Clemastine and metformin extend the window of NMDA receptor surface expression in ageing oligodendrocyte precursor cells.

In the central nervous system, oligodendrocyte precursor cells (OPCs) proliferate and differentiate into myelinating oligodendrocytes throughout life, allowing for ongoing myelination and myelin repair. With age, differentiation efficacy decreases and myelin repair fails; therefore, recent therapeutic efforts have focused on enhancing differentiation. Many cues are thought to regulate OPC differentiation, including neuronal activity, which OPCs can sense and respond to via their voltage-gated ion channels and glutamate receptors. However, OPCs' density of voltage-gated ion channels and glutamate receptors differs with age and brain region, and correlates with their proliferation and differentiation potential, suggesting that OPCs exist in different functional cell states, and that age-associated states might underlie remyelination failure. Here, we use whole-cell patch-clamp to investigate whether clemastine and metformin, two pro-remyelination compounds, alter OPC membrane properties and promote a specific OPC state. We find that clemastine and metformin extend the window of NMDAR surface expression, promoting an NMDAR-rich OPC state. Our findings highlight a possible mechanism for the pro-remyelinating action of clemastine and metformin, and suggest that OPC states can be modulated as a strategy to promote myelin repair.

Bottom-Up Generated Height Gauges for Silicon-Based Nanometrology.

Steady progress in integrated circuit design has forced basic metrology to adopt silicon lattice parameter as a secondary realization of the SI meter that lacks convenient physical gauges for precise surface measurements at a nanoscale. To employ this fundamental shift in nanoscience and nanotechnology, we propose a set of self-organized silicon surface morphologies as a gauge for height measurements within the whole nanoscale (0.3-100 nm) range. Using 2 nm sharp atomic force microscopy (AFM) probes, we have measured the roughness of wide (up to 230 µm in diameter) singular terraces and the height of monatomic steps on the step-bunched and amphitheater-like Si(111) surfaces. For both types of self-organized surface morphology, the root-mean-square terrace roughness exceeds 70 pm but has a little effect on step height measurements having 10 pm accuracy for AFM technique in air. We implement a step-free 230-µm-wide singular terrace as a reference mirror in an optical interferometer to reduce the systematic error of height measurements from >5 nm to about 0.12 nm, which allows visualizing 136-pm-high monatomic steps on the Si(001) surface. Then, using a "pit-patterned" extremely wide terrace with dense but counted monatomic steps in a pit wall, we have optically measured mean Si(111) interplanar spacing (313.8 ± 0.4 pm) that agrees well with the most precise metrological data (313.56 pm). This opens up avenues for the creation of silicon-based height gauges using bottom-up approaches and advances optical interferometry among techniques for metrology-grade nanoscale height measurements.

Assessing the Effects of Cytoprotectants on Selective Neuronal Loss, Sensorimotor Deficit and Microglial Activation after Temporary Middle Cerebral Occlusion.

Although early reperfusion after stroke salvages the still-viable ischemic tissue, peri-infarct selective neuronal loss (SNL) can cause sensorimotor deficits (SMD). We designed a longitudinal protocol to assess the effects of cytoprotectants on SMD, microglial activation (MA) and SNL, and specifically tested whether the KCa3.1-blocker TRAM-34 would prevent SNL. Spontaneously hypertensive rats underwent 15 min middle-cerebral artery occlusion and were randomized into control or treatment group, which received TRAM-34 intraperitoneally for 4 weeks starting 12 h after reperfusion. SMD was assessed longitudinally using the sticky-label test. MA was quantified at day 14 using in vivo [11C]-PK111195 positron emission tomography (PET), and again across the same regions-of-interest template by immunofluorescence together with SNL at day 28. SMD recovered significantly faster in the treated group (p = 0.004). On PET, MA was present in 5/6 rats in each group, with no significant between-group difference. On immunofluorescence, both SNL and MA were present in 5/6 control rats and 4/6 TRAM-34 rats, with a non-significantly lower degree of MA but a significantly (p = 0.009) lower degree of SNL in the treated group. These findings document the utility of our longitudinal protocol and suggest that TRAM-34 reduces SNL and hastens behavioural recovery without marked MA blocking at the assessed time-points.

Oligodendrocyte Progenitor Cells Become Regionally Diverse and Heterogeneous with Age.

Oligodendrocyte progenitor cells (OPCs), which differentiate into myelinating oligodendrocytes during CNS development, are the main proliferative cells in the adult brain. OPCs are conventionally considered a homogeneous population, particularly with respect to their electrophysiological properties, but this has been debated. We show, by using single-cell electrophysiological recordings, that OPCs start out as a homogeneous population but become functionally heterogeneous, varying both within and between brain regions and with age. These electrophysiological changes in OPCs correlate with the differentiation potential of OPCs; thus, they may underlie the differentiational differences in OPCs between regions and, likewise, differentiation failure with age.

Stimulation and Artifact-Suppression Techniques for In Vitro High-Density Microelectrode Array Systems.

We present novel voltage stimulation buffers with controlled output current, along with recording circuits featuring adjustable high-pass cut-off filtering to perform efficient stimulation while actively suppressing stimulation artifacts in high-density microelectrode arrays. Owing to the dense packing and close proximity of the electrodes in such systems, a stimulation through one electrode can cause large electrical artifacts on neighboring electrodes that easily saturate the corresponding recording amplifiers. To suppress such artifacts, the high-pass corner frequencies of all available 2048 recording channels can be raised from several Hz to several kHz by applying a "soft-reset" or pole-shifting technique. With the implemented artifact suppression technique, the saturation time of the recording circuits, connected to electrodes in immediate vicinity to the stimulation site, could be reduced to less than 150 µs. For the stimulation buffer, we developed a circuit, which can operate in two modes: either control of only the stimulation voltage or control of current and voltage during stimulation. The voltage-only controlled mode employs a local common-mode feedback operational transconductance amplifier with a near rail-to-rail input/output range, suitable for driving high-capacitive loads. The current/voltage controlled mode is based on a positive current conveyor generating adjustable output currents, whereas its upper and lower output voltages are limited by two feedback loops. The current/voltage controlled circuit can generate stimulation pulses up to 30 µA with less than ±0.1% linearity error in the low-current mode and up to 300 µA with less than ±0.2% linearity error in the high-current mode.

Effects of hyperoxia on 18F-fluoro-misonidazole brain uptake and tissue oxygen tension following middle cerebral artery occlusion in rodents: Pilot studies.

PURPOSE: Mapping brain hypoxia is a major goal for stroke diagnosis, pathophysiology and treatment monitoring. 18F-fluoro-misonidazole (FMISO) positron emission tomography (PET) is the gold standard hypoxia imaging method. Normobaric hyperoxia (NBO) is a promising therapy in acute stroke. In this pilot study, we tested the straightforward hypothesis that NBO would markedly reduce FMISO uptake in ischemic brain in Wistar and spontaneously hypertensive rats (SHRs), two rat strains with distinct vulnerability to brain ischemia, mimicking clinical heterogeneity. METHODS: Thirteen adult male rats were randomized to distal middle cerebral artery occlusion under either 30% O2 or 100% O2. FMISO was administered intravenously and PET data acquired dynamically for 3hrs, after which magnetic resonance imaging (MRI) and tetrazolium chloride (TTC) staining were carried out to map the ischemic lesion. Both FMISO tissue uptake at 2-3hrs and FMISO kinetic rate constants, determined based on previously published kinetic modelling, were obtained for the hypoxic area. In a separate group (n = 9), tissue oxygen partial pressure (PtO2) was measured in the ischemic tissue during both control and NBO conditions. RESULTS: As expected, the FMISO PET, MRI and TTC lesion volumes were much larger in SHRs than Wistar rats in both the control and NBO conditions. NBO did not appear to substantially reduce FMISO lesion size, nor affect the FMISO kinetic rate constants in either strain. Likewise, MRI and TTC lesion volumes were unaffected. The parallel study showed the expected increases in ischemic cortex PtO2 under NBO, although these were small in some SHRs with very low baseline PtO2. CONCLUSIONS: Despite small samples, the apparent lack of marked effects of NBO on FMISO uptake suggests that in permanent ischemia the cellular mechanisms underlying FMISO trapping in hypoxic cells may be disjointed from PtO2. Better understanding of FMISO trapping processes will be important for future applications of FMISO imaging.

Normobaric hyperoxia markedly reduces brain damage and sensorimotor deficits following brief focal ischaemia.

'True' transient ischaemic attacks are characterized not only clinically, but also radiologically by a lack of corresponding changes on magnetic resonance imaging. During a transient ischaemic attack it is assumed that the affected tissue is penumbral but rescued by early spontaneous reperfusion. There is, however, evidence from rodent studies that even brief focal ischaemia not resulting in tissue infarction can cause extensive selective neuronal loss associated with long-lasting sensorimotor impairment but normal magnetic resonance imaging. Selective neuronal loss might therefore contribute to the increasingly recognized cognitive impairment occurring in patients with transient ischaemic attacks. It is therefore relevant to consider treatments to reduce brain damage occurring with transient ischaemic attacks. As penumbral neurons are threatened by markedly constrained oxygen delivery, improving the latter by increasing arterial O2 content would seem logical. Despite only small increases in arterial O2 content, normobaric oxygen therapy experimentally induces significant increases in penumbral O2 pressure and by such may maintain the penumbra alive until reperfusion. Nevertheless, the effects of normobaric oxygen therapy on infarct volume in rodent models have been conflicting, although duration of occlusion appeared an important factor. Likewise, in the single randomized trial published to date, early-administered normobaric oxygen therapy had no significant effect on clinical outcome despite reduced diffusion-weighted imaging lesion growth during therapy. Here we tested the hypothesis that normobaric oxygen therapy prevents both selective neuronal loss and sensorimotor deficits in a rodent model mimicking true transient ischaemic attack. Normobaric oxygen therapy was applied from the onset and until completion of 15 min distal middle cerebral artery occlusion in spontaneously hypertensive rats, a strain representative of the transient ischaemic attack-prone population. Whereas normoxic controls showed normal magnetic resonance imaging but extensive cortical selective neuronal loss associated with microglial activation (present both at Day 14 in vivo and at Day 28 post-mortem) and marked and long-lasting sensorimotor deficits, normobaric oxygen therapy completely prevented sensorimotor deficit (P < 0.02) and near-completely Day 28 selective neuronal loss (P < 0.005). Microglial activation was substantially reduced at Day 14 and completely prevented at Day 28 (P = 0.002). Our findings document that normobaric oxygen therapy administered during ischaemia nearly completely prevents the neuronal death, microglial inflammation and sensorimotor impairment that characterize this rodent true transient ischaemic attack model. Taken together with the available literature, normobaric oxygen therapy appears a promising therapy for short-lasting ischaemia, and is attractive clinically as it could be started at home in at-risk patients or in the ambulance in subjects suspected of transient ischaemic attack/early stroke. It may also be a straightforward adjunct to reperfusion therapies, and help prevent subtle brain damage potentially contributing to long-term cognitive and sensorimotor impairment in at-risk populations.

Neuronal activity regulates remyelination via glutamate signalling to oligodendrocyte progenitors.

Myelin regeneration can occur spontaneously in demyelinating diseases such as multiple sclerosis (MS). However, the underlying mechanisms and causes of its frequent failure remain incompletely understood. Here we show, using an in-vivo remyelination model, that demyelinated axons are electrically active and generate de novo synapses with recruited oligodendrocyte progenitor cells (OPCs), which, early after lesion induction, sense neuronal activity by expressing AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid)/kainate receptors. Blocking neuronal activity, axonal vesicular release or AMPA receptors in demyelinated lesions results in reduced remyelination. In the absence of neuronal activity there is a ∼6-fold increase in OPC number within the lesions and a reduced proportion of differentiated oligodendrocytes. These findings reveal that neuronal activity and release of glutamate instruct OPCs to differentiate into new myelinating oligodendrocytes that recover lost function. Co-localization of OPCs with the presynaptic protein VGluT2 in MS lesions implies that this mechanism may provide novel targets to therapeutically enhance remyelination.

Astrocyte response to motor neuron injury promotes structural synaptic plasticity via STAT3-regulated TSP-1 expression.

The role of remote astrocyte (AC) reaction to central or peripheral axonal insult is not clearly understood. Here we use a transgenic approach to compare the direct influence of normal with diminished AC reactivity on neuronal integrity and synapse recovery following extracranial facial nerve transection in mice. Our model allows straightforward interpretations of AC-neuron signalling by reducing confounding effects imposed by inflammatory cells. We show direct evidence that perineuronal reactive ACs play a major role in maintaining neuronal circuitry following distant axotomy. We reveal a novel function of astrocytic signal transducer and activator of transcription-3 (STAT3). STAT3 regulates perineuronal astrocytic process formation and re-expression of a synaptogenic molecule, thrombospondin-1 (TSP-1), apart from supporting neuronal integrity. We demonstrate that, through this new pathway, TSP-1 is responsible for the remote AC-mediated recovery of excitatory synapses onto axotomized motor neurons in adult mice. These data provide new targets for neuroprotective therapies via optimizing AC-driven plasticity.

A comparison of four PET tracers for brain hypoxia mapping in a rodent model of stroke.

INTRODUCTION: Severe brain hypoxia in the territory of the occluded artery is a key feature of ischemic stroke. This region can be imaged using positron emission tomography (PET) and the standard hypoxia radiotracer (18)F-fluoromisonidazole ((18)F-FMISO). However, the utility of (18)F-FMISO is limited by its slow accumulation in the lesion. Therefore, this study investigated three hypoxia-sensitive radiotracers, namely the nitroimidazole (18)F-fluoroazomycin arabinoside ((18)F-FAZA) and two (64)Cu bis(thiosemicarbazone) complexes ((64)Cu-ATSM and (64)Cu-ATSE), expected to have improved pharmacokinetic profiles relative to (18)F-FMISO, in a rodent model of ischemic stroke. METHODS: In anaesthetised Wistar rats, the distal middle cerebral artery was permanently occluded by electrocoagulation, the radiotracers administered intravenously and animals PET scanned for up to 3hours, followed by T2-weighted magnetic resonance imaging to map the infarct. RESULTS: As expected, late and prominent (18)F-FMISO retention was observed despite lower tracer delivery into the affected region. Time-activity curves revealed that both (64)Cu-ATSM and (64)Cu-ATSE showed rapid entry and efflux from the brain, but did not show significant accumulation in the lesion. (18)F-FAZA showed limited brain penetration, and accumulation in the lesion was inconsistent, low and as slow as (18)F-FMISO. CONCLUSIONS: This study suggests further development of these radiotracers as hypoxia markers for ischemic stroke may not be warranted.

Characterizing infarction and selective neuronal loss following temporary focal cerebral ischemia in the rat: a multi-modality imaging study.

BACKGROUND AND PURPOSE: Current models dictate that, depending on occurrence of early reperfusion, the ischemic penumbra either undergoes or escapes infarction (i.e., "pan-necrosis"). However, tissue outcome following temporary middle-cerebral artery occlusion (tMCAo) in rodents can also include selective neuronal loss (SNL), which even if subtle may impede functional recovery. In order to explore the pathophysiology of ischemic stroke, determine potential therapeutic targets and monitor effects of therapy, in vivo imaging surrogates of these varied histopathological outcomes applicable in the clinical setting would be useful. Although hyperintense signal on T(2)-weighted MRI in the chronic post-stroke stage is considered a reliable surrogate of tissue infarction, SNL is not associated with T(2)W abnormal signal. In the clinical setting, the neuron-specific PET ligand (11)C-flumazenil (FMZ) has been used to identify both pan-necrosis and peri-infarct SNL, but this inference has not been histopathological confirmed so far. Here we investigated the late tissue sequelae of tMCAo in the rodent using in vivo T(2)W MRI and FMZ-PET against post mortem immunohistochemistry as gold standard. METHODS: Adult spontaneously hypertensive rats (SHRs) underwent 45 min distal-clip middle-cerebral artery occlusion and, 28 days later, FMZ-PET and T(2)W-MRI, immediately followed by immunohistochemistry for neuronal loss (NeuN), activated microglia and astrocytosis. Based on standard histopathological definitions, ischemic lesions were classified into pan-necrosis, partial infarction or SNL. NeuN changes and FMZ binding across the whole hemisphere were quantified in the same set of 44 regions-of-interest according to previously validated protocols; linear regressions between these two measures were carried out both within and across subjects. RESULTS: Both cortical pan-necrosis/partial infarction and SNL were present in all rats except one, where SNL was isolated and extensive. Infarction/partial infarction, but not SNL, was associated with T(2)W hyperintense signals and cortical atrophy. In contrast, FMZ binding was decreased in all types of lesions including SNL, in proportion with NeuN staining intensity both within (p<0.05 to <0.001) and across (p<0.001) subjects, including the subject that showed pure SNL (p=0.01). CONCLUSION: This novel study revealed three main facts: i) long-term histopathological cortical changes following 45 min tMCAo in SHRs included all three of SNL, partial infarction and frank infarction; ii) T2W MRI showed conspicuous high signal lesions for complete or partial infarction, but no changes for SNL; and iii) FMZ-PET was sensitive to all three types of tMCAo-induced histopathological changes, including isolated SNL, suggesting it is a valid surrogate for the histological sequelae of focal cerebral ischemia. In addition, the finding of almost universal completed cortical infarction at 28 days differed from our previous findings at 14-day survival using the same model and rat strain, where SNL was the almost exclusive outcome, possibly representing delayed infarct maturation. Prospective studies are needed to investigate this interesting possibility.

Validation and quantification of [18F]altanserin binding in the rat brain using blood input and reference tissue modeling.

The 5-hydroxytryptamine type 2a (5-HT(2A)) selective radiotracer [(18)F]altanserin has been subjected to a quantitative micro-positron emission tomography study in Lister Hooded rats. Metabolite-corrected plasma input modeling was compared with reference tissue modeling using the cerebellum as reference tissue. [(18)F]altanserin showed sufficient brain uptake in a distribution pattern consistent with the known distribution of 5-HT(2A) receptors. Full binding saturation and displacement was documented, and no significant uptake of radioactive metabolites was detected in the brain. Blood input as well as reference tissue models were equally appropriate to describe the radiotracer kinetics. [(18)F]altanserin is suitable for quantification of 5-HT(2A) receptor availability in rats.