Advances in imaging and analysis of 4 fluorescent components through the rat cortical column.

BACKGROUND: Immunofluorescent staining coupled with axial optical sectioning allows for assessment of native three-dimensional structure of brain tissue. Typical challenges of analyzing network structure include limitations driven by magnification/field of view, spatial resolution, tissue thickness, staining quality of dense cell types, data quantifiability and the quantity of simultaneous staining targets. NEW METHOD: This manuscript demonstrates many methodological advancements. Software-aided alignment of the cortical slice and stereotaxic atlas maximizes ROI-identification accuracy. Tissue compression during antigen retrieval enhances epitope availability without damaging tissue. A thorough factorial experiment focusing on Smi-311 staining highlights the enhancements in image quality from our extended staining protocol. Mosaic scanning techniques and subsequent four-channel alignment ensures high data quality. RESULTS: Cortical column datasets [800µm x 3000µm x 70µm] utilizing sequential optical sectioning were successfully generated from three rats. Each rat provided three coronal sections in each of two regions, M1 and S1BF, from which data cubes were generated per hemisphere, totaling 36 high-magnification four-color datasets. COMPARISON WITH EXISTING METHOD(S): Typical confocal assessments of brain tissue do not utilize such thick tissue slices nor collect entire cortical columns from the cortical surface to the grey/white interface at a resolution that can map fine filamentous processes. The simultaneous collection of our four specific structural markers - neuronal, astrocytic, vascular and nuclear - is novel and the quantitative optimization of staining protocols through a factorial design rare. CONCLUSIONS: Building upon this preliminary success in protocol development, future work will encompass volumetric modeling and quantitative analysis of regional network architecture.

Smart-phone, paper-based fluorescent sensor for ultra-low inorganic phosphate detection in environmental samples.

A major goal of environmental agencies today is to conduct point-of-collection monitoring of excess inorganic phosphate (Pi) in environmental water samples for tracking aquatic "dead zones" caused by algae blooms. However, there are no existing commercial devices which have been miniaturized and are suitable for the point-of-need-testing ("PONT") that is required to fully map a large region, such as the Florida Everglades. To solve this challenge, a reflection-mode fluorescence-sensing apparatus was developed, leveraging an environmentally sensitive fluorophore (MDCC) bound to a bacterial phosphate-binding protein to generate a fluorescent optical signal proportional to the concentration of (Pi) present. The combined end-to-end integrated sensor system had a response time of only 4 s, with minimal effects of common interfering agents and a linear range spanning from 1.1 to 64 ppb. To support ease-of-use during PONT, the platform incorporated disposable wax-printed paper strip sample pads and a smartphone camera detection system. Since the EPA threshold is currently 30 ppb to prevent eutrophication, this system serves as a rapid test of whether a region is compliant.

Histological Characterization of the Irritative Zones in Focal Cortical Dysplasia Using a Preclinical Rat Model.

Current clinical practice in focal epilepsy involves brain source imaging (BSI) to localize brain areas where from interictal epileptiform discharges (IEDs) emerge. These areas, named irritative zones, have been useful to define candidate seizures-onset zones during pre-surgical workup. Since human histological data are mostly available from final resected zones, systematic studies characterizing pathophysiological mechanisms and abnormal molecular/cellular substrates in irritative zones-independent of them being epileptogenic-are challenging. Combining BSI and histological analysis from all types of irritative zones is only possible through the use of preclinical animal models. Here, we recorded 32-channel spontaneous electroencephalographic data from rats that have focal cortical dysplasia (FCD) and chronic seizures. BSI for different IED subtypes was performed using the methodology presented in Bae et al. (2015). Post-mortem brain sections containing irritative zones were stained to quantify anatomical, functional, and inflammatory biomarkers specific for epileptogenesis, and the results were compared with those obtained using the contralateral healthy brain tissue. We found abnormal anatomical structures in all irritative zones (i.e., larger neuronal processes, glioreactivity, and vascular cuffing) and larger expressions for neurotransmission (i.e., NR2B) and inflammation (i.e., ILß1, TNFα and HMGB1). We conclude that irritative zones in this rat preclinical model of FCD comprise abnormal tissues disregarding whether they are actually involved in icto-genesis or not. We hypothesize that seizure perpetuation happens gradually; hence, our results could support the use of IED-based BSI for the early diagnosis and preventive treatment of potential epileptic foci. Further verifications in humans are yet needed.

Electrochemical Lateral Flow Paper Strip for Oxidative-Stress Induced DNA Damage Assessment.

The phrase "oxidative-stress induced DNA damage" is commonly used in both the scientific literature and common media outlets, and is frequently linked to detrimental elements of aging as well as the onset of illnesses. Due to the growing focus on this topic, a clear need has emerged to develop a quantitative, low-cost methodology to allow for periodic monitoring of oxidative-stress induced DNA damage within individuals. Recent literature examining the link between oxidative stress and the onset of various cancers has made monitoring an even more pressing need. The mechanism of oxidative-stress induced DNA damage originates in chronic inflammation, which in turn activates various transcription factors and diseases that influence the onset of tumor development, chemoresistance, radioresistance, and other harmful cellular processes. While current technologies that aim to provide quantitative metrics require extremely expensive equipment and significant technical expertise, our laboratory has designed a low-cost methodology utilizing a combination of carbon nanotubes, paper electrodes, and immunochromatographic strips.

Peptide modified polymer poly (glycerol- dodecanedioate co-fumarate) for efficient control of motor neuron differentiation.

Neural tissue engineering is one of the most promising approaches for healing nerve damage, which bypasses the limits of contemporary conventional treatments. In a previous study, we developed a fibrous scaffold via electrospinning poly (glycerol dodecanedioate) (PGD) and gelatin that mimics the structure of a native extracellular matrix (ECM) for soft tissue engineering application. In this study, fumaric acid (FA) was incorporated into the PGD synthesis process, which produced a PGD derivative referred to as poly (glycerol dodecanedioate co-fumarate) (PGDF). This introduced a new functional group, a double bond, into the polymer thus providing new modification possibilities. Arg-Gly-Asp-Cys (RGDC) and laminin peptides were chosen as biomolecules to modify the fiber and facilitate cell attachment and differentiation efficiency. The release of FA into the medium was quantified to investigate the bioreactivity of the derived scaffolds. In combination with UV crosslinking, the developed PGDF fiber mats were able to withstand degradation processes for up to 2 months, which ensures that neural tissue engineering applications are viable. Cell viability and motor neuron differentiation efficiency were demonstrated to be significantly improved with the addition of FA, RGDC and laminin peptides.

(68)Ga-NOTA-CHSg and (99m)Tc-CHSg Labeled Microspheres for Lung Perfusion and Liver Radiomicrospheres Therapy Planning.

Fast biodegradable (12 h < half-life < 48 h) radioactive labeled microspheres are needed for PET and SPECT lung perfusion and radiomicrosphere therapy planning. An emulsion method was used to create 30.1 ±4.8 µ m size range microspheres with biodegradable Chitosan glycol (CHSg). Microspheres were characterized and labeled with (99m)Tc or (68)Ga as an alternative to MAA in perfusion PET and SPECT studies. Surface decoration of CHSg microspheres with p-SCN-Bn-NOTA was performed to increase (68)Ga in vivo stability. (99m)Tc was labeled directly to the CHSg microspheres. Labeling yield and in vitro radiochemical stability were evaluated. In vitro CHSg microsphere degradation half-life was ~24 hours in porcine blood. Labeled microspheres were injected into Sprague Dawley rats and biodistribution was determined after 2 and 4 hours. Both (99m)Tc-CHSg and (68)Ga-NOTA-CHSg were quickly allocated in the lungs after injection. (99m)Tc-CHSg showed 91.6 ± 6.5% and 83.2 ± 4.1% of the decay corrected injected activity remaining in the lungs after 2 and 4 hours, respectively. For the obtained (68)Ga-NOTA-CHSg microspheres, lung allocation was very high with 98.9 ± 0.2% and 95.6 ± 0.9% after 2 and 4 hours, respectively. The addition of p-SCN-Bn-NOTA acts as a radioprotectant eliminating the released (68)Ga activity from the lungs to the bladder protecting the other organs.