Photophysical properties of Na+ -indicator dyes suitable for quantitative two-photon fluorescence-lifetime measurements.

Two-photon microscopy (2PM) offers great potential in fluorescence imaging of intracellular Na+ dynamics of live cells. A severe drawback, however, is that quantitative ratioing of fluorescence intensities at different wavelengths [possible in one-photon imaging with the classical Na+ -indicator dye sodium-binding benzofuran isophtalate (SBFI)] is not practical in 2PM. We aimed at establishing 2PM-based time-correlated fluorescence lifetime measurements as an alternative method for quantifying Na+ dynamics. We compared the photophysical properties of the four Na+ -sensitive fluorescent indicator dyes SBFI, CoroNa Green, Sodium Green and Asante NaTRIUM Green-2 (ANG-2) in cuvette calibrations. All four dyes showed Na+ -dependent intensity changes, with ANG-2 having the most favourable properties for 2PM. All dyes but SBFI showed significant changes in their fluorescence lifetime upon Na+ binding, again with ANG-2 being the most promising dye. We found that, unfortunately, the fluorescence lifetime of ANG-2 is not only affected by Na+ but also by protons, K+ and dye impurities, rendering a quantitative description of the individual lifetime components impractical. However, a simplified calibration procedure, based on a published approach for Ca2+ imaging, allowed relating lifetimes to Na+ concentration. Using ANG-2 and the simplified calibration will allow quantitative two-photon Na+ imaging with millimolar sensitivity. LAY DESCRIPTION: Dynamic changes of ion concentrations, which play crucial roles in cellular physiology, can be monitored with appropriate fluorescent indicator dyes. For intracellular sodium ions (Na+ ), certain dyes even allow quantitative measurements with standard microscopic techniques. However, for two-photon microscopy, which allows resolving cells deep in intact tissue, imaging solutions that are fully quantitative are lacking. For the four commercially available Na+ dyes 'SBFI', 'CoroNa Green', 'Sodium Green', and 'Asante NaTRIUM Green-2' (ANG2) we analyzed whether their fluorescent lifetime (LT), i.e., the nanosecond decay of emission of photons after a pulsed excitation, could serve as a quantitative measure of intracellular Na+ . Pulsed excitation in the femtosecond range is an inherent feature of two-photon microscopy and, in combination with fast, single-photon counting microscopes, allows for easy-to-implement LT microscopy. We found that Sodium Green and ANG2 showed strong Na+ -dependent changes in the fluorescence LT, while SBFI showed no, and CoroNa Green only small changes. ANG2, as the brightest dye, was further characterized regarding effects of protons and potassium ions (K+ ), both also present in cells at significant concentrations, on the fluorescence LT. We found that the LT of ANG2 is affected in a predictable manner by Na+ , K+ , and protons. However, our data reveal that the commercial dye must also contain impurities with unexpected Na+ - and K+ -binding characteristics, rendering a quantitative description of the individual lifetime components impractical. We, therefore, adapted a simplified calibration procedure, based on a published approach for Ca2+ imaging, that allows relating the average lifetime to Na+ concentration. With this simplified calibration procedure, ANG2 is well suited for quantitative two-photon Na+ imaging with millimolar sensitivity.

Co-occurrence of two cases of progressive multifocal leukoencephalopathy in a natalizumab "infusion group".

We observed two cases of progressive multifocal leukoencephalopathy (PML) that occurred in the same "infusion group". The group consisted of four patients with relapsing-remitting multiple sclerosis (RRMS) who had been treated with natalizumab (NAT) in the same medical practice for more than four years at the same times and in the same room, raising concerns about viral transmission between members of the infusion group. DNA amplification and sequence comparison of the non-coding control region (NCCR) of JC virus (JCV) present in cerebrospinal fluid (CSF) samples from PML patients #1 and #2 revealed that the amplified JCV sequences differed from the JCV archetype. The NCRR of the viral DNA was unique to each patient, arguing against the possibility of viral transmission between patients. Statistical considerations predict that similar co-occurrences of PML are likely to happen in the future.

Peri-infarct blood-brain barrier dysfunction facilitates induction of spreading depolarization associated with epileptiform discharges.

Recent studies showed that spreading depolarizations (SDs) occurs abundantly in patients following ischemic stroke and experimental evidence suggests that SDs recruit tissue at risk into necrosis. We hypothesized that BBB opening with consequent alterations of the extracellular electrolyte composition and extravasation of albumin facilitates generation of SDs since albumin mediates an astrocyte transcriptional response with consequent disturbance of potassium and glutamate homeostasis. Here we show extravasation of Evans blue-albumin complex into the hippocampus following cortical photothrombotic stroke in the neighboring neocortex. Using extracellular field potential recordings and exposure to serum electrolytes we observed spontaneous SDs in 80% of hippocampal slices obtained from rats 24 h after cortical photothrombosis. Hippocampal exposure to albumin for 24 h through intraventricular application together with serum electrolytes lowered the threshold for the induction of SDs in most slices irrespective of the pathway of stimulation. Exposing acute slices from naive animals to albumin led also to a reduced SD threshold. In albumin-exposed slices the onset of SDs was usually associated with larger stimulus-induced accumulation of extracellular potassium, and preceded by epileptiform activity, which was also observed during the recovery phase of SDs. Application of ifenprodil (3 µM), an NMDA-receptor type 2 B antagonist, blocked stimulus dependent epileptiform discharges and generation of SDs in slices from animals treated with albumin in-vivo. We suggest that BBB opening facilitates the induction of peri-infarct SDs through impaired homeostasis of K+.