Real Time Imaging and Dynamics of Hippocampal Zn2+ under Epileptic Condition Using a Ratiometric Fluorescent Probe

Zinc, the essential trace element in human body exists either in the bound or free state, for both structural and functional roles. Insights on Zn2+ distribution and its dynamics are essential in view of the fact that Zn2+ dyshomeostasis is a risk factor for epileptic seizures, Alzheimer’s disease, depression, etc. Herein, a bipyridine bridged bispyrrole (BP) probe is used for ratiometric imaging and quantification of Zn2+ in hippocampal slices. The green fluorescence emission of BP shifts towards red in the presence of Zn2+. The probe is used to detect and quantify the exogenous and endogenous Zn2+ in glioma cells and hippocampal slices. The dynamics of chelatable zinc ions during epileptic condition is studied in the hippocampal neurons, in vitro wherein the translocation of Zn2+ from presynaptic to postsynaptic neuronal bodies is imaged and ratiometrically quantified. Raman mapping technique is used to confirm the dynamics of Zn2+ under epileptic condition. Finally, the Zn2+ distribution was imaged in vivo in epileptic rats and the total Zn2+ in rat brain was quantified. The results favour the use of BP as an excellent Zn2+ imaging probe in biological system to understand the zinc associated diseases and their management.


In Vitro Cytotoxicity Assay
The cellular cytotoxicity of BP was tested on C6 glioma cells seeded into a 96-well plate (1x10 4 cells per well). After 26 h incubation, the cells were further incubated with fresh DMEM media (100 μl per well) containing BP in different concentrations of 0.09, 0.18, 0.375, 0.75, 1.5, 3.125, 6.25, 12.5, 25 and 50 µM for 24 and 48 h, respectively. Then the cells were washed with the culture media, and 100 μl of fresh DMEM containing 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT-10 µl, 5 mgml −1 ) was added to each well followed by incubation for 4 h to allow the formation of formazan dye. The insoluble MTT formazan crystals were dissolved with DMSO (100 μl). The absorbance intensity at 570 nm was measured using a microplate reader (Synergy H1 hybrid multi-mode microplate reader, Bio-Tek). The relative cell viability (%) for each sample related to the control well was finally calculated.

Hippocampal Slice Preparation
For the preparation of hippocampal slices, standard procedure with minor modifications was adopted 1 . Adult male and female Sprague-Dawley rats weighing 250-300 g were anesthetized by isoflurane inhalation and decapitated. The brains were quickly removed and placed in ice-cold artificial cerebrospinal fluid (ACSF) containing (mM): NaCl 125, NaHCO 3 25, KCl 2.5, NaH 2 PO 4 1.25, MgCl 2 1, D-glucose 10, CaCl 2 2; saturated with 95% O 2 and 5% CO 2 at pH 7.4. The transverse slices of 400 µm thickness were prepared using Vibroslice NVSL with manually advanced tissue bath. All slice manipulation was done using phosphate free saline (PFS), because the presence of phosphate can precipitate zinc and thus limit the availability of free Zn 2+ for detection. PFS was reported to cause no effect on transmission at the Schaffer collateral CA1 synapse suggesting that the neurons have substantial reserves of phosphate 2 , 3 . The PFS composition (mM) was: NaCl 125, NaHCO 3 26, KCl 2.5, MgSO 4 1.3, D-glucose 10, CaCl 2 2; continuously bubbled with 95% O 2 and 5% CO 2 , pH 7.4 3 .

Zn 2+ detection in acute hippocampal slices
Slices were separated into three groups and incubated with (i) PFS, (ii) PFS and 50 µM of CaEDTA, which is a classic extracellular zinc chelator, for 10-15 min and (iii) PFS and 50 µM of TPEN, which is an intracellular Zn 2+ chelator, for 20 min at 37 o C. For Zn 2+ quantification, a minimum of three control and Zn 2+ chelated slices were incubated with BP and were imaged with a Rolera-XR Mono Fast 1394 Cooled digital camera (QImaging).
Relative fluorescence intensity measured from three regions of interest for each slice was processed using ImageJ and quantified by the formula, CTCF=Integrated Density -(Area of selection x Mean fluorescence of background).

Quantification of Zn 2+ in whole brain:
Adult Sprague-Dawley rats (>300 g) were euthanized and the brain was excised.
Whole brain was weighed, incubated with BP probe (80 µM) and equally cut into 22 sections.
Each section was weighed separately and imaged along with the solution having different concentration of zinc chloride incubated with BP probe in 96 well black plates. The quantification of free Zn 2+ in whole brain of adult Sprague-Dawley rat was done by calculating the fluorescence pixel intensity of a particular section of brain assuming that the fluorescence from all brain sections are same. The pixel intensity of separate wells was measured by drawing ROI using living image software of IVIS. The ratio of pixel intensity of sample (different Zn 2+ concentration) to pixel intensity of reference (BP alone) was plotted with different concentration of Zn 2+ . From the average pixel intensity of brain section, the concentration of Zn 2+ in particular section was calculated using calibration plot. By multiplying with the number of sections, the amount of Zn 2+ in whole brain was calculated.

In vivo studies: Establishing epileptic rat model
All animals used in this study were maintained under a 12/12h light/dark cycle at room temperature. Adult female Sprague-Dawley rats weighing 150 g were used as (1) control (n=3), injected with saline and (2) epilepsy model (n=6). Animals in the second group were given a single intra-peritoneal injection of pilocarpine hydrochloride (350mg/kg, Sigma), freshly dissolved in 0.9% sterile saline and control rats received an injection of equal volume of sterile saline. The injection protocols were similar to those described previously 4,5 .
To increase the survival rate, the pilocarpine treated animals received an intra-peritoneal injection of 0.5 mg/kg midazolam 1 h after the onset of status epilepticus (at stage IV and V) and when severe seizure was observed. Animals were observed under video-recording with iBall Face2Face C8 (Rev.3.0) camera for next 24 h for the signs of seizure activity and the seizures were classified according to Racine's scale (1972) and previous reports 6,7 : Normal activity of animal as stage 0, motionless stage for the first 10 min after pilocarpine injection as stage I, stiffened tail, head nodding and facial clonus as stage II, jerks in forelimb and whole body continuous clonic seizure as stage III, severe whole body continuous clonic seizure with rearing as stage IV and tonic-clonic seizure with falling or jumping due to loss of balance as stage V.

Intra-carotid artery infusion, Hyperosmolar BBB disruption and Zn 2+ detection
The protocol was adapted from previous reports 8,9 with slight modification according to the needs. Control and epileptic rats were anesthetized by intra-peritoneal injection of ketamine @ 100mg/kg BW and xylazine @ 7mg/kg BW. After aseptic precautions, a paramedian longitudinal incision was made in the jugular furrow and left common carotid artery was exposed. The carotid artery was carefully separated out and 3-0 silk suture was passed underneath so as to lift the vessel and control bleeding. A sterile 26G cannula was then inserted into the lumen common carotid artery by direct puncture, with tip of the cannula directed towards the carotid bifurcation and was secured with silk sutures. Thereafter a ligature was placed proximal to the cannula. A small volume of heparinized PFS was flushed into the cannula to prevent blood coagulation and to confirm the forward flow towards the carotid bifurcation. The hub of the cannula was filled with PFS to minimize the introduction of air and 1.5 ml of PFS was infused through it for 1 min. PFS was aerated with 95% O 2 -5% CO 2 , filtered through a 0.22 µm membrane filter (Millex-GP, no. SLGP033RS; Millipore Corp.) and warmed to 37°C prior to injection. To disturb the BBB, sterile 20% wt/vol mannitol (Mark Biosciences Ltd.) was warmed to 37°C and injected through the cannula into the external carotid artery for 30 s at a rate of 0.25 ml/kg/s. This dose has been reported to create a reversible BBB disruption without neuronal damage. Figure S1: SERS spectra of BP with or without ZnCl 2       Epileptic animals were anesthetized after 24 h and taken for intra-carotid artery cannulation and BBB disruption for imaging.