with an increase in cation absorption and/or anion secretion. Forskolin mediated ion transport was inhibited by CFTR inhibitor II GlyH-101 when added on either side of the membrane. No change in TER was observed. No effect on forskolin mediated ion flux was observed when T16Ainh-A01, apamin or fluoxetine were added. Forskolin stimulated transport is partially inhibited by 1 mM BaCl2. Barium chloride is a general inhibitor of K+ channels. No change in TER was observed.

<p>  </p> <p>The choroid plexus (CP) in brain ventricles secrete cerebrospinal fluid (CSF) that bathes the adjacent subventricular zone (SVZ); the latter is the largest neurogenic region in adult brain harboring neural stem/progenitor cells (NSPCs) and supplies newborn neurons to the olfactory bulb (OB) for normal olfaction. We discovered the presence of a CP-SVZ regulatory (CSR) axis in which the CP, by secreting small extracellular vesicles (sEVs), regulated adult neurogenesis in the SVZ and maintained olfaction. The proposed CSR axis was supported by 1) differential neurogenesis outcomes in the OB when animals treated with intracerebroventricular (ICV) infusion of sEVs collected from the CP of normal or manganese (Mn)-poisoned mice, 2) progressively diminished SVZ adult neurogenesis in mice following CP-targeted knockdown of SMPD3 to suppress CP sEV secretion, and 3) compromised olfactory performance in these CP-SMPD3-knockdown mice. Collectively, our findings demonstrate the biological and physiological presence of this sEV-dependent CSR axis in adult brains.</p>

Acetazolamide (AZA), the only drug approved for treatment of hydrocephalus, is effective in only 25-30% of patients while its effect on fluid flow in the choroid plexus (CP) is unknown. The drug reversibly inhibits Aquaporin 4 (AQP4), the most highly expressed „water pore‟ in the brain, and it is postulated that it reduces cerebrospinal fluid (CSF) production by modulating AQP1 (mostly found in the apical membrane of the CP). In this study, we sought to elucidate the effect of AZA on AQP1 and fluid flow in CP. Primary CP culture from p10 Sprague-Dawley rats and TRCSF-B cell line were grown on Transwell permeable supports, treated with 100µM AZA or 100µM Vinpocetine (previously shown to increase AQP1 levels), and tested by: a) Fluid assays using TRITC-labeled Dextran to assay direction and extent of fluid flow; b) Immunoblot, Immunocytochemistry (ICC), and RT-PCR for AQP1 expression. Immnoblots and ICC analyses showed that AQP1 protein levels decrease in a delayed manner (lowest at 12 hours) with AZA treatment. The reduction in AQP1 protein was transient and preceded by a reduction in mRNA levels (lowest at 6 hours). Transwell fluid assays indicate a shift in fluid flow at 2 hours, prior to the changes in AQP1 mRNA or protein. Alteration of fluid flow by AZA (in both primary culture and TR-CSFB) is similar to Vinpocetine‟s effect in primary culture. Together with druginduced alterations in AQP1 levels, these data suggest independent mechanisms behind fluid flow and AQP1 expression.

Indiana University-Purdue University Indianapolis (IUPUI) / Pediatric hydrocephalus is a complex neurological condition associated with a pathological accumulation of cerebrospinal fluid (CSF), typically within the brain ventricular system. Pediatric hydrocephalus can be primary (due to genetic abnormalities or idiopathic causes), or secondary to injuries such as hemorrhage, trauma, or infection. The current permanent treatment paradigms for pediatric hydrocephalus are exclusively surgical and include the diversion of CSF via shunt or ventriculostomy. These surgical interventions are wrought with failures, burdening both the United States healthcare system and patients with repeat neurosurgical procedures. Thus, the development of nonsurgical interventions to treat hydrocephalus represents a clinically unmet need. To study hydrocephalus, we use a genetic rat model of primary neonatal hydrocephalus, the Tmem67P394L mutant. In several proof-of-concept studies, we identify antagonism of the transient receptor potential vanilloid 4 (TRPV4) channel and associated upstream regulatory kinase, serum-andglucocorticoid-induced kinase 1 (SGK1) as therapeutics for the treatment of hydrocephalus. Using in vitro models of the choroid plexus epithelium, the tissue which produces CSF, we show compelling proof-of-mechanism for TRPV4 antagonism and SGK1 inhibition at preventing CSF production. Therefore, the studies in this dissertation provide substantive evidence on the role of TRPV4 in the choroid plexus in health and disease.