Institute of the Royal Academy of Arts and science

Amsterdam, Netherlands

Institute of the Royal Academy of Arts and science

Amsterdam, Netherlands
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Ehlert E.M.,Institute of the Royal Academy of Arts and science | Eggers R.,Institute of the Royal Academy of Arts and science | Verhaagen J.,Institute of the Royal Academy of Arts and science
BMC Neuroscience | Year: 2010

Background: After a spinal cord lesion, axon regeneration is inhibited by the presence of a diversity of inhibitory molecules in the lesion environment. At and around the lesion site myelin-associated inhibitors, chondroitin sulfate proteoglycans (CSPGs) and several axon guidance molecules, including all members of the secreted (class 3) Semaphorins, are expressed. Interfering with multiple inhibitory signals could potentially enhance the previously reported beneficial effects of blocking single molecules. RNA interference (RNAi) is a tool that can be used to simultaneously silence expression of multiple genes. In this study we aimed to employ adeno-associated virus (AAV) mediated expression of short hairpin RNAs (shRNAs) to target all Semaphorin class 3 signaling by knocking down its receptors, Neuropilin 1 (Npn-1) and Neuropilin 2 (Npn-2).Results: We have successfully generated shRNAs that knock down Npn-1 and Npn-2 in a neuronal cell line. We detected substantial knockdown of Npn-2 mRNA when AAV5 viral vector particles expressing Npn-2 specific shRNAs were injected in dorsal root ganglia (DRG) of the rat. Unexpectedly however, AAV1-mediated expression of Npn-2 shRNAs and a control shRNA in the red nucleus resulted in an adverse tissue response and neuronal degeneration. The observed toxicity was dose dependent and was not seen with control GFP expressing AAV vectors, implicating the shRNAs as the causative toxic agents.Conclusions: RNAi is a powerful tool to knock down Semaphorin receptor expression in neuronal cells in vitro and in vivo. However, when shRNAs are expressed at high levels in CNS neurons, they trigger an adverse tissue response leading to neuronal degradation. © 2010 Ehlert et al; licensee BioMed Central Ltd.


Van Der Meer J.N.,Institute of the Royal Academy of Arts and science | Van Der Meer J.N.,Leibniz Institute for Neurobiology | Van Hest G.,Philips | Plattel G.-J.,Philips | And 3 more authors.
Magnetic Resonance Materials in Physics, Biology and Medicine | Year: 2014

Object: While pseudo-continuous arterial spin labeling (pCASL) is a promising imaging technique to visualize cerebral blood flow, it is also (acoustically) very loud during labeling. In this paper, we reduced the labeling loudness on our scanner by increasing the interval between the RF pulses from the literature standard of 1.0 ms. We also propose recommendations to reduce the loudness on scanners of the same type at other sites. Materials and methods: First, the sound pressure level (SPL) was both simulated and measured as a function of the labeling interval (1.0-1.8 ms) and longitudinal position in the scanner (-10 to +10 cm, relative to isocenter). Subsequently, we selected the labeling interval with the lowest overall SPL for the SPL-optimized pCASL sequence. Nine volunteers were scanned to compare raw signal intensity, temporal signal-to-noise ratio (tSNR) and labeling efficiency between the SPL-optimized and the standard PCASL sequence. Results: Sound pressure level measurements on our scanner showed that loudness was reduced by 6.5 dB at the approximate location of the ear by adjusting the labeling interval to 1.4 ms. Furthermore, image quality was not affected, since no significant differences in signal intensity, tSNR and labeling efficiency were observed. Conclusion: By increasing the pCASL labeling interval, acoustic noise in the pCASL sequence was reduced with 6.5 dB, while image quality was preserved. © 2013 ESMRMB.


Fagoe N.D.,Institute of the Royal Academy of Arts and science | Eggers R.,Institute of the Royal Academy of Arts and science | Verhaagen J.,Institute of the Royal Academy of Arts and science | Verhaagen J.,Vrije Universtiteit Amsterdam | Mason M.R.J.,Institute of the Royal Academy of Arts and science
Gene Therapy | Year: 2014

Adeno-associated viral (AAV) vectors based on serotype 5 are an efficient means to target dorsal root ganglia (DRG) to study gene function in the primary sensory neurons of the peripheral nervous system. In this study, we have developed a compact AAV dual promoter vector composed of the cytomegalovirus (CMV) and chicken beta-actin (CAG) promoters in a back-to-back configuration with a shared enhancer, and show efficient expression of two proteins simultaneously in DRG neurons. We demonstrate how this is useful for experiments on axonal regeneration, by co-expressing a gene of interest and an axonal marker. Using a farnesylated form of eGFP, which is actively transported along axons, we show superior long-distance labelling of axons of DRG neurons compared with normal eGFP. Additionally, we have efficiently transduced lumbar DRG neurons by injecting the AAV dual promoter vector into the dorsal intrathecal space, which is a less invasive delivery method. In summary, we have developed an AAV dual promoter vector designed for simultaneous expression of a gene of interest and a fluorescent protein to label long-distance axonal projections, which allows specific quantification of axons from transduced neurons after injury. © 2014 Macmillan Publishers Limited.


Afshari F.T.,University of Cambridge | Kwok J.C.,University of Cambridge | Andrews M.R.,University of Cambridge | Blits B.,Institute of the Royal Academy of Arts and science | And 4 more authors.
Brain | Year: 2010

Retinal pigment epithelial cell malfunction is a causative feature of age-related macular degeneration, and transplantation of new retinal pigment epithelial cells is an attractive strategy to prevent further progression and visual loss. However, transplants have shown limited efficacy, mainly because transplanted cells fail to adhere and migrate onto pathological Bruch's membrane. Adhesion to Bruch's membrane is integrin-mediated. Ageing of Bruch's membrane leads to a decline in integrin ligands and, added to this, wet age-related macular degeneration leads to upregulation of anti-adhesive molecules such as tenascin-C. We have therefore investigated whether manipulation of integrin function in retinal pigment epithelial cells can restore their adhesion and migration on wet age-related macular degeneration-damaged Bruch's membrane. Using spontaneously immortalized human retinal pigment epithelial cells (adult retinal pigment epithelium-19), we show that adhesion and migration on the Bruch's membrane components is integrin-dependent and enhanced by integrin-activating agents manganese and TS2/16. These allowed cells to adhere and migrate on low concentrations of ligand, as would be found in aged Bruch's membrane. We next developed a method for stripping cells from Bruch's membrane so that adhesion and migration assays can be performed on its surface. Integrin activation had a moderate effect on enhancing retinal pigmented epithelial cell adhesion and migration on normal human and rat Bruch's membrane. However, on Bruch's membrane prepared from human wet age-related macular degeneration-affected eyes, adhesion was lower and integrin activation had a much greater effect. A candidate molecule for preventing retinal pigmented epithelial interaction with age-related macular degeneration-affected Bruch's membrane is tenascin-C which we confirm is present at high levels in wet age-related macular degeneration membrane. We show that tenascin-C is anti-adhesive for retinal pigmented epithelial cells, but after integrin activation, they can adhere and migrate on it using alphaVbeta3 integrin. Alternatively, we find that transduction of retinal pigmented epithelial cells with alpha9 integrin, a tenascin-C-binding integrin, led to a large increase in alpha9beta1-mediated adhesion and migration on tenascin-C. Both expression of alpha9 integrin and integrin activation greatly enhanced the ability of retinal pigment epithelial cells to adhere to tenascin-rich wet age-related macular degeneration-affected Bruch's membranes. Our results suggest that manipulation of retinal pigment epithelial cell integrins through integrin activating strategies, or expression of new integrins such as alpha9, could be effective in improving the efficacy of retinal pigment epithelial cell transplantation in wet age-related macular degeneration-affected eyes.


van der Meer J.N.,Institute of the Royal Academy of Arts and science | Heijtel D.F.,Institute of the Royal Academy of Arts and science | van Hest G.,Institute of the Royal Academy of Arts and science | Plattel G.-J.,Institute of the Royal Academy of Arts and science | And 4 more authors.
Magma (New York, N.Y.) | Year: 2014

OBJECT: While pseudo-continuous arterial spin labeling (pCASL) is a promising imaging technique to visualize cerebral blood flow, it is also (acoustically) very loud during labeling. In this paper, we reduced the labeling loudness on our scanner by increasing the interval between the RF pulses from the literature standard of 1.0 ms. We also propose recommendations to reduce the loudness on scanners of the same type at other sites.MATERIALS AND METHODS: First, the sound pressure level (SPL) was both simulated and measured as a function of the labeling interval (1.0-1.8 ms) and longitudinal position in the scanner (-10 to +10 cm, relative to isocenter). Subsequently, we selected the labeling interval with the lowest overall SPL for the "SPL-optimized" pCASL sequence. Nine volunteers were scanned to compare raw signal intensity, temporal signal-to-noise ratio (tSNR) and labeling efficiency between the SPL-optimized and the standard PCASL sequence.RESULTS: Sound pressure level measurements on our scanner showed that loudness was reduced by 6.5 dB at the approximate location of the ear by adjusting the labeling interval to 1.4 ms. Furthermore, image quality was not affected, since no significant differences in signal intensity, tSNR and labeling efficiency were observed.CONCLUSION: By increasing the pCASL labeling interval, acoustic noise in the pCASL sequence was reduced with 6.5 dB, while image quality was preserved.


Borchardt V.,Leibniz Institute for Neurobiology | Borchardt V.,Clinical Affective Neuroimaging Laboratory | Lord A.R.,Clinical Affective Neuroimaging Laboratory | Lord A.R.,QIMR Berghofer Medical Research Institute | And 16 more authors.
Human Brain Mapping | Year: 2016

Resting-state fMRI studies have gained widespread use in exploratory studies of neuropsychiatric disorders. Graph metrics derived from whole brain functional connectivity studies have been used to reveal disease-related variations in many neuropsychiatric disorders including major depression (MDD). These techniques show promise in developing diagnostics for these often difficult to identify disorders. However, the analysis of resting-state datasets is increasingly beset by a myriad of approaches and methods, each with underlying assumptions. Choosing the most appropriate preprocessing parameters a priori is difficult. Nevertheless, the specific methodological choice influences graph-theoretical network topologies as well as regional metrics. The aim of this study was to systematically compare different preprocessing strategies by evaluating their influence on group differences between healthy participants (HC) and depressive patients. We thus investigated the effects of common preprocessing variants, including global mean-signal regression (GMR), temporal filtering, detrending, and network sparsity on group differences between brain networks of HC and MDD patients measured by global and nodal graph theoretical metrics. Occurrence of group differences in global metrics was absent in the majority of tested preprocessing variants, but in local graph metrics it is sparse, variable, and highly dependent on the combination of preprocessing variant and sparsity threshold. Sparsity thresholds between 16 and 22% were shown to have the greatest potential to reveal differences between HC and MDD patients in global and local network metrics. Our study offers an overview of consequences of methodological decisions and which neurobiological characteristics of MDD they implicate, adding further caution to this rapidly growing field. © 2016 Wiley Periodicals, Inc.


PubMed | Institute of the Royal Academy of Arts and science
Type: Journal Article | Journal: Molecular therapy : the journal of the American Society of Gene Therapy | Year: 2010

For many experiments in the study of the peripheral nervous system, it would be useful to genetically manipulate primary sensory neurons. We have compared vectors based on adeno-associated virus (AAV) serotypes 1, 2, 3, 4, 5, 6, and 8, and lentivirus (LV), all expressing green fluorescent protein (GFP), for efficiency of transduction of sensory neurons, expression level, cellular tropism, and persistence of transgene expression following direct injection into the dorsal root ganglia (DRG), using histological quantification and qPCR. Two weeks after injection, AAV1, AAV5, and AAV6 had transduced the most neurons. The time course of GFP expression from these three vectors was studied from 1 to 12 weeks after injection. AAV5 was the most effective serotype overall, followed by AAV1. Both these serotypes showed increasing neuronal transduction rates at later time points, with some injections of AAV5 yielding over 90% of DRG neurons GFP(+) at 12 weeks. AAV6 performed well initially, but transduction rates declined dramatically between 4 and 12 weeks. AAV1 and AAV5 both transduced large-diameter neurons, IB4(+) neurons, and CGRP(+) neurons. In conclusion, AAV5 is a highly effective gene therapy vector for primary sensory neurons following direct injection into the DRG.


PubMed | Institute of the Royal Academy of Arts and science
Type: Journal Article | Journal: Magma (New York, N.Y.) | Year: 2014

While pseudo-continuous arterial spin labeling (pCASL) is a promising imaging technique to visualize cerebral blood flow, it is also (acoustically) very loud during labeling. In this paper, we reduced the labeling loudness on our scanner by increasing the interval between the RF pulses from the literature standard of 1.0 ms. We also propose recommendations to reduce the loudness on scanners of the same type at other sites.First, the sound pressure level (SPL) was both simulated and measured as a function of the labeling interval (1.0-1.8 ms) and longitudinal position in the scanner (-10 to +10 cm, relative to isocenter). Subsequently, we selected the labeling interval with the lowest overall SPL for the SPL-optimized pCASL sequence. Nine volunteers were scanned to compare raw signal intensity, temporal signal-to-noise ratio (tSNR) and labeling efficiency between the SPL-optimized and the standard PCASL sequence.Sound pressure level measurements on our scanner showed that loudness was reduced by 6.5 dB at the approximate location of the ear by adjusting the labeling interval to 1.4 ms. Furthermore, image quality was not affected, since no significant differences in signal intensity, tSNR and labeling efficiency were observed.By increasing the pCASL labeling interval, acoustic noise in the pCASL sequence was reduced with 6.5 dB, while image quality was preserved.

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