B LETÍN Asociación Médica de Puerto Rico

CONTENIDO 3

MENSAJE DEL PRESIDENTE Rolance Chavier Roper, MD

Editorial Article/Articulo Editorial 4

Editorial

José Ginel Rodríguez, MD, FAAP

Original Articles/Articulos originales

62

Failure To Pass Meconium

68

Instrucciones para los autores / Instructions to Authors

Luis Lizardo Sánchez MS, Humberto Lugo-Vicente, MD

Case Reports / Reporte de Casos 69

Late Presentation Of Familial Mediterranean Fever: A Case Report

72

Rectal Stricture: A Rare Presentation Of Primary Amyloidosis

76

Development Of Grave’s Disease Seven Months After Hashimoto’s Thyroiditis: A Rare Occurrence

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Membrane Potential And Ph-Dependent Accumula- tion Of Decynium-22 (1,1’-Diethyl-2,2’-Cyanine Iodi de) Flourencence Through Oct Transporters In As- trocytes

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Prevalence Of Drug Resistance And Associated Mutations In A Population Of Hiv-1+ Puerto Ricans In 2005

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Effects Of Gravitational Perturbation On The Expression Of Genes Regulating Metabolism In Jurkat Cells

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Hidden In Plain Sight: Macrophage Activation Syndrome Complicating Adult Onset Still’s Disease

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83 Cardiovascular Events During General Elections In Bayamon, Puerto Rico

Acute Hepatitis A Infection: A Predisposing Factor For Severe Leptospirosis

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Gender Differences In Social And Developmental Factors Affecting Puerto Rican Adolescents During The Early Stage

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Changes In The Socio-Demographics, Risk Behaviors, Clinical And Immunological Profile Of A Cohort Of The Puerto Rican Population Living With Hiv: An Update Of The Retrovirus Research Center (1992-2008)

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Variation Of The Posterior Cerebral Artery And Its Embryological Explanation: A Cadaveric Study















Mikhail Inyushin, Yuri Kucheryaykh, Lilia Kucheryavykh, Priscilla Sanabria, Carlos Jiménez-Rivera Irina Strugano- va Misty Eaton, Serguei Skatchkov

Luis A. Cubano PhD, Luz Cumba, Lycely del C. Sepúlve- da-Torres, Nawal Boukli, Eddy Ríos-Olivares

Kanika Singh, Luis Cubano PhD, Marian Lewis









Arnaldo E. Pérez-Mercado MD, Gerónimo Maldonado- Martínez MPH, José Rivera del Rio MD, Robert F. Hunter Mellado MD

Christine Miranda MPHE, Diana M. Fernandez EdD, Geronimo Maldonado MPH, Raul O. Ramon MS, Miriam Velázquez MS, Angel M. Mayor MD, Robert F. Hunter- Mellado, MD

Wilson R. Veras T. MD, Gary W. Elhert MD

Clinical Studies Support A Role For Trem-Like Transcript-1 During The Progression Of Sepsis

Limaris Russe Gomez MD, Robert Hunter Mellado MD

Jaime Rodríguez Santiago MD, Jennifer Oppenheimer Catalá MD, José Ramírez Rivera MD

Wilfredo Eddy Bravo-Llerena MD, Rodrigo J. Valderrabano-Wagner MD, Juan Quevedo- Quevedo MD, Luis M. Reyes-Ortiz MD

Lourdes Benitez MD, Salvador Vila MD, Robert Hunter Mellado MD

Juan M. Quevedo Quevedo MD, Rodrigo Valderrabano Wagner MD, Wilfredo Bravo Llerena MD, Melba Colón Quintana MD, José Ramírez Rivera MD

86

Semblanza del Dr. Rafael Fernandez Feliberti

88

CEM Credits / Questions

Wanda I. Figueroa Cosme MD, Christine Miranda MPHE, 90 Diana M. Fernandez EdD, Johanna Maysonet BSHE, Raul O. Ramon MS

Review Articles / Articulos de Reseña 59



CEM Credits / Answers

Catalogado en Cumulative Index e Index Medicus Listed in Cumulative Index and Index Medicus No. ISSN-0004-4849 Registrado en Latindex -Sistema Regional de Información en Línea para Revistas Científicas de América Latina, el Caribe, España y Portugal

BOLETÍN - Asociación Médica de Puerto Rico Ave. Fernández Juncos Núm. 1305 P.O.Box 9387 - SANTURCE, Puerto Rico 00908-9387 Tel.: (787) 721-6969 - Fax: (787) 724-5208 e-mail:[email protected] Web site: www.asociacionmedicapr.org Web site para el paciente: www.saludampr.org

Portada: Dr. Rafael Fernández Feliberti y fachada del edificio de la Asociación Médica de Puerto Rico

Omar Esponda MD, Jessica Morales BS, Alexandra Aguilar, Michael Gomez, A. Valance Washington PhD

Diseño Gráfico e Ilustración digital de cubierta realizados por Juan Carlos Laborde en el Departamento de Informática de la AMPR E-mail: [email protected]

JUNTA DE DIRECTORES Dr. Rolance G. Chavier Roper Presidente Dra. Wanda G. Velez Andujar Presidente Distrito Sur Presidente Consejo de Educación Médica

Dr. Raúl G. Castellanos Bran Presidente Electo Dr. Eduardo Rodríguez Vázquez Presidente Saliente

Dr. José C. Román de Jesus Presidente Consejo Ético Judicial

Dr. Pedro Zayas Santos Secretario

Dra. Hilda Rivera Tubéns Presidente Consejo Relaciones y Servicios Públicos

Dr. José R. Villamil Rodríguez Tesorero

Dr. Salvador Torros Romeu Presidente Consejo Servicios Médicos

Dra. Hilda Ocasio Maldonado Vicepresidente

Dr. Jaime M. Diaz Hernandez Presidente Consejo Salud Pública y Bienestar Social

Dr. Natalio Izquierdo Encarnación Vicepresidente Dr. Raúl A. Yordán Rivera Vicepresidente Dr. Arturo Arché Matta Presidente Cámara Delegados

Dr.a. Ilsa Figueroa Presidente Consejo Política Pública y Legislación Dr. Eugenio R. Barbosa del Valle Presidente Comité de Planes Prepagados y Seguros Médicos Dr. Héctor L. Cáceres Delgado Presidente Comité Afiliación y Credenciales

Dr. Juan Rodríguez del Valle Vicepresidente Cámara de Delegados

Dr. Ney Modesti Tañon Presidente Comité Ad-hoc de Compañerismo

Dr. Gonzalo González Liboy Delegado AMA

Dr. José A. Rodríguez Ruiz Presidente Comité de Historia, Cultura y Religión

Dr. Rafael Fernández Feliberti Delegado Alterno AMA Presidente del Comité Asesor del Presidente

Dr. Luis A. Román Irizarry Presidente Comité Médico Impedido

Dr. Benigno López López Presidente Distrito Este

Dra. Luisa Marrero Santiago Presidente Comité de Seguros

Dr. Ángel E. Michel Terrero Presidente Distrito Sur

Dr. José I. Gerena Díaz Presidente Comité Ad-hoc Clínicas Multifásicas

Dra. Mildred R. Arché Matta Presidente Distrito Central

Dr. Félix N. Cotto González Presidente Comité Ad-hoc de Reclutamiento y Servicios al Médico Joven

JUNTA EDITORA Humberto Lugo Vicente, MD Presidente Luis Izquierdo Mora, MD

Juan Aranda Ramírez, MD

Melvin Bonilla Félix, MD

Francisco J. Muñiz Vázquez, MD

Carlos González Oppenheimer, MD

Walter Frontera, MD

Eduardo Santiago Delpin, MD

Mario. R. García Palmieri, MD

Francisco Joglar Pesquera, MD

Natalio Izquierdo Encarnación, MD

Yocasta Brugal, MD

José Ginel Rodríguez, MD

President Message/Mensaje del Presidente

Dr. Rolance G. Chavier Roper

Presidente de la Asociación Médica de Puerto Rico

Distinguidos colegas:

E



s para mi un honor presentar el tercer número del corriente año de nuestra revista científica Boletín. En esta ocasión, el contenido de los artículos científicos es producto del esfuerzo de la facultad de la Escuela de Medicina Universidad Central del Caribe con la colaboración de algunos de sus estudiantes. Nos llena de esperanzas el ver la dedicación con la que futuras generaciones de médicos, se esfuerzan por lograr la excelencia en su entrenamiento. La calidad de los artículos que van a leer, habla por sí sola. La educación médica es cada vez más importante y debemos destacar que nuestra pequeña isla de 100 x 35 millas cuenta con cuatro (4) escuelas de medicina acreditadas por el Comité de Enlace de Educación Médica (LCME, por sus siglas en inglés). Debemos sentirnos orgullosos de este logro, ya que hay otras jurisdicciones donde apenas hay una. Ante esta situación, es importante que organizaciones como la Asociación Médica de Puerto Rico sigan apoyando la educación médica a todos los niveles. Mediante esta publicación, cooperamos con las universidades y programas postgraduados ofreciendo un importante foro para la divulgación de resultados de investigaciones clínicas, que son tan necesarias para mantener las acreditaciones de los talleres académicos. La Asociación Médica de Puerto Rico, además, realiza múltiples actividades que apoyan la formación de nuevos galenos. Este año 2010, hemos celebrado cuatro (4) grandes clínicas multifásicas de salud. Los municipios de Moca, Añasco, Adjuntas y Jayuya, han recibido el servicio gratuito de docenas de miembros de nuestra Asociación Médica, que han provisto servicio a mas de 3,500 pacientes. En estas actividades, hemos sido apoyados especialmente por estudiantes de la Escuela de Medicina San Juan Bautista, quienes Asociación Médica de Puerto Rico

han estado presentes demostrando lo que es verdadera vocación de servicio. Aprovechando que este numero está tan relacionado a la educación médica, hemos querido dedicarlo a un medico excepcional, que ha sido un pasado presidente de nuestra institución y a su vez, ha estado ligado a la capacitación de médicos, específicamente en el área de ortopedia. Se trata del Dr. Rafael Fernández Feliberti, ícono del profesionalismo y la seriedad a la que debe aspirar todo médico. El Dr. Fernández, quien fue director de ortopedia y del programa de residencia en ortopedia de la Universidad de Puerto Rico recinto de ciencias médicas, por más de treinta anos, se mantiene activo aportando a esta institución y forjando el futuro de nuestra profesión. Más adelante, podrán disfrutar de una breve semblanza de nuestro amigo y mentor. Finalmente, hago un llamado a todos los lectores a que activen su membresía en nuestra institución. Hoy en día, es más importante que nunca pertenecer a una organización que está tan activa y cubre tantos flancos a la vez como la nuestra. En educación médica, servicio público y a la comunidad, servicio a su membresía, publicaciones, medios de comunicación electrónica, defensa de los médicos en “issues de importancia” y tecnología, estamos a la vanguardia. Pronto, estaremos ampliando nuestros servicios y ofreciendo una excelente alternativa a la hora de entrar al mundo de “record” medico electrónico. Nuestro norte será la credibilidad, la calidad del producto y servicio y el costo efectividad... 3

Editorial Article/Articulo Editorial

UNIVERSIDAD CENTRAL DEL CARIBE APARTADO 60327 BAYAMON, P.R. 00960-6032

José Ginel Rodríguez, MD, FAAP Presidente y Decano de Medicina Universidad Central del Caribe

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a publicación en el Boletín de la Asociación Médica de Puerto Rico de varios proyectos de investigación realizados en la Escuela de Medicina de la Universidad Central del Caribe (UCC) nos llena de mucho orgullo y esperanza. La Escuela de Medicina de la UCC es una institución sin fines de lucro fundada en el 1976. Al presente se encuentra acreditada por el LCME. Esta institución desde sus comienzos ha demostrado su compromiso con la investigación. La labor investigativa de nuestros científicos ha sido reconocida en y fuera de Puerto Rico por su dedicación y por sus iniciativas en temas que resultan en beneficio de la salud de los pacientes en Puerto Rico y los Estados Unidos. Un gran número de nuestros investigadores han recibido fondos de instituciones nacionales como National Institute of Health, mejor conocido como el NIH.

académico, nuestros logros son significativos. Los logros se demuestran con publicaciones propuestas logradas y nuevos RO1, como por ejemplo el grant del Dr. Serguei Skatchkov en el área de Neurociencias. Este es nuestro segundo RO1 y nos llena de mucha esperanza por los cuatro (4) proyectos pilotos en el área de enfermedades degenerativas. Estos proyectos fortalecerán la agenda de investigación del RO1 de la Dra. María Bykovskaia, facultad del Departamento de Neurociencias, el nuevo doctorado en Biología Celular Molecular así como el Doctorado en Neurociencias el cual próximamente será parte de nuestra oferta académica y de investigación.

En estos momentos presido esta universidad y es de mucho orgullo compartir con Puerto Rico y el mundo esta publicación que representa el entusiasmo y el esfuerzo de la buena investigación que se logra uniendo las capacidades Los artículos seleccionados para este bole- de la facultad, directores y estudiantes. La UCC tín de la Asociación Médica asignado a la Escuela comparte con ustedes los artículos que cuidadode Medicina de la UCC demuestra la productividad samente han sido seleccionados. Los temas de de la investigación en nuestro quehacer científico. éstos son amplios y representan la diversidad de La diversidad de la investigación en la UCC y el temas, así como los planteamientos científicos infuturo de la misma es prometedora. La realidad novadores que confiamos redunden en la salud es que a pesar del clima de la investigación, los del paciente y el bienestar de poblaciones que así factores internos y externos que inciden la agen- lo necesitan. da de invetigación de cualquier centro médico, 4

Asociación Médica de Puerto Rico

Original Articles/Articulos originales

Membrane Potential And Ph-Dependent Accumulation Of Decynium-22 (1,1’-Diethyl-2,2’-Cyanine Iodide) Flourencence Through Oct Transporters In Astrocytes Mikhail Inyushin1 Yuri Kucheryaykh2 Lilia Kucheryavykh2 Priscilla Sanabria1 Carlos Jiménez-Rivera3 Irina Struganova4 Misty Eaton2 Serguei Skatchkov1,2, From the Departments of Physiology1 and Biochemistry2, Universidad Central del Caribe, School of Medicine, Bayamón, PR; Department of Physiology3, University of Puerto Rico, Medical Sciences Campus, San Juan, PR and Department of Physical Sciences4, Barry University, Miami, FL. Address reprints requests to: Serguei Skatchkov, PhD, Department of Physiology and Department of Biochemistry, Universidad Central del Caribe, School of Medicine, P.O. Box 60327, Bayamón PR 00960-6032. E-mail: sergueis50@ yahoo.com

INTRODUCTION Monoamines play important roles in several disorders such as Parkinson’s disease, Alzheimer’s disease and drug addiction. There are several families of monoamine transporters in the CNS: (i) transporters with high affinity but low capacity (like DAT, NET) and (ii) transporters with low affinity but high capacity (like organic cation transporters; OCTs) (1-3). Non-neuronal uptake through OCTs was found in glia and glial cells outnumber neurons about ten times in the brain. This suggests a prominent role of glial cells in the regulation of extracellular monoamines. The members of the organic cation transporter (OCT) family can move endogenous monoamines across cell membranes (4, 5). 1,1'-Diethyl-2,2'-cyanine iodide (decynium22) is an organic cation widely used as a blocker for OCT transporters since it has a very high affinity for all members of this family in both human and rat cells (6, 7). Importantly, it has little effect Asociación Médica de Puerto Rico

ABSTRACT 1,1'-Diethyl-2,2'-cyanine iodide (decynium22; D22) is a potent blocker of the organic cation family of transporters (EMT/OCT) known to move endogenous monoamines like dopamine and norepinephrine across cell membranes. Decynium22 is a cation with a relatively high affinity for all members of the OCT family in both human and rat cells. The mechanism through which decynium22 blocks OCT transporters are poorly understood. We tested the hypothesis that denynium22 may compete with monoamines utilizing OCT to permeate the cells. Using the ability of D22 to aggregate and produce fluorescence at 570 nm, we measured D22 uptake in cultured astrocytes. The rate of D22 uptake was strongly depressed by acid pH and by elevated external K+. The rate of uptake was similar to that displayed by 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP+), a well established substrate for OCT and high-affinity Na+dependent monoamine transporters. These data were supported by measurement of electrogenic uptake using whole cell voltage clamp recording. Decynium22 depressed norepinephrine, but not glutamate uptake. These data are also consistent with the described OCT transporter characteristics. Taken together, our results suggest that decynium22 accumulation might be a useful instrument to study monoamine transport in the brain, and particularly in astrocytes, where they may play a prominent role in monoamine uptake during brain dysfunction related to monoamines (like Parkinson disease) and drug addiction. Index Words: astrocytes, OCT transporter, decynium22, ASP+, fluorescence

on high affinity monoamine transporters such as the dopamine transporter (DAT) and norepinephrine transporter (NET) (6, 8). This makes decynium22 an important tool to distinguish between the transporter families. This substance has been shown to block uptake of 1-Methyl-4-phenylpyridinium (MPP), a prototypical substrate for specific non-neuronal transporter system (uptake 2) mediated by the OCT3 transporter in cultured rat astrocytes (9). 5

The mechanisms through which the quinoline derivatives, decynium22 and quinine, block the OCT are poorly understood. It has been shown that quinine is a competitive blocker for the rat OCT2 transporter from the cytoplasmic side (10), but the same authors have found that quinine is a noncompetitive blocker for the rat OCT1/OCT2 transporter from the extracellular side (11). This prompted the question of whether quinoline derivatives can enter the cell. Arndt et al., (11) measured radiolabeled-quinine uptake in Xenopus oocytes and established that it does go inside the cell, but found no corresponding substrate-coupled current. As the OCT transport is known to be electrogenic, they proposed that the quinine molecule can be transported into the cell in an uncharged form by simple diffusion. The same paper claims that decynium22 also is a noncompetitive blocker for OCT transporters from outside of the cell, but Schömig et al., (8), the group who first introduced decynium22 as potent blocker of OCT family transporters, established that decynium22 is a competitive blocker. The possibility that decynium22 may be captured by the cells has not been evaluated. The fluorescence of this dye in monomeric form is practically absent, however it forms molecular aggregates (J-aggregates) characterized by very strong superradiant emission with a maximum near 570-580 nm. (12-14). Therefore, if decynium22 is captured by the cells it may form J-aggregates that can be measured as fluorescence in astrocytes. Thus, we tested the hypothesis that decynium22 may compete with monoamines using OCT and permeate glial cells. We found that, indeed, incubation with decynium22 caused a time dependent increase of fluorescence in astrocytes. Pharmacological characterization of decynium22 uptake in astrocytes suggested that uptake was through OCT transporters. Preliminary data were reported in abstract form (15).

METHODS Materials Used: 1,1’-Diethyl-2,2’-cyanine iodide (decyinium22); 4-(4-(dimethylamino)-styryl)-N-methylpyridinium (ASP+) and all other substances used were purchased from Sigma Chemical Co. (St. Louis, MO). Astrocyte primary cultures: Primary cultures of astrocytes were prepared from the neocortex of 1-2 day old rats. Brains were removed after decapitation and the meninges stripped away to minimize fibroblast contamination. The forebrain cortices were collected and dissociated using the stomacher blender method. The cell suspension was then allowed to filter by gravity only through a #60 sieve and then through a #100 sieve. After centrifugation, the cells were suspended in modified Eagle’s medium (containing 30 mM glucose, 2 mM glutamine, 1 mM pyruvate and 10% fetal bovine serum, 100 units/ml of penicillin/streptomycin) and plated on uncoated 75 cm2 flasks at a density of 100,000 cells/cm2. The medium was exchanged with fresh culture medium about every 5 days. At confluence (about 12-14 days), the mixed glial cultures were treated with 50 mM leucine methylester (pH 7.4) for 60 min to effectively kill microglia. Cultures were then allowed to recover for at least one day in growth medium 6

prior to reseeding. Astrocytes were dissociated by trypsinization and reseeded onto coverglasses at least two days prior to the imaging experiments. The purity of the astrocyte cultures was greater than 95% as assessed by immunocytochemical staining for glial fibrillary acidic protein (GFAP; data not shown). The “Principles of laboratory animal care” (NIH publication No. 85-23, revised 1985) were followed. Microscopy and image analysis: On the day of the experiment, a coverglass with astrocytes was mounted in a closed imaging chamber (Warner Inst, UT). After the chamber was connected to the perfusion system, cells were immediately mounted on an Olympus 1X70 microscope with Lambda DG4 computer-driven excitation system, and the microscope was focused on the center of the monolayer of cells. During the measurement, cells remained in a closed chamber and could be perfused with experimental solutions. The external buffer was composed of (in mM): 145 NaCl, 2 KCl, 2.5 CaCl2, 2.5 MgCl2, 10 HEPES(Na), pH 7.4. In some experiments, KCl was substituted by NaCl, the appropriate monoamine substrates and inhibitors were added or the pH was adjusted to 6.4 or 8.4. Background autofluorescence was established by collecting images prior to the addition of 1mM decynium22 or 2mM of ASP+. Time-series fluorescent images were recorded using a 40x oil immersion objective. The excitation was 480/30x nm; the emitted light was filtered with a 495- to 575-nm band pass filter (mean 535 nm). The gain (contrast) and offset (brightness) for the DCC camera tube was set to avoid detector saturation at the concentration used in these experiments (1 µM of decynium22 and 2mM of ASP+). The acquisition rate was 1 frame every 10 sec to prevent photobleaching of decynium22 and ASP+ sequestered inside the cell, with exposure of 100 or 200 msec. The fluorescent images were processed using MetaMorph imaging software (Universal Imaging Corp., Downington, PA). Fluorescence accumulation was established by measuring the average pixel intensity of fluorescent images of a cell identified from the differential interference contrast (DIC) image. Decynium22 or ASP+ accumulation were defined as the fluorescence of the cells minus background fluorescence by automatic background subtraction (standard feature of MetaMorph). In the current experiments, background fluorescence was negligible. Whole cell recordings. Membrane currents were measured with the single electrode whole-cell patch-clamp technique. Cells were visualized using a Nikon 300 inverted microscope (Nikon, Japan). Two Narishige hydraulic micromanipulators (Narishige, MMW-203, Japan) were used for (1) voltage-clamp recording, and (2) positioning a micropipette with 30 - 50 µm tip diameter for application of test solutions. A five valve system for fast drug application (MS Concentration Clamp; VS-2001, Vibraspec, PA), controlled by a second computer, was connected to the outlets. Electrodes from hard glass (GC-150-10 glass tubing, Clark Electromedical Instruments, England) Asociación Médica de Puerto Rico

were pulled in four steps using a Sutter P-97 puller (Novato, CA). After filling with intracellular solution (ICS) containing (in mM): KCl 141, MgCl2 1, CaCl2 1, EGTA 10, HEPES 10, Na2ATP 3, pH adjusted to 7.2 with NaOH/HCl, they had resistances of 4-6 MW; after cell penetration, the access resistance was 10-15 MW, compensated by at least 75%. The extracellular solution (ECS) contained (in mM): NaCl 138, CaCl2 2, MgCl2 1.9, and HEPES 10. High frequencies (>1 kHz) were cut off, using an Axopatch-200B amplifier and a CV-203BU headstage, and digitized at 5 kHz through a DigiData 1200A interface (Axon Instruments, USA). The pClamp 7 and 9 (Axon Instrument, USA) software packages were used for data acquisition and analysis. Statistics: GraphPad Prism was used to present and analyze imaging data. Linear regressions and an unpaired t-test were used to show the difference in the slope of accumulation. Calculation of correlation coefficients (Pearson r) was used to show similarity between accumulation of ASP+ and decynium22. Confidence levels of 0.95 were used in this study. Figure 1.

RESULTS Due to the expected low fluorescence yield (estimated to be 0.001), we did not anticipate substantial fluorescence of decynium22 (16). Initially, we planned experiments using decynium22 as a non-fluorescent substance to block accumulation of 4-(4-(dimethylamino)-styryl)-N-methylpyridinium (ASP+), a well-established substrate for OCT (17), and high-affinity Na+-dependent monoamine transporters (18), inside cultured astrocytes. Surprisingly, we discovered that after being perfused with a solution containing 1mM decynium22, astrocytes accumulated the fluorescence of decynium22 (Fig. 1a). One possible way to explain the strong fluorescence of decynium22 can be aggregation of the dye inside the astrocytes. While the fluorescence of this dye in monomeric form is very weak, it is known to form molecular aggregates (J-aggregates) characterized by very strong superradiant emission with a maximum near 570-580 nm (1214). The parameters we used (see Methods) are suitable for the excitation of fluorescence of J-aggregates of decynium22 as 480 nm falls near a short wavelength peak of the excitation spectrum of these aggregates Figure 1. Accumulation of 1,1'-diethyl-2,2'cyanine iodide (decynium22 or D22) in cultured cortical astrocytes during perfusion with buffer containing 1 µM of D22.

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a. From left to right: Differential Interference Contrast (DIC) image of cultured astrocytes (40x Oil immersion); fluorescence after 5 min of perfusion with D22; fluorescence after 15 min of perfusion with D22.

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Asociación Médica de Puerto Rico

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b. Graph showing mean (± S.D.) fluorescence with time after application of Decynium22 (1 µM) to cultured cortical astrocytes (n = 14 astrocytes). 7

The accumulation curve of decynium22 can be divided into three parts: very fast initial growth, then a linear part and, finally, saturation. In the majority (95%) of cells, accumulation of 1 µM decynium22 was nearly linear during at least 15 minutes (i.e., without saturation) (Fig. 1b). The accumulation timescale was different for different astrocytes, some had no accumulation of decynium22, and some had relatively fast accumulation that leads to the brighter final image of these cells (Fig. 1a, 15 min of accumulation). Very few cells showed fast saturation. In order to determine the influence of membrane potential on decynium22 accumulation, we examined the effect of increased external K+ on decynium22 accumulation using HEPES buffer with Na+ substituted by K+. Increasing external K+ depolarizes the membrane potential of astrocytes. Application of 1 µM decynium22 in the following buffer (in mM): 145 KCl,

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2 KCl, 2.5 CaCl2, 2.5 MgCl2, 10 HEPES(Na) significantly reduced decynium22 uptake (p2.0: Up-regulation;