Randy Alan Nessler

Derivation of blood-brain barrier endothelial cells from human pluripotent stem cells

The blood-brain barrier (BBB) is crucial to the health of the brain and is often compromised in neurological disease. Moreover, because of its barrier properties, this endothelial interface restricts uptake of neurotherapeutics. Thus, a renewable source of human BBB endothelium could spur brain research and pharmaceutical development. Here we show that endothelial cells derived from human pluripotent stem cells (hPSCs) acquire BBB properties when co-differentiated with neural cells that provide relevant cues, including those involved in Wnt/β-catenin signaling. The resulting endothelial cells have many BBB attributes, including well-organized tight junctions, appropriate expression of nutrient transporters and polarized efflux transporter activity. Notably, they respond to astrocytes, acquiring substantial barrier properties as measured by transendothelial electrical resistance (1,450 ± 140 Ω cm2), and they possess molecular permeability that correlates well with in vivo rodent blood-brain transfer coefficients.

A Claudin-9-Based Ion Permeability Barrier Is Essential for Hearing

Hereditary hearing loss is one of the most common birth defects, yet the majority of genes required for audition is thought to remain unidentified. Ethylnitrosourea (ENU)–mutagenesis has been a valuable approach for generating new animal models of deafness and discovering previously unrecognized gene functions. Here we report on the characterization of a new ENU–induced mouse mutant (nmf329) that exhibits recessively inherited deafness. We found a widespread loss of sensory hair cells in the hearing organs of nmf329 mice after the second week of life. Positional cloning revealed that the nmf329 strain carries a missense mutation in the claudin-9 gene, which encodes a tight junction protein with unknown biological function. In an epithelial cell line, heterologous expression of wild-type claudin-9 reduced the paracellular permeability to Na+ and K+, and the nmf329 mutation eliminated this ion barrier function without affecting the plasma membrane localization of claudin-9. In the nmf329 mouse line, the perilymphatic K+ concentration was found to be elevated, suggesting that the cochlear tight junctions were dysfunctional. Furthermore, the hair-cell loss in the claudin-9–defective cochlea was rescued in vitro when the explanted hearing organs were cultured in a low-K+ milieu and in vivo when the endocochlear K+-driving force was diminished by deletion of the pou3f4 gene. Overall, our data indicate that claudin-9 is required for the preservation of sensory cells in the hearing organ because claudin-9–defective tight junctions fail to shield the basolateral side of hair cells from the K+-rich endolymph. In the tight-junction complexes of hair cells, claudin-9 is localized specifically to a subdomain that is underneath more apical tight-junction strands formed by other claudins. Thus, the analysis of claudin-9 mutant mice suggests that even the deeper (subapical) tight-junction strands have biologically important ion barrier function.

Diabetic Peripheral Neuropathies: A Morphometric Overview

La diabetes es considerada hoy una de las principales amenazas para la salud humana en el siglo 21 y muchos investigadores se dedican a investigar la fisiopatologia de la enfermedad, con otras visiones sobre el manejo de sus principales complicaciones. Dado que la comprension de la fisiopatologia de las principales complicaciones de la diabetes y sus procesos subyacentes es obligatorio, modelos experimentales de la enfermedad pueden ser utiles ya que permiten el reconocimiento de los primeros mecanismos implicados en las complicaciones a largo plazo de la diabetes. El compromiso de los nervios perifericos es muy frecuente en la diabetes mellitus y se ha documentado que un tercio de los pacientes diabeticos tiene neuropatia periferica. La prevalencia es desconocida y los informes varian de 10% a 90% en los pacientes diabeticos, en funcion de los criterios y metodos utilizados para definir la neuropatia. En esta revision, se presentan los modelos experimentales mas comunes de diabetes y se discuten los hallazgos patologicos en los principales nervios perifericos. Ademas, se destacan las visiones presentadas por la morfometria de la investigacion la neuropatia diabetica.

Ultrastructural and morphometric alterations in the aortic depressor nerve of rats due to long term experimental diabetes: effects of insulin treatment.

Most of the reports about an altered baroreflex attribute this condition to the diabetic efferent neuropathy of the aortic depressor nerve (ADN) (afferent arm of the baroreflex less explored). We evaluated the ADN ultrastructural alterations caused by long term experimental diabetes and the effects of insulin treatment. Wistar rats (N=14) received a single intravenous injection of streptozotocin (40 mg/kg) 12 weeks before the experiment. Control animals (N=9) received vehicle (citrate buffer). Insulin treated animals (N=8) received a single subcutaneous injection of insulin daily. Under pentobarbital anesthesia the ADNs were isolated and had their spontaneous activity recorded. Afterwards, proximal and distal segments of the nerves were prepared for transmission electron microscopy study. Morphometry of the unmyelinated fibers was carried out with the aid of computer software. ADN of the diabetic animals showed axonal atrophy for myelinated fibers, with more pronounced alterations of the myelin sheath, such as myelin infolding and out folding, presence of myelin balls and very thin myelin sheath in relation to the axonal size, particularly for the small myelinated fibers becoming evident. No differences were observed in myelinated fiber number and their density, as well as on the fascicular area. Unmyelinated fiber number was significantly larger in the diabetic group while fiber diameter was significantly smaller compared to control. This result suggests axonal atrophy or, if associated to the larger number of fibers present in this group, could indicate fiber sprouting. These alterations were more evident in the distal segments of the nerves and were moderated by insulin treatment.

Renal nerve ultrastructural alterations in short term and long term experimental diabetes

BackgroundDespite the evidence that renal hemodynamics is impaired in experimental diabetes, associated with glomeruli structural alterations, renal nerves were not yet investigated in experimental models of diabetes and the contribution of nerve alterations to the diabetic nephropathy remains to be investigated. We aimed to determine if ultrastructural morphometric parameters of the renal nerves are affected by short term and/or long term experimental diabetes and if insulin treatment reverses these alterations. Left renal nerves were evaluated 15 days or 12 weeks (N = 10 in each group) after induction of diabetes, with a single injection of streptozotocin (STZ). Control rats (N = 10 in each group) were injected with vehicle (citrate buffer). Treated animals (N = 10 in each group) received a single subcutaneous injection of insulin on a daily basis. Arterial pressure, together with the renal nerves activity, was recorded 15 days (short-term) or 12 weeks (long-term) after STZ injection. After the recordings, the renal nerves were dissected, prepared for light and transmission electron microscopy, and fascicle and fibers morphometry were carried out with computer software.ResultsThe major diabetic alteration on the renal nerves was a small myelinated fibers loss since their number was smaller on chronic diabetic animals, the average morphometric parameters of the myelinated fibers were larger on chronic diabetic animals and distribution histograms of fiber diameter was significantly shifted to the right on chronic diabetic animals. These alterations began early, after 15 days of diabetes induction, associated with a severe mitochondrial damage, and were not prevented by conventional insulin treatment.ConclusionsThe experimental diabetes, induced by a single intravenous injection of STZ, in adult male Wistar rats, caused small fiber loss in the renal nerves, probably due to the early mitochondrial damage. Conventional treatment with insulin was able to correct the weight gain and metabolic changes in diabetic animals, without, however, correcting and / or preventing damage to the thin fibers caused by STZ-induced diabetes. The kidney innervation is impaired in this diabetic model suggesting that alterations of the renal nerves may play a role in the development of the diabetic nephropathy.

Transmission Electron Microscopy Studies of the Vestibulocochlear Nerve in Chronic Diabetic Rats

Se ha descrito ampliamente en la literatura que los pacientes diabeticos presentan discapacidad auditiva. En estos pacientes, a pesar de las alteraciones histologicas de las estructuras del oido interno, asi como en modelos experimentales de diabetes, que mejoran nuestro conocimiento, la evaluacion histologica del nervio vestibulococlear no ha sido realizada. Se describen y comparan las alteraciones ultraestructurales entre un nervio espinal y uno craneal en ratas con diabetes cronica inducida. Fueron utilizadas 12 ratas Wistar machos, de 42 dias de edad, alimentadas con dieta estandar. Los animales diabeticos inducidos (n = 6) se mantuvieron en ayuno por 12 horas antes de ser inyectados por via intraperitoneal con estreptozotocina (STZ - 60mg/kg) en una sola dosis. Los animales control (n = 6) solo recibieron inyeccion de 0.01 mol/l buffer, citrato pH 4,5. Diez semanas despues de la inyeccion de STZ, los animales fueron perfundidos intracardiacamente con solucion de Karnovsky. Los nervios vestibulococlear derecho e izquierdo fueron disecados y procesados histologicamente para ser incluidos en resina epoxy. Las muestras fueron estudiadas con microscopio electronico de transmision. Fueron observadas facilmente, grandes fibras mielinizadas con signos morfologicos de atrofia axonal en los nervios vestibulococlear. Estos resultados sugieren que la diabetes cronica inducida por STZ en ratas causo alteraciones en las fibras mielinicas y celulas del neurilema, compatible, con los signos y sintomas clasicos de la diabetes. Alteraciones morfologicas del nervio vestibulococlear en la diabetes son descritas por primera vez, lo que aporta informacion para una mejor comprension de por que hay cambios en la audicion en los pacientes diabeticos.

An Update On Rodent Model Studies In Diabetic Neuropathy: A Brief Communication

The diseases of the peripheral nerves are quite common and diversified, are directly related to several factors, ranging from the imbalance related to good nutrition and adequate needs of nutrients, going to the injuries caused by drugs or mechanisms external to the human organism. Diabetes is a complex syndrome that affects and kills millions of people worldwide. We demonstrated through the experimental model of diabetes using STZ@ in single intraperitoneal dose of 60 mg / kg, that both a purely motor nerve can be directly affected as well as a special afferent nerve (cranial nerve), this comparison showed us that there is a possibility of having a new type of mixed type neuropathy, this may be related to the amount of the dose involved, such as the time of disease progression, but more studies need to be done for definitive confirmation. We can extrapolate the original results to understand the mechanisms of diabetes in humans, although it does not yet have an experimental model of type II diabetes, more related to eating disorders, the STZ application simulates the effects of type I or insulin dependent diabetes, with more serious and deleterious effects mainly the more distal portions of the nerves. Prevention and food control are very important, especially those related to the mechanisms that involve carbohydrate metabolism and its peripheral resistance. The original results commented here are relevant for the continuous study of this serious but old illness, but quite current in the medical and therapeutic clinic. Keywords: Experimental Model of Diabetes; Sensory and Motor Peripheral Nerves; Peripheral Nerve Injuries; Neuropathies; STZ; Rats.

Applications of Microscopy to Genetic Therapy of Cystic Fibrosis and Other Human Diseases

Gene therapy has become an extremely important and active field of biomedical research. Microscopy is an integral component of this effort. This chapter presents an overview of imaging techniques used in our facility in support of cystic fibrosis gene therapy research. Instrumentation used in these studies includes light and confocal microscopy, transmission electron microscopy, and scanning electron microscopy. Techniques outlined include negative staining, cryo-electron microscopy, three-dimentional reconstruction, enzyme cytochemistry, immunocytochemistry, and fluorescence imaging.