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Bimodal treatment for ischemic stroke and high definition whole brain microscopic imaging (BISTAR)

Project type PN-II-RU-TE-2014 (UEFISCDI), contract no 160/01.10.2015

Project director: Professor, MD, PhD Pirici Nicolae Daniel, MD, PhD, Department of Morphological Sciences, Chair of Scientific Research Methodology, UMF Craiova

Scientific Report (01.01.2016- 31.12.2017)

 Objectives of the execution phase

During this project the proposed experiments were carried out and the analysis of the data considered for publication was finalized. We have achieved the following objectives and activities:

Project management

Working meetings between partners; establishing the final work protocols, evaluating the results for publication and drawing up the final scientific report.

Initially, within these meetings, the BISTAR project objectives were discussed, and if these still constitute the most up-to-date elements, taking into account the time elapsed since the initial proposal for the competition. Based on bibliographic documentation, the team updated their EndNote reference database and the publications show that there are no reports yet of the use of the selective aquaporin inhibitor (AQP4) (commercially called TGN-020) in the treatment of ischemic stroke to address histopathology of stroke and the treatment combined with a stimulator of the endogenous glutamate capture. (Huber et al., 2012; Igarashi et al., 2011; Igarashi et al., 2013; Nakamura et al., 2011; Suzuki et al., 2013). In conclusion, the overall objectives of this project have retained their scientific priority and topicality.

Following discussions between team members it was maintained the idea of working on Spraque-Dawley rats, since the success of the operation by occlusion of the middle cerebral artery is greater than in mice, and the anatomopathological material (encephalic) can be better harnessed due to the volume and the fact that using the rat as the antigenic target in immunohistochemistry tests does not require changing well-established anti-rabbit and anti-mouse amplification systems in our laboratory (Pirici et al., 2009). It was decided to maintain the number of experimental animals, according to the proposed plan in the submitted project, for all large study groups. The dose of TGN-020 was extrapolated from the literature as 200 mg/kg dissolved in physiological saline buffer (Igarashi et al., 2011), the dose being superior to the proven effective concentrations in another model (Huber et al., 2009) or for another technique (Nakamura et al., 2011).

The project director had already contacted a manufacturer able to synthesize the inhibitor (http://www.uorsy.com), because the TGN-020 product [N- (1,3,4-thiadiazol-2-yl) pyridine-3- carboxamide] available for example at Sigma-Aldrich has a prohibitive price for the amount required in this project (over 4g), and furthermore, this neutral form may present solubility problems in physiological saline. Immediately after winning the project, the above producer was contacted and a 5g synthesis order (first trance) from a Cl salt showing an increased solubility in the physiological saline [N- (1,3,4- thiadiazol-2-yl) pyridine-3-carboxamide dihydrochloride]. The product had a high priority in the purchase list for this step and the order has already been launched.

Signing an agreement with the Ethics Committee of UMF Craiova; creating a dedicated webpage for the project.

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After the first meeting between the team members, and following the decision to maintain the number of animals and the proposed working methodology from the submitted form of the project, a request for endorsement of the project was made by the Ethics Commission of UMF Craiova. (26.11.2015). At the UMF Craiova site a dedicated page was created for the project, which will be updated periodically as it evolves (http://www.umfcv.ro/bistar).

During the final meeting of the team members (24.09.2017), the articles published during the project were reviewed, the status of the patent application was evaluated, and it was agreed that the data still available and unfilled in the publications should be further processed in the same team in order to future publications and new applications for projects on this subject.

Testing and optimization of the working techniques, the actual development of the experiments

Completing the testing and optimization of 3DISCO technique (IHC-enabled three-dimensional imaging of solvent-cleared organs).

We first continued testings initiated in the first phase of the project, where we compared practically on rat brain tissue, the two most widely used methods of tissue clarification: iDisco and 3Disco techniques (Erturk et al., 2012; Renier et al. 2014). Given that 3Disco technique is faster and does not lead to excessive volume changes in the tissue undergoing treatment, we choose it in the end. Finally, fully optimized protocol included a process for permeabilization of the rat brain in detergent, of immunostaining with primary antibodies (we tested Aquaporin 4 antibody, Santa Cruz, sc-20812, 1:1000; Solanum tuberosa – biotinylated, Vector Laboratories, B-1165, 1:1000; GFAP, Dako, Z0334, 1:20.000), then with secondary antibodies (we tested anti-rabbit Alexa 555, 596, and streptavidin-Alexa 596, Molecular Probes, 1:1000), and then 3DISCO clarification and immersion in the immersion solution (dibenzyl ether). Final tests were done both at UMF Craiova by assessing sliced tissue between the glass slide and the coverslip, or immersed in dibenzyl ether in a chamber made of dental acrylic resin (Figure 1); and using a light-sheet microscope, during the mobility that has been supported by this project in the Department of Anatomy at the University of Southampton, led by Prof Roxana Carare, MD, PhD (Figure 1)

Because it represented the most important technical challenge of tissue processing within this project, we evaluated the two most widely used methods of tissue clarification: iDisco and 3Disco techniques (Erturk et al., 2012; Renier et al. 2014), and a preliminary practical comparison of the iDisco and 3Disco methods (Erturk et al., 2012; Renier et al., 2014), along with immunofluorescence and microscopic imaging tests [we mention that besides the Nikon 90i (Nikon, Apidrag, Romania), which is in the endowment of UMF Craiova, a two-photon laser microscope was recently put into operation which allows the evaluation of immunomarked targets on the clarified tissues]. In order to perform imaging itself within the whole brain through the lightsheet microscopy technique, Dr. Roxana Octavia Carare (University of Southampton, UK) has been in constant contact with both groups. We mention that the chemicals required for these clarification protocols also had a high priority in the procurement list for this phase.

We first continued testings initiated in the first phase of the project, where we compared practically on rat brain tissue, the two most widely used methods of tissue clarification: iDisco and 3Disco techniques (Erturk et al., 2012; Renier et al. 2014). Given that 3Disco technique is faster and does not lead to excessive volume changes in the tissue undergoing treatment, we choose it in the end. Finally, fully optimized protocol included a process for permeabilization of the rat brain in detergent, of immunostaining with primary antibodies (we tested Aquaporin 4 antibody, Santa Cruz, sc-20812, 1:1000; Solanum tuberosa – biotinylated, Vector Laboratories, B-1165, 1:1000; GFAP, Dako, Z0334, 1:20.000), then with secondary antibodies (we tested anti-rabbit Alexa 555, 596, and streptavidin-Alexa 596, Molecular Probes, 1:1000), and then 3DISCO clarification and immersion in the immersion solution (dibenzyl ether). Final tests were done both at UMF Craiova by assessing sliced tissue between the glass slide and the coverslip, or immersed in

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dibenzyl ether in a chamber made of dental acrylic resin; and using a light-sheet microscope, during the mobility that has been supported by this project in the Department of Anatomy at the University of Southampton, led by Prof Roxana Carare, MD, PhD.

When traveling at the University of Southampton, Dr. Pirici Daniel gave a presentation regarding the scientific interest of the group from Craiova in the department of Southampton, and later we scanned control brains processed in Craiova yielding high resolution 3D renderings, the total volume of data collected exceeding 100GB. This thus confirmed that the clarification was made even in the midle of the tissue and diffusion of primary and secondary antibodies was also complete.

Back in Craiova, data visualization was not possible until we purchase a powerful computer with sufficient processing power to open these large stacks of seriate images. For this we used a freeware software package dedicated to large volumes Vaa3d (Mac OS X) http://home.penglab.com/proj/vaa3d/Vaa3D/, that allowed rendering volumetric data with the ability to perform virtual tissue sections and to measure the intensity of immunofluorescent markers (Figure 1).

Figure 1. Example of three-dimensional renderings of AQP4 views on a rat cerebral microvasculature (Alexa Fluor 596).

Moreover, during the evaluation process we used new physicochemical methods such as forced diffusion of solutions within the studied tissue, thus increasing the stability of the immunolabellings, the volume of samples processed, and reducing processing times. It was also proposed to carry out an automated tissue processing protocol, thus increasing the repeatability and stability of the protocol, and after these testings, on the 29.09.2016 a proposal has been filed to the State Office for Inventions and Trademarks (OSIM) for a patent application (number A/00686). On 30.05.2017, the summary of the patent application was published in the Official Industrial Property Bulletin (BOPI), with the end of 2017 being granted the patent itself.

We intend to achieve precise correlations between data volume as obtained by immunolabellings / clarification and the actual ex-vivo volumes of the brains treated or untreated after middle cerebral artery occlusion (MCAO) and the onset of ischemic stroke. Thus we aquired a 3D scanner (Einscan S, http://www.einscan.com) able to scan real world objects in a digital format, with a resolution of less than 0.1mm. Scanning process is extremely fast, so it can scan the tissue immediately after removal from the body without delaying the fixation in neutral buffered formalin, and it will be essential for subsequent volumetric correlations. From our knowledge, the proposal of using a 3D scanner in neuropathology on an animal model is the first of this kind (Figure 2). For three dimensional rendering, virtual sectioning and quantitative analysis (volumetric) of the data we used the freeware package Autodesk Meshmixer (Mac OS X, http://www.meshmixer.com).

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Figure 2. Examples of 3D scan of a rat brain (left) with final volumetric measurement (right).).

Volumetric analysis was performed on the brains of animals treated with the AQP4 inhibitor (or untreated) after induction of stroke, and at 3 days of survival there were significant volume reductions of both the infarct area and the ipsilateral or contralateral hemisphere in treated animals (Figure 3) (Popescu et al., 2017).

Figure 3. The infarct volume of contralateral and ipsilateral hemispheres are reduced after treatment with TGN-020, at 3 days after the ischemic event.

Using the 3D scanner we then showed that the volume of the entire previous brain was reduced in the treated animals compared to the control animals (Figure 4) (Popescu et al., 2017).

Figure 4. Total brain volume is reduced after treatment with TGN-020, at 3 days after the ischemic event.

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During the project, a 3D UP BOX plus (Tiertime Corp.) 3D printer was purchased, which together with a free 3D drawing package (Autodesk Tinkercad, https://www.tinkercad.com) proved to be a real help in designing and making viewing chambers for clarified tissue fragments (0.1mm print resolution). The viewing chamber, designed with variable heights (any value between 3 mm and 1 cm), is made of solvent-resistant material used to clarify the organ (polylactic acid, PLA), and allows the specimen to be viewed in the final immersion solution directly on a fluorescence microscope or on a microscope in the system 2 photons from the endwoment of UMF Craiova biobas (Figure 5).

Besides the fact that PLA 3D printing is the only way to make these incubation rooms in their own mode, depending on the experiment, the acquisition of the 3D scanning/printing system also allows for the realization of didactic models: the picture below is illustrated the result of scanning a mouse skull, and of 3D printing of a high-resolution model that was 8 times enlarged (Figure 5).

Figure 5. The UP BOX 3D printer, along with various PLA filament designs, teaching models and viewing camera for optical microscopy

Immunohistochemistry testing and optimization procedures

We have developed all working processes, optimizing every step, from taking fresh tissue and processing it for paraffin inclusion and sectioning, optimizing primary and secondary antibody dilutions. We decided that all immunodetection techniques would rely on the use of an anti-rabbit or mouse peroxidase-conjugated and adsorbed to rat immunoglobulins (Nikirei Histofine polymer system, http://www.nichirei.co.jp/bio/englis), in order to ensure the maximum adsorption specificity of the immunostaining.

In order to visualize the vascular endothelial cells we have also optimized a lectin (a plant antibody capable of detecting carbohydrates residues of the endothelial cells (Solanum tuberosa - biotinilated, Vector Laboratories, B-1165). This lectin was then used in both studies of immunohistochemistry on fixed tissue, and organ clearing.

Neuropathological and morphometric analysis of changes in the haemato-encephalic barrier is one of the objectives of this study, and along with experience in immunohistochemical work on rat tissue (including the use of the above mentioned lectin), this expertise allowed our group to collaborate with a group from the University of Magdeburg (Germany), focused on changes in cerebral microvasculature of Alzheimer's disease (Held et al., 2017; Pirici et al., 2017), and with an experimental neuropathology group from University of Antwerp, Belgium, focusing on the study of neuroinflammation in stroke.

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In order to quantify astrocytes branching (astrocyte is the main buffer of water in the CNS), we intend to use an algorithm to analyze 3D fractal morphology [Fractal Analysis System version 3.4.7, developed by Hiroyuki Sasaki, PhD, National Agriculture and Food Research Organization (NARO), Japan], and improvement of our theoretical and practical experience allowed meantime the writing and publication of a book chapter invited by Springer Science.

Establishing animals’ groups.

Optimizing surgical techniques and establishing the groups of animals were the main targets at this stage of the project. After studying the literature, and the fact that the AQP4 inhibitor treatment targets the over-acute and acute phases of the stroke, it was decided for histological and behavioral assessments at 3 and 7 days post stroke. (Figure 6).

Figure 6. The lots of rats needed in this project.

We further optimized the administration protocol of TGN-020 intraperitoneally at a concentration of 100 mg/kg at 15 minutes after induction of stroke, the key difference from the only existing published study being administration after and not before stroke, this being the only a therapeutically feasible option to be considered (Igarashi et al., 2011).

Chosen GLT-1 activator (ceftriaxone, 200 mg/kg, intraperitoneally) was administered daily within the first 7 days after OACM (first administration at one hour after OACM), recent studies showing stable GLT-1 growth for 3 months after this administration (Rothstein et al., 2005).

We adapted in the same time the surgical technique of inducing the ischemic stroke. Classical model uses a temporary occlusion of ACM by clamping or using a monofilament, overlapped with the common carotid artery occlusion, followed by reperfusion of all three arteries. Not only that reperfusion does not occur in most cases of ischemic stroke in humans (where besides a thrombolytic therapy instituted quickly, an embolus cannot be revascularized efficiently spontaneously), and restoring an increased blood flow in an ischemic area will induce endothelial oxidative damage with subsequent hemorrhagic transformation. In these conditions, we implemented a simplified technique and much closer to the pathophysiology of ischemic stroke in humans, namely MCA occlusion by electrocauterisation only of the MCA under the microscope with a digital soldering stations and a tip of 0.2mm (Davis et al., 2013; Manley et al., 2000). To ensure the high specificity of this technique, ACM was lifted from the

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leptomeninges using a micromanipulator, and then electrocauterised in 2 points with the heated tip, thus ensuring a safe thrombosis of the vessel, with the lack of any injuries to the leptomeninges.

Further histopathological analysis confirmed that inducing ischemic stroke by this technique will be lead to minimal hemorrhagic transformation in the temporal cortex, without inclusion of the structures beneath the corpus callosum (thalamus, striatum) (Figure 7).

Figure 7. Histopathological confirmation of cortical ischemic stroke. The image is exemplified by a rat that survives for 7 days, and necrosis liquefaction occurs in the affected parenchyma

Initiation of the integrative pathological analysis

Histopathological and behavior analysis after the treatment TGN-020 showed, first of all, no major histological changes in the brain, kidney, retina, intestine, and the same pattern of AQP4 immunoexpression, without changes in behavior of the treated animals compared to untreated ones. In the same train of ideas, there are studies in the literature showing lack of histopathological changes in animal models following administration of higher doses of TGN-020, or even the use of this blocker with imaging purpose in humans, which requires passing rigorous toxicity tests (Huber et al., 2012; Suzuki et al., 2013). However, the present study is the first to evaluate AQP4 expression in the tissue. (Figure 8).

Figure 8. Lack of AQP4 expression changes after a single dose, exemplified here in an animal with a 3-day survival.

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Motility assessment by rotarod test in the animals treated with TGN-020 compared to untreated showed larger scores for animals treated compared with the untreated animals, both at 3 and 7 days after stroke. Statistically significant differences were recorded for investigations at 6 rpm to 7 days (p <0.05) of survival, proving that the initial administration is reflected in the further evolution of the animals (Figure 9).

Figura 9 – Evolution of the rotarod test score in animals treated/untreated with TGN-020 (data are represented as mean ± SE).

For animals treated with Ceftriaxone or Ceftriaxone + TGN-020, an improvement in motor performance was observed 6 days after stroke, with significant differences for the double treated group at only 3 rpm (Figure 10).

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Figure 10 – Evolution of the rotarod test score in animals treated/untreated with TGN-020 (data are represented as mean ± SE).

From histopatologic point of view, we firstly aimed to confirm decrease of edema in the treated animals, and for this purpose we compared vascular densities based on immunolabeling for laminin. Treated animals showed a peak of vascular density at 3 days (reflecting the effect of therapy) for all areas studied (scar glial, cortex perilesional peri-scar glial and the cortex of the contralateral hemisphere), with significantly higher values compared with untreated animals (p <0.05) (Figure 11). At 7 days untreated animals still showed edema in all studied areas, while rats treated with TGN-020 showed similar values (statistically no significant differences) of normal vascular densities recorded in sham animals without inducing a stroke.

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Figuea 11 – Evolution of the rotarod test score in animals treated/untreated with TGN-020 (data are represented as mean ± SE).

We followed also peri-lesional glial scar density by calculating the area of the signal given by the astrocytes immunoreactivity for GFAP. The study confirmed that although the reaction of glial increased overall from 3 to 7 days (as expected, given the organization of the injury in time through a glial scar with increasing density), the density of astrocytes in the scar it was lower for TGN-020 treated animals (p <0.01) (Figure 12). In areas located perilesional (except the scar itself), the treatment effect was more efficient at 3 days (p <0.05), without significant differences in the contralateral cortex.

Figura 12 – Glial reaction density is reduced in animals treated with TGN-020 comparative to untreated animals (data are represented as mean ± SE).

Moreover, astrocytes morphology seems to be differed also in treated animals, gemistocytic phenotype being elongated compared to the astrocytes in untreated rats (Figure 13)

In order to quantify subtle differences that appeared in astrocytes morphology, we used a 3D fractal analysis algorithm which analyzed an image stack of binarized representative serial optical sections captured in the full thickness of the tissue. This analysis revealed already lower values in astrocytes taken from glial scar of the treated animals compared to untreated animals, suggesting a simpler morphology (shorter and less branched processes) of these cells, compatible with water accumulation inside them and swelling of their extensions. Both classes of astrocytes showed higher values of fractal dimension (FD) when compared with the rare astrocytes in the brains of rats without MCAO (Figure 13)

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Figure 13 – Different phenotype of the glial reaction around the area of necrosis in the case of untreated animals as compared with treated ones. The images represent XYZ projections on image stacks processed

by deconvolution, and the values represent FD of the cellular silhouettes

To evaluate the involvement of the TGN-020 treatment in cells survival, we counted immunopositive cells for the caspase-3 pro-apoptotic marker in the perilesional area using a multispectral acquisition camera (Nuance FX, Perkin Elmer), capable of select and separate wavelengths of transmitted light (Figure 14). Using spectral separation, we were thus able to quantify only the cells that expressed caspase-3 in the nuclei, cytoplasmic expression being not a sign of entering into the execution phase of apoptosis.

Figure 14 – Quantification of a positive cell for caspase 3 is done only based on the nuclear signal (green arrow), multispectral separation providing individual analysis of hemtoxylin and immunohistochemical

signal (DAB).

Caspase-3 positive cells showed lower levels in perilesional areas in treated animals compared to untreated ones, emphasizing the clear role of TGN-020 in cells survival after stroke (P <0.01) (Figure 15).

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Figure 15– Density profile of pro-apoptotic cells (positive for caspase 3) is larger in untreated

animals compared with those injected with TGN-020 (data are represented as mean ± SE).

Immunohistochemistry for rat endogenous albumin used as a control for edema and elevated permeability of blood-brain barrier showed a pronounced diffusion in the areas of necrosis, and in perilesional areas the number of albumin-positive cells was higher in untreated animals compared with treated ones (ongoing statistical analysis) (Figure 16).

Figure 16– Perilesional infiltration with endogenous albumin, glial reaction and AQP4 expression are lower in treated animals compared to untreated, both at 3 and 7 days of survival.

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Perhaps the most important observation of the study, was that albumin was not only present in neuropil and cell membranes, but also in the thickness and around the vascular basement membranes and this occurred both in arterioles and capillaries (Figure 17). The phenomenon seems to have more emphasis in the TGN-020 treated animals brains, the storage of albumin was more intense and in the vessel walls of treated animals. In addition, in the arterioles, albumin appeared to be locate also to the basement membrane that surrounds the smooth muscle cells (Figure 17).

Figure 17 – Perilesional infiltration with endogenous albumin in the basal membranes of arterioles with wall positive for smooth muscle actin.

We quantified further the thickness of the vascular basement membranes by direct measurement of the thickness of the immunolabeling for albumin/laminin, and preliminary results showed not only a prevalence of albumin in the thickness of the vascular walls in animals treated with TGN-020, but also that thickened membranes basement were present in all the areas examined in the animals treated with TGN-020 (Figure 18).

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Figure 18 –Evaluation of basal vascular membrane thicknesses in treated / untreated animals 3 days after stroke (data is represented as mean ± SE).

This result coincides with a functional theoretical prediction recently proposed in the literature, based on identifying a distinct lamination to the perivascular basement membranes (Morris et al., 2016) and are being evaluated in the Journal Neurobiology of Disease. Based on experience gained in vascular density and collagen IV analysis, we also participated in clarifying the association of vascular functional lesions (translated by increased permeability for serum proteins) with the initial deposition of Aβ in the brains of a hypertensive rat animal model (Held et al. et al., 2017, Pirici et al., 2017).

Quantitative analysis

According to the initial proposal, in order to quantify the AQP4 expression in the treated/ untreated brains, we made sections of 15μm, from which we separated by laser dissection (using the Olimpus system in the endowment of the Department of Morphology of UMF Craiova, MMI GmbH, Eching, Germany), the infarct area, the rest of the ipsilateral hemisphere, and the contralateral hemisphere. The sections were then homogenized in a free RNA medium and frozen at -30 ° C for westerblot quantitative analysis.

Starting with this project, our department has benefited from the acquisition of a Nuance Fx multispectral camera (Perkin Elmer, Hopkinton, MA, USA) capables of separating spectral signals into microscopic light transmission and fluorescence in the 400-700nm band in steps 10nm on microscopic slides. This device represents the first certified system noticed in literature (Fiore et al., 2012) (for research and diagnosis purposes) for quantitative measurements directly on the slide, the advantage over tissue homogenization (Western blotting and Elisa techniques) is the preservation of histological location data of the analyzed signal.

The Nuance Fx camera and software package were tested for the first time at UMF Craiova in the present project, resulting in a quantification of the area and signal intensity (integrated optical density) of AQP4 in perilesional area of TGN-020 treated/untreated animals, the level of caspase expression 3 in these brains, and a measuring of the the density of nerve endings of the enteric nervous system in digestive tumors (intra-institutional collaboration activity (Ciurea et al., 2017)). Analysis of the AQP4 expression level in TGN-020 treated/untreated animals recorded, as expected, insignificant differences between the AVC and sham model, confirming that the inhibitor does not reduce its expression but modulate its activity (Figure 19).

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Figure 19 – AQP4 levels shows little increase in animals with stroke compared to sham animals

In addition, UMF Craiova has begun the acquisition of a fully automated WES (Protein Simple, California, USA) capillary westernblot system, unique in Romania, which allows electrophoresis, immunomotorization and comparison of 1-25 samples simultaneously for antigenic targets with molecular weights between 2-440 kDa and pg level sensitivity, completely independent of the user, the total working time being only 3 hours. Our frozen samples will be the first tests of this device, and the first PhD student of the project manager will handle these optimizations.

After initiating the integration of these data, we confirmed for the first time in literature that the AQP4 TGN-020 inhibitor reduces cerebral edema, and we showed that it decreases glial reaction and reduces apoptosis in the brains of animals treated with a single dose of the inhibitor, administrated at 15 minutes after inducing the ischemic stroke

A very interesting incidental observation, was that albumin seems stuck in the thickness of basement membranes of the vessels of treated animals, resulting in their increased thickness, a phenomenon that appears more intense compared to untreated animals (where this retention can be attributed on the inability of the ischemic tissue to adjust the balance of total water).

TGN-020 treatment apparently works by blocking the passage of interstitial fluid from the neuropil in the perivascular space, and implications of such a modulation could be used as a therapeutic target in stroke, but also in other pathologies. The association of Ceftriaxone with TGN-020 leads to an improvement in the motor clinical evolution of animals 6 days after stroke, but without volumetric changes in the area of necrosis or telencephalic volume.

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Acquisitions of equipment and supplies

-Fluorescence LED source for the Nikon 90i microscope from the Research Center for Microscopic, Morphology and Immunology (UMF Craiova). The recent emergence of these universal illumination modules to replace classic fluorescence lamps brings countless advantages (lifetime of 25,000h, minimal heat dissipation, constant intensity, can be stopped/started instantly, light intensity control 0-100% in steps of 1%, do not require additional motorization in a microscope, etc.). The source will ensure the continuity of this microscope and its utility in the microscopic imaging of the project (meanwhile the same microscope was equipped with the first multi-spectral acquisition room in the country (Nuance FX, Perkin Elmer, West Midlands - UK)], making it an essential element within any chemical/fluorescence quantitative immunoassay on fixed tissue and not only (Fiore et al., 2012).

- motorized stage for the Nikon 90i microscope from the Research Center for Microscopic Morphology and Immunology (UMF Craiova) and upgrading of the control software and image analysis, so we can scan entire slides in high resolutions in both transmitted light and in reflected light (fluorescence). We will thus be able to realize the evaluation of cerebral edema and the reporting of the area changes on hemisphere lesion compared to the contralateral hemispheres. Before the upgrade, this work was achieved by merging picture with picture manually, the method requiring long times and without being able to correct the problem of marginal illumination. Currently this is the only microscope in UMF Craiova able to scan entire sections, and it can be the starting point and support for other projects

-three-dimensional white light scanner EinScan S. During the documentation phase of the project we identified freeware software versions compatible with files created by this tool, software that allows the sectioning in reconstructed material, together with surface and volume measurements. Its role will be to measure more precisely the brain volume sampled immediately, to assess changes in volume before and after applying the techniques of organ clarification. This is the only accessible scanner on the market that allows color scanning.

-The UP BOX Plus 3D printer that allows the printing of solid objects with a maximum size of 255x205x205cm with ABS or PLA filament at a resolution of 0.1mm. This printer is one of the most automated products on the market today with a closed enclosure, heated printing plate, automatic calibration and wi-fi connectivity. The device has already been successfully used in the manufacture of microscopic viewing enclosures for clarified parts and for didactic anatomical models for students interested in microsurgery on the animal model used in this study

-a portable computer for image analysis MacBook Pro (16 GB RAM, Intel Core i7, Intel Iris Graphics 6100, 1TB SSD), which is currently the only system available to open and analyze the data volume generated in the examination in light sheet microscopy, taken at the University of Southampton. All software for the analysis of images in this project are running under MacOS, and were already identified in the preliminary stage of this acquisition;

- ultraportable computer for documentation and presentation Dell Inspiron 5378, with all necessary facilities for data visualization and statistical analysis, bibliographic documentation, and dedicated presenter function (folding touch screen and "screen mirror-ing");

-temperature and humidity sensor with GSM alarm for the antibodies refrigerator. Being a GSM terminal it is independent from the local internet network;

-chemicals and consumables necessary for immunohistochemistry protocols and care of the animals (primary antibodies, secondary animals, Proxxon mini-drills for cutting the calvaria during the MCAO, other consumables);

-electronic soldering station with digital temperature controlled thermostat ST-50D Toolcraft that ensures punctate electrocauterisation (with tips of 0.2mm) of the MCA;

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-voltage sources (Ni-Mh batteries, battery charger) necessary to custom build mechanical lab stirrers that can be kept in incubators and refrigerators without an external power supply at 220V. Clarification organ protocols were improved and simplified by devising of these instruments;

-software licenses (X7 EndNote software for references management; BitDefender antivirus, Microsoft Office MacOS) necessary for the proper organization and protection of the data and results. Rezults

At this stage of the project the following results were obtained:

- Establishing and confirming final work protocols. - Obtaining the agreement of the Ethics Committee of UMF Craiova. - Creating and updating a dedicated webpage for the project (http://www.umfcv.ro/bistar) - Optimizing the immunolabeling protocol and organ clarification and all immunohistochemistry

protocols and image analysis required for this project - Setting up all the lots of studied animals with motor tests, tissue processing and archiving of the

organs in paraffin blocks. Any histopathological and molecular analysis is possible from these archived fixed blocks and processed in a controlled and constantly optimized protocol.

- Filing a patent application at OSIM in terms of developing an automated tool for clarifying organ Pirici Ionica, Pirici Nicolae Daniel, Muresanu Fior Dafin, Mogoanta Laurentiu, Margaritescu Claudiu, Balseanu Tudor Adrian, Bogdan Catalin. Automated device for clarification process and immunolabeling in block of the anatomical and pathological specimens. OSIM patent application registered under number A / 00686 of 09/29/2016.

- Publication of four articles and a book chapter: Held F, Morris AW, Pirici D, Niklass S, Sharp MM, Garz C, Assmann A, Heinze HJ, Schreiber F, Carare RO, Schreiber S. Vascular basement membrane alterations and β-amyloid accumulations in an animal model of cerebral small vessel disease. Clin Sci (Lond). 2017 Mar 27, doi: 10.1042/CS20170004. [Epub ahead of print]

Popescu ES, Pirici I, Ciurea RN, Bălşeanu TA, Cătălin B, Mărgăritescu C, Mogoantă L, Hostiuc S, Pirici D. Three-dimensional organ scanning reveals brain edema reduction in a rat model of stroke treated with an aquaporin 4 inhibitor. Rom J Morphol Embryol. 2017;58(1):59-66.

Ciurea RN, Rogoveanu I, Pirici D, Târtea GC, Streba CT, Florescu C, Cătălin B, Puiu I, Târtea EA, Vere CC. B2 adrenergic receptors and morphological changes of the enteric nervous system in colorectal adenocarcinoma. World J Gastroenterol. 2017 Feb 21;23(7):1250-1261.

Pirici D, Stanaszek L, Garz C, Niklass S, Heinze HJ, Kalinski T, Attems J, Schreiber S. Common impact of chronic kidney disease and brain microhemorrhages on cerebral Aβ pathology in SHRSP. Brain Pathol. 2016 Apr 8. doi: 10.1111/bpa.12384. [Epub ahead of print]

Pirici D., Mogoanta L., Ion D.A., Kumar-Singh S. Capitol: Fractal Analysis in Neurodegenerative Diseases. Carte: The Fractal Geometry of the Brain, Editor: Antonio di leva (Springer Science), New York, 2016; 9 pagini, ISBN 978-1-4939-3995-4

- Submitting an article for evaluation by the journals "Neurobiology of Disease" and "Stroke": Jan Boddaert, Kenny Bielen, Bart 'S Jongers, Tom Raju, Laetitia Yperzeele, Patrick Cras, Daniel Pirici, and Samir Kumar-Singh. CD8 signaling in microglia/macrophages involved in brain M1 polarization after cerebral ischemia in a rat stroke model

Pirici I, Balsanu TA, Margaritescu C, Divan T, Vitalie V, Pirici D, Mogoanta L, Carare RO, Muresanu DF. Short-term inhibition of aquaporin-4 improves the outcome of ischaemic stroke and modulates brain paravascular drainage pathways. Submitted to Neurobiology of Disese

- Six presentations at symposiums and congresses: Pirici D., Pirici I., Balsanu T.A., Bogdan C., Tudorica V., Vacaras V., Muresanu D. Inhibiting aquaporin 4 water transporter improves the outcome of ischemic stroke and inhibits perivascular

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flow through the basement membranes. The 3rd Congress of the European-Academy-of-Neurology, June 24-26, Amsterdam, The Nederlands, published in European Journal of Neurology, 2017; 24 (S1):618, PR3016

Pirici D. (invited speaker), Muresanu D.F., Pirici I., Tudorica V., Balsanu A., Bogdan C. Modulation of water buffering as a treatment option for oedema in ischemic stroke. The 6th Teaching Course on Neurorehabilitation, RoNeuro Brain Days, June 1-3, 2016, Cluj-Napoca

Balsanu A., Carare R., Pirici D., Pirici I., Bogdan C., Mogoanta L., Margaritescu C. Practical application and optimisation of whole-organ clearing for light-sheet microscopy. 14th National Symposium of Microscopic Morphology with International Participation, May 4-6, 2016, Tirgu Mures, published in Acta Medica Marisiensis, 2016; 62(3):44

Pirici D,. Balsanu A., Bogdan C., Pirici I., Margaritescu C., Mogoanta L. Modulation of water buffering in the brain as a treatment option for oedema in ischemic stroke. 14th National Symposium of Microscopic Morphology with International Participation, May 4-6, 2016, Tirgu Mures, published in Acta Medica Marisiensis, 2016; 62(3):28

Balsanu A., Pirici D,. Bogdan C., Pirici I., Margaritescu C., Mogoanta L. Aquaporin water diffusion control in acute ischemic stroke. The 7th Conference of the National Neuroscience Society of Romania (SNN), November 24-26, Bucharest, published in Fiziologia, 2016; suppl.1: 16

Margaritescu C., Bogdan C., Pirici I., Balseanu A., Mogoanta L., Ofiteru AM., Pirici D. Evaluarea volumului cerebral dupa tratamentul cu un inhibitor de aquaporina 4 pe un model animal de accident vascular cerebral. Al XV-lea simpozion national de morfologie microscopica cu participare internationala, Mai 24-27, 2017, Baile Felix-Oradea.

In conclusion, this project has provided not only an opportunity to build collaborations with

prestigious neuropathology centers from Europe, to improve our material base with reagents and state-of-the-art equipment (scanner and 3D printer, automation of the research microscope within of our department at UMF Craiova), but especially gave us the opportunity to offer a framework of scientific activity for new PhD students and young researchers from the preclinical field at UMF Craiova.

24.09.2017 Project director, Professor, MD, PhD Pirici Nicolae-Daniel

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Reference List 

 

Ciurea, R.N., Rogoveanu,  I., Pirici, D., Tartea, G.C., Streba, C.T., Florescu, C., Catalin, B., Puiu,  I., Tartea, E.A., and Vere, C.C.  (2017). B2 adrenergic  receptors and morphological changes of  the enteric nervous system in colorectal adenocarcinoma. World J Gastroenterol 23, 1250‐1261. 

Davis, M.F.,  Lay,  C.,  and  Frostig,  R.D.  (2013).  Permanent  cerebral  vessel  occlusion  via  double ligature and transection. J Vis Exp. 

Erturk,  A.,  Becker,  K.,  Jahrling, N., Mauch,  C.P., Hojer,  C.D.,  Egen,  J.G., Hellal,  F.,  Bradke,  F., Sheng, M., and Dodt, H.U.  (2012). Three‐dimensional  imaging of solvent‐cleared organs using 3DISCO. Nat Protoc 7, 1983‐1995. 

Fiore, C., Bailey, D., Conlon, N., Wu, X., Martin, N., Fiorentino, M., Finn, S., Fall, K., Andersson, S.O.,  Andren, O.,  et  al.  (2012).  Utility  of multispectral  imaging  in  automated  quantitative  scoring  of immunohistochemistry. J Clin Pathol 65, 496‐502. 

Held,  F., Morris, A.W.,  Pirici, D., Niklass,  S.,  Sharp, M.M., Garz,  C., Assmann, A., Heinze, H.J., Schreiber,  F.,  Carare,  R.O.,  et  al.  (2017). Vascular  basement membrane  alterations  and  beta‐amyloid accumulations in an animal model of cerebral small vessel disease. Clinical science. 

Huber, V.J., Tsujita, M., and Nakada, T.  (2009).  Identification of aquaporin 4  inhibitors using  in vitro and in silico methods. BioorgMedChem 17, 411‐417. 

Huber,  V.J.,  Tsujita,  M.,  and  Nakada,  T.  (2012).  Aquaporins  in  drug  discovery  and pharmacotherapy. Mol Aspects Med 33, 691‐703. 

Igarashi, H., Huber, V.J., Tsujita, M., and Nakada, T. (2011). Pretreatment with a novel aquaporin 4 inhibitor, TGN‐020, significantly reduces ischemic cerebral edema. NeurolSci 32, 113‐116. 

Igarashi, H., Tsujita, M., Suzuki, Y., Kwee,  I.L., and Nakada, T.  (2013).  Inhibition of aquaporin‐4 significantly increases regional cerebral blood flow. Neuroreport 24, 324‐328. 

Manley, G.T., Fujimura, M., Ma, T., Noshita, N., Filiz, F., Bollen, A.W., Chan, P., and Verkman, A.S. (2000). Aquaporin‐4 deletion  in mice reduces brain edema after acute water  intoxication and  ischemic stroke. Nat Med 6, 159‐163. 

Morris, A.W., Sharp, M.M., Albargothy, N.J., Fernandes, R., Hawkes, C.A., Verma, A., Weller, R.O., and Carare, R.O.  (2016). Vascular basement membranes as pathways  for  the passage of  fluid  into and out of the brain. Acta Neuropathol 131, 725‐736. 

Nakamura,  Y.,  Suzuki,  Y.,  Tsujita,  M.,  Huber,  V.J.,  Yamada,  K.,  and  Nakada,  T.  (2011). Development of a Novel Ligand,  [C]TGN‐020,  for Aquaporin 4 Positron Emission Tomography  Imaging. ACS ChemNeurosci 2, 568‐571. 

Pirici, D., Mogoanta, L., Kumar‐Singh, S., Pirici, I., Margaritescu, C., Simionescu, C., and Stanescu, R. (2009). Antibody elution method for multiple  immunohistochemistry on primary antibodies raised  in the same species and of the same subtype. JHistochemCytochem 57, 567‐575. 

Pirici, D., Stanaszek, L., Garz, C., Niklass, S., Heinze, H.J., Kalinski, T., Attems, J., and Schreiber, S. (2017).  Common  Impact  of  Chronic  Kidney  Disease  and  Brain Microhemorrhages  on  Cerebral  Abeta Pathology in SHRSP. Brain Pathol 27, 169‐180. 

Popescu, E.S., Pirici,  I., Ciurea, R.N., Balseanu, T.A., Catalin, B., Margaritescu, C., Mogoanta, L., Hostiuc, S., and Pirici, D.  (2017). Three‐dimensional organ scanning reveals brain edema reduction  in a rat  model  of  stroke  treated  with  an  aquaporin  4  inhibitor.  Romanian  journal  of  morphology  and embryology = Revue roumaine de morphologie et embryologie 58, 59‐66. 

Renier, N., Wu,  Z.,  Simson, D.J.,  Yang,  J.,  Ariel,  P.,  and  Tessier‐Lavigne, M.  (2014).  iDISCO:  A Simple, Rapid Method to Immunolabel Large Tissue Samples for Volume Imaging. Cell 159, 896‐910. 

Rothstein,  J.D., Patel,  S., Regan, M.R., Haenggeli, C., Huang,  Y.H., Bergles, D.E.,  Jin,  L., Dykes, H.M., Vidensky, S., Chung, D.S., et al. (2005). Beta‐lactam antibiotics offer neuroprotection by increasing glutamate transporter expression. Nature 433, 73‐77. 

Suzuki, Y., Nakamura, Y., Yamada, K., Huber, V.J., Tsujita, M., and Nakada, T. (2013). Aquaporin‐4 positron emission tomography imaging of the human brain: first report. J Neuroimaging 23, 219‐223.