Exosome-associated AAV vector as a robust and convenient neuroscience tool.

Adeno-associated virus (AAV) vectors are showing promise in gene therapy trials and have proven to be extremely efficient biological tools in basic neuroscience research. One major limitation to their widespread use in the neuroscience laboratory is the cost, labor, skill and time-intense purification process of AAV. We have recently shown that AAV can associate with exosomes (exo-AAV) when the vector is isolated from conditioned media of producer cells, and the exo-AAV is more resistant to neutralizing anti-AAV antibodies compared with standard AAV. Here, we demonstrate that simple pelleting of exo-AAV from media via ultracentrifugation results in high-titer vector preparations capable of efficient transduction of central nervous system (CNS) cells after systemic injection in mice. We observed that exo-AAV is more efficient at gene delivery to the brain at low vector doses relative to conventional AAV, even when derived from a serotype that does not normally efficiently cross the blood-brain barrier. Similar cell types were transduced by exo-AAV and conventionally purified vector. Importantly, no cellular toxicity was noted in exo-AAV-transduced cells. We demonstrated the utility and robustness of exo-AAV-mediated gene delivery by detecting direct GFP fluorescence after systemic injection, allowing three-dimensional reconstruction of transduced Purkinje cells in the cerebellum using ex vivo serial two-photon tomography. The ease of isolation combined with the high efficiency of transgene expression in the CNS, may enable the widespread use of exo-AAV as a neuroscience research tool. Furthermore, the ability of exo-AAV to evade neutralizing antibodies while still transducing CNS after peripheral delivery is clinically relevant.

Angiotensin II induced cerebral microvascular inflammation and increased blood-brain barrier permeability via oxidative stress.

Although hypertension has been implicated in the pathogenesis of vascular disease, its role in inflammatory responses, especially in brain, remains unclear. In this study we found key mechanisms by which angiotensin II (AngII) mediates cerebral microvascular inflammation. C57BL/6 male mice were subjected to slow-pressor dose of AngII infusion using osmotic mini-pumps at a rate of 400 ng/kg/min for 14 days. Vascular inflammation in the brain was evaluated by analysis of leukocyte-endothelial interaction and blood-brain barrier (BBB) permeability. Results from intravital microscopy of pial vessels in vivo, revealed a 4.2 fold (P<0.05, compared to vehicle) increase in leukocyte adhesion on day 4 of AngII infusion. This effect persisted through day 14 of AngII infusion, which resulted in a 2.6 fold (P<0.01, compared to vehicle) increase in leukocyte adhesion. Furthermore, evaluation of BBB permeability by Evans Blue extravasation showed that Ang II significantly affected the BBB, inducing 3.8 times (P<0.05, compared to vehicle) higher permeability. Previously we reported that AngII mediated hypertension promotes oxidative stress in the vasculature. Thus, we used the superoxide scavenger; 4-hydroxy-TEMPO (Tempol) to determine whether AngII via oxidative stress could contribute to higher leukocyte adhesion and increased BBB permeability. Tempol was given via drinking water (2 mmol) on day 4th following Ang II infusion, since oxidative stress increases in this model on day 4. Treatment with Tempol significantly attenuated the increased leukocyte/endothelial interactions and protected the BBB integrity on day 14 of AngII infusion. In conclusion, AngII via oxidative stress increases cerebral microvasculature inflammation and leads to greater immune-endothelial interaction and higher BBB permeability. This finding may open new avenues for the management of nervous system pathology involving cerebrovascular inflammation.

Neurotrophins prevent HIV Tat-induced neuronal apoptosis via a nuclear factor-kappaB (NF-kappaB)-dependent mechanism.

HIV-1 associated dementia is thought to be caused by neuronal damage and death in response to the production of soluble neurotoxic factors by virally infected mononuclear phagocytes. These neurotoxins include HIV-1 Tat. The ability of neurotrophins to promote cell survival prompted us to examine whether neurotrophins might also be capable of opposing the pro-apoptotic effects of Tat. Here, we show that Tat-induced neuronal apoptosis in primary cultures of rat cerebellar granule cells and in neuronally differentiated human SK-N-MC cells is profoundly inhibited by brain-derived neurotrophic factor, nerve growth factor and activity-dependent neurotrophic factor nonamer peptide. These neurotrophins activated the transcription factor NF-kappaB, and inhibition of NF-kappaB activation using a super-repressor IkappaB-alpha mutant was found to block the survival-promoting activity of the neurotrophins. Reporter gene assays and immunoblot experiments revealed that the neurotrophins also up-regulated the expression of Bcl-2, at both the transcriptional and protein levels. Overexpression of the super-repressor IkappaB-alpha mutant prevented this induction of Bcl-2 expression. Moreover, overexpression of either Bcl-2, alone, or the RelA subunit of NF-kappaB, alone, protected neurons from Tat-induced apoptosis. These findings suggest that the activation of NF-kappaB by neurotrophic factors may promote survival of neurons exposed to Tat, via regulation of anti-apoptotic genes including Bcl-2.

Activation of glycogen synthase kinase 3 beta (GSK-3beta) by platelet activating factor mediates migration and cell death in cerebellar granule neurons.

Children with vertically acquired HIV-1 can present with a rapidly progressive encephalopathy and neuronal apoptosis in the first 12-18 months of life. Furthermore, abnormal prenatal platelet activating factor (PAF) signalling may result in lissencephaly, a disorder of neuronal migration. PAF, produced from human immunodeficiency virus type 1 (HIV-1) -infected brain-resident macrophages, induces neuronal apoptosis in cultured cerebellar granule neurons (CGNs) in part by activating glycogen synthase kinase 3 beta (GSK-3beta). However, PAF can also inhibit migration of CGNs that are dispersed and allowed to reaggregate. Therefore, we investigated the biological effects following activation of GSK-3beta by PAF, and whether these effects were dependent on the culture conditions of the CGNs. We show here that activation of neuronal GSK-3beta by PAF is receptor-specific, with similar kinetics of activation in both monolayer cultures of CGNs that have ceased to migrate and reaggregate cultures of CGNs that are actively migrating. However, PAF receptor activation in reaggregated CGNs inhibits neuronal migration and induces approximately half the level of neuronal apoptosis compared with PAF-treated CGN cultures that have ceased to migrate. PAF-mediated inhibition of neuronal migration in reaggregated CGNs or induction of apoptosis in CGNs that have ceased to migrate can be reversed by either PAF receptor antagonists, or the GSK-3beta inhibitors lithium or valproic acid, in a dose-dependent manner. Abnormal PAF signalling that results in GSK-3beta overactivation may represent a common mechanism for pathological defects in neuronal migration in the prenatal period and neuronal apoptosis in the postnatal period.

Manejo de las fracturas del complejo órbito-cigómato-malar en 70 pacientes / Management of orbitozygomatic fractures in 70 patients

En esta comunicación preliminar se relata la experiencia clínica del manejo de las fracturas órbito-cigomático-malar en el Hospital Instituto de Seguridad del Trabajo, entre los años 1989 y 1995. Se consideran 70 pacientes y se analizan: 1º Tipos de accidentes; 2º Criterios para intervenir quirúrgicamente. En general descrito en la literatura; 3º Frecuencia de los distintos tipos de fracturas; 4º Vías de abordaje. Se discuten: el uso de los diferentes tipos de materiales de osteosíntesis, oportunidad de la intervención quirúrgica y, por último, se concluye que el tratamiento ideal debe ser lo más conservador posible y el trabajo en un equipo multidisciplinario