Background: Current therapy does not eradicate ocular morbidity and visual disability following retinopathy of prematurity. Anti-vascular endothelial growth factor treatment provides a potentially new treatment. Methods: Infants with birth weight <1,500g meeting established criteria for ROP treatment were recruited in 87 neonatal and ophthalmology centres in 26 countries. We performed a randomised, multicentre, open-label, 3arm, parallel-group study evaluating efficacy and safety of intravitreal injection of ranibizumab 0•2mg or ranibizumab 0•1mg against laser therapy. The primary outcome was treatment success, defined as survival with no active retinopathy, unfavourable structural outcomes or the need for an additional treatment modality at or before 24 weeks. Findings: Treatment success occurred in 56/70 (80%) infants receiving ranibizumab 0•2mg compared with 57/76 (75%) receiving ranibizumab 0•1mg and 45/68 (66%) infants following laser therapy. The odds ratio of a successful outcome following ranibizumab 0•2mg compared with laser therapy was 2•19 (95% confidence interval 0•99-4•82; p=0•051). One infant had an unfavourable structural outcome following ranibizumab 0•2mg, compared to five following ranibizumab 0•1mg and seven after laser therapy. Ranibizumab 0•2mg was effective in both Zone I and Zone II disease. Ranibizumab 0•1mg offered no advantage over 0•2 mg. Death, serious and non-serious systemic and ocular adverse events were evenly distributed between the three groups. Interpretation: In the treatment of retinopathy of prematurity, ranibizumab 0•2mg was effective with fewer unfavourable ocular outcomes than laser therapy and with an acceptable short-term safety profile. Funding: Novartis Pharma; RAINBOW ClinicalTrials.gov number, NCT02375971.
Gene expression analysis implicates an increasing number of novel genes in the brain as potential targets for the treatment of neurological and psychiatric disorders. Frequently, these genes are ubiquitously expressed in the brain and, thus, may contribute to a pathophysiological state through actions in several brain nuclei. Current strategies employing genetically modified animals for in vivo validation of such targets are time-consuming and often limited by developmental adaptations. Somatic gene manipulation using viral-mediated RNA interference (RNAi) has emerged recently, although restricting the target validation to specific brain nuclei. We investigated whether nonviral infusion of short interfering RNA (siRNA) into the ventricular system would enable a sequence-specific gene knockdown. The temporality and extent of siRNA-induced down-regulation were analyzed by targeting a transgene, EGFP, in mice overexpressing EGFP. Extensive knockdown of EGFP was observed, especially in regions adjacent or dorsoventrally and mediolaterally distant to the infusion site (dorsal third ventricle), with lesser knockdown in more distal regions. We challenged our RNAi approach to generate a specific knockdown of an endogenous gene, encoding the dopamine transporter (DAT) in regions (ventral midbrain) far distal to the infusion site. DAT-siRNA infusion in adult mice produced a significant down-regulation of DAT mRNA and protein in the brain and also elicited a temporal hyperlocomotor response similar to that (but delayed) obtained upon infusion of GBR-12909, a pharmacologically selective DAT inhibitor. Application of this nonviral RNAi approach may accelerate target validation for neuropsychiatric disorders that involve a complex interplay of gene(s) from various brain regions.T he burgeoning use of microarray analyses to detect potential target genes relevant to neuropsychiatric disorders necessitates the validation of such targets in vivo (1-3). The approach of genetically modifying animals (knockouts or transgenics) for target validation often is limited by developmental adaptations and genetic compensation that may mask the establishment of a clear phenotype. Further, such methodology is laborious and timeconsuming and not applicable for high-throughput in vivo validation of the large number of hits generated from modern microarray and proteomic target-identification strategies. Additional approaches using ribozymes and antisense oligodeoxynucleotides for somatic gene manipulation also suffer from drawbacks of eliciting nonspecific actions. RNA interference (RNAi) recently has emerged as a potentially superior alternative to the traditional approaches for assessing gene function in adult animals (4).RNAi is a cellular surveillance mechanism that responds to double-stranded RNA (dsRNA) by destroying cytoplasmic mRNAs containing sequences homologous to the dsRNA trigger (5). dsRNAs longer than 30 nt introduced in mammalian cells can induce an undesirable IFN response to produce cell death (6, 7). Conversely, short interfer...
Formation and extinction of aversive memories in the mammalian brain are insufficiently understood at the cellular and molecular levels. Using the novel metabotropic glutamate receptor 7 (mGluR7) agonist AMN082, we demonstrate that mGluR7 activation facilitates the extinction of aversive memories in two different amygdala-dependent tasks. Conversely, mGluR7 knockdown using short interfering RNA attenuated the extinction of learned aversion. mGluR7 activation also blocked the acquisition of Pavlovian fear learning and its electrophysiological correlate long-term potentiation in the amygdala. The finding that mGluR7 critically regulates extinction, in addition to acquisition of aversive memories, demonstrates that this receptor may be relevant for the manifestation and treatment of anxiety disorders.
Selective serotonin reuptake inhibitors (SSRIs) are widely used antidepressant drugs that increase the extracellular levels of serotonin by blocking the reuptake activity of the serotonin transporter (SERT). Although SSRIs elevate brain serotonergic neurotransmission acutely, their full therapeutic effects involve neurochemical adaptations that emerge following chronic drug administration. The adaptive downregulation of SERT has recently been implicated in the therapeutic response of SSRIs. Interestingly, studies using SERT-knockout mice reveal somewhat paradoxical depression-related effects, probably specific to the downregulation of SERT during early development. However, the behavioral significance of SSRI-mediated downregulation of SERT during adulthood is still unknown. We investigated whether somatic gene manipulation, triggered by infusing short interfering RNA (siRNA) into the ventricular system, would enable the downregulation of SERT in the adult mouse brain. Infusing the SERT-targeting siRNA, for 2 weeks, significantly reduced the mRNA levels of SERT in raphe nuclei. Further, a significant, specific and widespread downregulation of SERT-binding sites was achieved in the brain. In contrast, 2-week infusion of the SSRI, citalopram, produced a widespread downregulation of SERT-binding sites, independent of any alterations at the mRNA level. Irrespective of their mechanisms for downregulating SERT in the brain, infusions of SERT-siRNA or citalopram elicited a similar antidepressant-related behavioral response in the forced swim test. These results signify a role for the downregulation of SERT in mediating the antidepressant action of SSRIs in adults. Further, these data demonstrate that siRNAinduced widespread knockdown of gene expression serves as a powerful tool for assessing the function of endogenous genes in the adult brain. The serotonin transporter (SERT) controls the temporal and spatial activity of extracellular serotonin by facilitating a rapid and high-affinity reuptake of this neurotransmitter into presynpatic terminals. 1 SERT is also a primary molecular target for the most widely prescribed antidepressant drugs, the selective serotonin reuptake inhibitors (SSRIs). 2 SSRIs readily inhibit SERT activity and elevate the serotonergic tone in the brain. However, full therapeutic benefits ensue only after chronic use of SSRIs, requiring the manifestation of long-term adaptations, secondary to the blockade of serotonin reuptake. 3,4 Emerging studies reveal downregulation of SERT as an adaptive consequence of sustained and high occupancy by chronically administered SSRIs. 5-8 The downregulation of SERT favorably potentiates the SSRI-mediated increase in the brain serotonergic neurotransmission. [6][7] Further, the lag time required by SSRIs to produce a substantial downregulation of SERT correlates well with the time needed for eliciting a full therapeutic response. Therefore, downregulation of SERT has been implicated in the antidepressant action of SSRIs. 7 Recently, SERT-deficient mice were used ...
A unique approach to achieve siRNA-induced knockdown of the serotonin transporter (SERT) mRNA as well as binding sites in the adult mouse brain. Vehicle, SERT-targeting short interfering (si)RNA, mismatch (mm)RNA or citalopram were infused for 2 weeks into the adult mouse brain, using a subcutaneously implanted osmotic minipump connected with a catheter to a cannula for infusion into the dorsal third ventricle. (Top) siRNA infusion significantly reduced the mRNA levels of SERT expressed in the dorsal and median raphe nuclei, DR and MR, respectively. This is illustrated by low-magnification, dark-field photomicrographs of emulsion-dipped slides following in situ hybridization with a SERT riboprobe on coronal brain sections of mice from each treatment group. (Bottom) Infusion of the SERT-targeting siRNA or citalopram led to an extensive downregulation of SERT-binding sites in the brain as compared with infusions of vehicle or mmRNA. Representative autoradiograms of specific [ 125 I]RTI-55 binding to SERT in coronal sections show maximum number of brain regions with siRNA-and citalopram-induced downregulation of SERT. For more information on this topic, please see the article by Thakkar et al on pages 782-789.
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