Test of neural inertia in humans during general anaesthesia.

BACKGROUND: Neural inertia is defined as the tendency of the central nervous system to resist transitions between arousal states. This phenomenon has been observed in mice and Drosophila anaesthetized with volatile anaesthetics: the effect-site concentration required to induce anaesthesia in 50% of the population (C50) was significantly higher than the effect-site concentration for 50% of the population to recover from anaesthesia. We evaluated this phenomenon in humans using propofol or sevoflurane (both with or without remifentanil) as anaesthetic agents. METHODS: Thirty-six healthy volunteers received four sessions of anaesthesia with different drug combinations in a step-up/step-down design. Propofol or sevoflurane was administered with or without remifentanil. Serum concentrations of propofol and remifentanil were measured from arterial blood samples. Loss and return of responsiveness (LOR-ROR), response to pain (PAIN), Patient State Index (PSI) and spectral edge frequency (SEF) were modeled with NONMEM®. RESULTS: For propofol, the C50 for induction and recovery of anaesthesia was not significantly different across the different endpoints. For sevoflurane, for all endpoints except SEF, significant differences were found. For some endpoints (LOR and PAIN) the difference was significant only when sevoflurane was combined with remifentanil. CONCLUSIONS: Our results nuance earlier findings with volatile anaesthetics in mice and Drosophila. Methodological aspects of the study, such as the measured endpoint, influence the detection of neural inertia. A more thorough definition of neural inertia, with a robust methodological framework for clinical studies is required to advance our knowledge of this phenomenon. CLINICAL TRIAL REGISTRATION: NCT 02043938.

Dexmedetomidine pharmacokinetic-pharmacodynamic modelling in healthy volunteers: 1. Influence of arousal on bispectral index and sedation.

BACKGROUND: Dexmedetomidine, a selective α 2 -adrenoreceptor agonist, has unique characteristics, such as maintained respiratory drive and production of arousable sedation. We describe development of a pharmacokinetic-pharmacodynamic model of the sedative properties of dexmedetomidine, taking into account the effect of stimulation on its sedative properties. METHODS: In a two-period, randomized study in 18 healthy volunteers, dexmedetomidine was delivered in a step-up fashion by means of target-controlled infusion using the Dyck model. Volunteers were randomized to a session without background noise and a session with pre-recorded looped operating room background noise. Exploratory pharmacokinetic-pharmacodynamic modelling and covariate analysis were conducted in NONMEM using bispectral index (BIS) monitoring of processed EEG. RESULTS: We found that both stimulation at the time of Modified Observer's Assessment of Alertness/Sedation (MOAA/S) scale scoring and the presence or absence of ambient noise had an effect on the sedative properties of dexmedetomidine. The stimuli associated with MOAA/S scoring increased the BIS of sedated volunteers because of a transient 170% increase in the effect-site concentration necessary to reach half of the maximal effect. In contrast, volunteers deprived of ambient noise were more resistant to dexmedetomidine and required, on average, 32% higher effect-site concentrations for the same effect as subjects who were exposed to background operating room noise. CONCLUSIONS: The new pharmacokinetic-pharmacodynamic models might be used for effect-site rather than plasma concentration target-controlled infusion for dexmedetomidine in clinical practice, thereby allowing tighter control over the desired level of sedation. CLINICAL TRIAL REGISTRATION: NCT01879865.

Dexmedetomidine pharmacodynamics in healthy volunteers: 2. Haemodynamic profile.

BACKGROUND: Dexmedetomidine, a selective α 2 -adrenoreceptor agonist, has unique characteristics, with little respiratory depression and rousability during sedations. We characterized the haemodynamic properties of dexmedetomidine by developing a pharmacokinetic-pharmacodynamic (PKPD) model with a focus on changes in mean arterial blood pressure (MAP) and heart rate. METHODS: Dexmedetomidine was delivered i.v. to 18 healthy volunteers in a step-up fashion by target-controlled infusion using the Dyck model. Exploratory PKPD modelling and covariate analysis were conducted in NONMEM. RESULTS: Our model adequately describes dexmedetomidine-induced hypotension, hypertension, and bradycardia, with a greater effective concentration for the hypertensive effect. Changes in MAP were best described by a double-sigmoidal E max model with hysteresis. Covariate analysis revealed no significant covariates apart from age on the baseline MAP in the population pharmacokinetic model used to develop this PKPD model. Simulations revealed good general agreement with published descriptive studies of haemodynamics after dexmedetomedine infusion. CONCLUSIONS: The present integrated PKPD model should allow tighter control over the desired level of sedation, while limiting potential haemodynamic side-effects. CLINICAL TRIAL REGISTRATION: NCT01879865.

HSP70 interacting protein prevents the accumulation of inclusions in polyglutamine disease.

Heat shock proteins (HSPs) are associated with the proteinaceous inclusions that characterise many neurodegenerative diseases. This suggests they may be associated with disease aetiology and/or represents an attempt to remove abnormal protein aggregates. In this study the adenoviral mediated over-expression of HSP70 interacting protein (HIP) alone was shown to significantly reduce inclusion formation in both an in vitro model of Spinal Bulbar Muscular Atrophy and a primary neuronal model of polyglutamine disease. Experiments to determine the mechanism of action showed that: denatured luciferase activity (a measure of protein refolding) was not increased in the presence of HIP alone but was increased when HIP was co-expressed with HSP70 or Heat Shock cognate protein 70 (HSC70); the expression of polyglutamine inclusions in cortical neurons mediated an increase in the levels of HSC70 but not HSP70. Our data suggest that HIP may prevent inclusion formation by facilitating the constitutive HSC70 refolding cycle and possibly by preventing aggregation. HIP expression is not increased following stress and its over-expression may therefore reduce toxic polyglutamine aggregation events and contribute to an effective therapeutic strategy.

cAMP responsive element-binding protein phosphorylation is necessary for perirhinal long-term potentiation and recognition memory.

We established the importance of phosphorylation of cAMP responsive element-binding protein (CREB) to both the familiarity discrimination component of long-term recognition memory and plasticity within the perirhinal cortex of the temporal lobe. Adenoviral transduction of perirhinal cortex (and adjacent visual association cortex) with a dominant-negative inhibitor of CREB impaired the preferential exploration of novel over familiar objects at a long (24 h) but not a short (15 min) delay, disrupted the normal reduced activation of perirhinal neurons to familiar compared with novel pictures, and impaired long-term potentiation of synaptic transmission in perirhinal slices. The consistency of these effects across the behavioral, systems, and cellular levels of analysis provides strong evidence for involvement of CREB phosphorylation in synaptic plastic processes within perirhinal cortex necessary for long-term recognition memory.

Assessing adenoviral hammerhead ribozyme and small hairpin RNA cassettes in neurons: inhibition of endogenous caspase-3 activity and protection from apoptotic cell death.

Antisense technology, including ribozyme and small interfering RNA, is being developed to mediate the down-regulation of specific intracellular genes. It was observed in this study that both antiluciferase ribozymes and short hairpin RNAs (shRNAs) could significantly reduce the activity of exogenously expressed luciferase in primary hippocampal neurons in a viral titer-dependent manner. shRNAs were more effective gene-silencing agents than ribozymes, although they exhibited some nonspecific gene-silencing effects at high viral titers. We also attempted to increase ribozyme efficacy by using a woodchuck hepatitis posttranscriptional regulatory element (WPRE) in the ribozyme expression cassette. The results showed that adenoviral vectors encoding specific ribozymes could silence the cellular expression of luciferase and endogenous procaspase-3 significantly. Furthermore, the antiprocaspase-3 ribozyme was shown to inhibit staurosporine-mediated cell death. The addition of a WPRE did not, however, increase or decrease ribozyme activity. As far as we are aware, this is the first example of adenovirally mediated delivery of hammerhead ribozymes being used to manipulate gene expression in primary neurons. The results therefore suggest that hammerhead ribozymes may be useful tools for studying neuronal gene function and have potential as therapeutic agents to treat CNS diseases.

Increased utility in the CNS of a powerful neuron-specific tetracycline-regulatable adenoviral system developed using a post-transcriptional enhancer.

BACKGROUND: In previous studies we have found that the tetracycline (Tet)-regulatable system functions best in recombinant adenoviral (Ad) vectors when the Tet transactivators and the Tet-regulatable element (TRE) are incorporated into separate viral vectors. However, such a dual vector system is disadvantaged by the need to use relatively high titres that may elicit an immune response. Therefore, to develop a system that could be used at low titres while mediating strong, tightly regulatable gene expression in the central nervous system (CNS), we incorporated the woodchuck hepatitis virus post-transcriptional enhancer (WPRE) into a neuron-specific Tet-regulatable Ad system. METHODS: The WPRE was incorporated into Ad vectors encoding the Tet-Off (tTA) transactivator driven by the synapsin-1 and CMV promoters and encoding the TRE driving EGFP expression (TRE)-EGFP. RESULTS: The addition of the WPRE to the neuron-specific Tet-regulatable system mediated a greater than three-fold increase in transgene expression in primary hippocampal neurons with no loss of gene regulation. The results also showed that the addition of the WPRE enhanced transgene expression in the CNS without the loss of neuron specificity and without affecting the ability to regulate transgene expression. CONCLUSIONS: We have further developed a tetracycline-regulatable neuron-specific expression system such that it can now be used at low titres with no loss of transgene expression or ability to regulate transgene expression. It should therefore be of significant value to studies investigating neuronal gene function and to those seeking to develop effective neuronal gene therapy strategies.

Optimizing regulatable gene expression using adenoviral vectors.

Inducible gene expression systems have typically encountered limitations, such as pleitropic effects of the inducer, basal leakiness, toxicity of inducing agents and low levels of expression. However, recently non-toxic, tightly regulated control of transgene expression has been reported for several systems, the most frequently cited being the tetracycline gene control system. We have found that the individual components of the Tet system [the Tet transactivators and tetracycline responsive element (TRE)] function optimally to control gene expression when they are incorporated into separate adenoviral vectors. Furthermore, incorporation of the Woodchuck hepatitis virus post-transcriptional enhancer (WPRE) allows a dual vector Tet-regulatable Ad system to be used at very low titres (2 x 10(4)) that elicit a minimal inflammatory response, with no loss of transgene expression or ability to regulate transgene expression. This and similar regulatable systems will benefit studies investigating neuronal gene function and those seeking to develop effective neuronal gene therapy strategies.

Adenoviral expression of CREB protects neurons from apoptotic and excitotoxic stress.

In this study we have used a molecular approach to manipulate CREB gene expression to study its role in the regulation of neuronal cell death. To achieve this, adenoviral (Ad) vectors encoding EGFP, CREB, and a powerful CREB dominant-negative, known as A-CREB were constructed. The over-expression of CREB but not A-CREB was found to protect primary hippocampal neurons from staurosporine-induced apoptosis, glutamate induced excitotoxicity and exposure to an in vitro ischaemic stress. Hence, manipulating CREB-regulated pathways may provide a means of delaying or preventing the neuronal cell death associated with ischaemic related injury, and in neurodegenerative diseases such as Huntington's and Alzheimer's disease.

Long-term transgene expression can be mediated in the brain by adenoviral vectors when powerful neuron-specific promoters are used.

BACKGROUND: Adenoviral (Ad) vectors are one of the most widely used tools for modelling gene therapy strategies. However, they have not been used in long-term models of neurological disease, as the period of time for which they mediate strong transgene expression is limited and/or variable. In this study we investigated the longevity of transgene expression in the brain when the powerful neuron-specific Ad-synapsin (Sy)-EGFP-woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) vector cassette is used at titres that do not elicit an immune response. METHODS: Adenoviral vectors expressing enhanced green fluorescent protein (EGFP) under the control of either the hCMV, hCMV-WPRE, Sy or Sy-WPRE promoter were constructed. These vectors were injected into the dentate gyrus region of hippocampus and transgene expression and immune cell infiltration assessed by fluorescence microscopy and immunocytochemical techniques, respectively. RESULTS: The quantitative analysis of EGFP expression showed that there was no significant change in synapsin or synapsin-WPRE driven transcription 9 months after injection when compared with expression levels obtained 3 days after injection. However, when the hCMV promoter or the hCMV-WPRE promoter cassette drove transgene expression, there was a dramatic fall in expression levels and very little expression was seen 9 months post-transfection. CONCLUSIONS: This study shows that non-integrating vectors can be used to mediate powerful, long-term episomal transgene expression in neurones. This work has important implications for neuronal gene therapy and is of relevance to studies investigating memory, behaviour and neuronal gene function.

Long-term replacement of a mutated nonfunctional CNS gene: reversal of hypothalamic diabetes insipidus using an EIAV-based lentiviral vector expressing arginine vasopressin.

Due to the complexity of brain function and the difficulty in monitoring alterations in neuronal gene expression, the potential of lentiviral gene therapy vectors to treat disorders of the CNS has been difficult to fully assess. In this study, we have assessed the utility of a third-generation equine infectious anemia virus (EIAV) in the Brattleboro rat model of diabetes insipidus, in which a mutation in the arginine vasopressin (AVP) gene results in the production of nonfunctional mutant AVP precursor protein. Importantly, by using this model it is possible to monitor the success of the gene therapy treatment by noninvasive assays. Injection of an EIAV-CMV-AVP vector into the supraoptic nuclei of the hypothalamus resulted in expression of functional AVP peptide in magnocellular neurons. This was accompanied by a 100% recovery in water homeostasis as assessed by daily water intake, urine production, and urine osmolality lasting for a 1-year measurement period. These data show that a single gene defect leading to a neurological disorder can be corrected with a lentiviral-based strategy. This study highlights the potential of using viral gene therapy for the long-term treatment of disorders of the CNS.

Adenoviral-mediated, high-level, cell-specific transgene expression: a SYN1-WPRE cassette mediates increased transgene expression with no loss of neuron specificity.

Viral vectors are excellent tools for studying gene function in the brain, although a limitation has been the ability to effectively target transgene expression to specific neuronal populations. This generally cannot be overcome by the use of neuron-specific promoters, as most are too large to be used with current viral vectors and expression from these promoters is often relatively weak. We therefore developed a composite expression cassette, comprising 495 bp of the weak human SYN1 (synapsin-1) promoter and 800 bp of the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). Studies in hippocampal cultures, organotypic cultures, and in vivo showed that the 3' addition of the WPRE to the SYN1 element greatly increased enhanced green fluorescent protein expression levels with no loss of neuronal specificity. In vivo studies also showed that transgene expression was enhanced with no loss of neuronal specificity in dentate-gyrus neurons for at least 6 weeks following transfection. Therefore, unlike most powerful promoter systems, which mediate expression in neurons and glia, this SYN1-WPRE cassette can target powerful long-term transgene expression to central nervous system neurons when delivered at relatively low titers of adenovirus. Its use should therefore facilitate both gene therapy studies and investigations of neuronal gene function.