Histochemistry of microinfarcts in the mouse brain after injection of fluorescent microspheres into the common carotid artery

The mouse model of multiple cerebral infarctions,established by injecting fluorescent microspheres into the common carotid artery,is a recent development in animal models of cerebral ischemia.To investigate its effectiveness,mouse models of cerebral infarction were created by injecting fluorescent microspheres,45–53μm in diameter,into the common carotid artery.Six hours after modeling,fluorescent microspheres were observed directly through a fluorescence stereomicroscope,both on the brain surface and in brain sections.Changes in blood vessels,neurons and glial cells associated with microinfarcts were examined using fluorescence histochemistry and immunohistochemistry.The microspheres were distributed mainly in the cerebral cortex,striatum and hippocampus ipsilateral to the side of injection.Microinfarcts were found in the brain regions where the fluorescent microspheres were present.Here the lodged microspheres induced vascular and neuronal injury and the activation of astroglia and microglia.These histopathological changes indicate that this animal model of multiple cerebral infarctions effectively simulates the changes of various cell types observed in multifocal microinfarcts.This model is an effective,additional tool to study the pathogenesis of ischemic stroke and could be used to evaluate therapeutic interventions.This study was approved by the Animal Ethics Committee of the Institute of Acupuncture and Moxibustion,China Academy of Chinese Medical Sciences(approval No.D2021-03-16-1)on March 16,2021.

Ratiometric fluorescence probe for accurate detection of Concanavalin A by coupling fluorescent microsphere with boric acid functionalized carbon dots

Accurate and sensitive strategies for Concanavalin A(Con A)sensing are conducive to the better cognition of various important biological and physiological processes.Here,by designing dextran-functionalized fluorescent microspheres(DxFMs)and boric acid-modified carbon dots(BCDs)as recognition unit and built-in signal reference respectively,a ratiometric fluorescent detection platform was proposed for Con A detection with high reliability.In this protocol,the BCDs/DxFMs precipitation was formed due to the covalent interactions between cis-diol of DxFMs and boronic acid groups of BCDs,thus only fluorescence of BCDs could be detected in the supernatant.When Con A was presented,it could bind to DxFMs through its carbohydrate recognition ability and suppress the subsequent assembly between DxFMs and BCDs,leading to the simultaneous capture of DxFMs and BCDs fluorescence in the supernatant.Since the BCDs content was superfluous,their fluorescence intensities were basically constant in all cases.Based on the unchanged BCDs fluorescence signal and target-dependent DxFMs fluorescence signal in supernatant,the ratiometric detection of Con A was realized.Under optimized conditions,this ratiometric fluorescent platform displayed a linear detection range from 0.125μg/mL to 12.5μg/mL with a detection limit of 0.089μg/mL.Moreover,satisfied analytical outcomes for Con A detection in serum samples were obtained,manifesting huge application potential of this ratiometric fluorescent platform in clinical diagnosis.

Preparation and performance of fluorescent polyacrylamide microspheres as a profile control and tracer agent

Polyacrylamide microspheres have been suc- cessfully used to reduce water production in reservoirs, but it is impossible to distinguish polyacrylamide microspheres from polyacrylamide that is used to enhance oil recovery and is already present in production fluids. In order to detect polyacrylamide microspheres in the reservoir pro- duced fluid, fluorescent polyacrylamide microspheres P（AM-BA-AMCO）, which fluoresce under ultraviolet irradiation, were synthesized via an inverse suspension polymerization. In order to keep the particle size distribu- tion in a narrow range, the synthesis conditions of the polymerization were studied, including the stirring speed and the concentrations of initiator, NaaCO3, and dispersant. The bonding characteristics of microspheres were deter- mined by Fourier transform infrared spectroscopy. The surface morphology of these microspheres was observed under ultraviolet irradiation with an inverse fluorescence microscope. A laboratory evaluation test showed that the fluorescent polymer microspheres had good water swelling capability, thus they had the ability to plug and migrate in a sand pack. The plugging rate was 99.8 % and the residual resistance coefficient was 800 after microsphere treatment in the sand pack. Furthermore, the fluorescent microspheres and their fragments were accurately detected under ultra- violet irradiation in the produced fluid, even though theyhad experienced extrusion and deformation in the sand pack.

Synthesis of Micron-size Functional Polystyrene Fluorescent Micro- spheres and their Adsorbability to Human Serum Albumin

Polystyrene microspheres with sulfo- or aldehyde- surface were synthesized through dispersion polymerization. Functional polystyrene fluorescent microspheres were prepared by the way of adding 2, 5-diphenyloxazole (PPO) into the reaction system directly and dying the blank microspheres in the ethanol solution of PPO. The influence of preparing matters on the encapsulating rate of PPO, and the influence of functional groups on the adsorbability to human serum albumin (HSA) were investigated.

Time-dependent effects of castration on the bladder function and histological changes in the bladder and blood vessels

We examined the effect of androgens on bladder blood flow （BBF）, bladder function and histological changes in castrated male rats. Male Wistar rats were classified into unoperated group （control group）, groups castrated at the age of 8weeks （group 8wPC） and groups castrated at the age of 4weeks （group 4wPC）. Each rat was used at the age of 20weeks. BBF was measured using fluorescent microspheres. Bladder cystometry was performed without anesthesia or restraint; the bladder was first irrigated with saline and then with 0.25% acetic acid （AA） solution. Maximum voiding pressure and voiding interval were measured. The bladder and lilac artery were histologically examined for differences in smooth muscle and quantity of collagen fiber to analyze the effect of castration on the smooth muscle content. No differences were noted in BBF following castration. The voiding intervals for all groups were shortened （P 〈 0.001） following AA irrigation. No significant difference was noted in the maximum voiding pressure. Histological changes were observed in bladder and lilac artery. Smooth muscle/collagen ratio at the bladder was lower in groups 8wPC and 4wPC compared to the control group （P 〈 0.01）, while that at the lilac artery was decreased in group 4wPC compared to the control group （P〈 0.001）. In conclusion, our findings indicate that castration does not alter BBF, but leads to histological changes in the bladder as well as its associated blood vessels.

Reversible changes in aqueous outflow facility, hydrodynamics, and morphology following acute intraocular pressure variation in bovine eyes

Background Elevated intraocular pressure （lOP） is primarily due to increased aqueous outflow resistance, but how aqueous outflow resistance is generated and regulated are still not fully understood. The aim of this study is to determine whether changes in outflow facility, outflow pattern, and morphology following acute lOP elevation were reversible when the lOP was returned to a normal level in bovine eyes using a two-color tracer technique to label outflow patterns within the same eye. Methods Twelve fresh enucleated bovine eyes were perfused with Dulbecco＇s phosphate buffer saline （PBS） containing 5.5 mmol/L glucose （DBG） at 30 mmHg first to establish the baseline outflow facility followed by a fixed volume of red fluorescent microspheres （0.5 μm, 0.002% v/v）. After the red tracer being replaced with DBG in the anterior chamber, perfusion was continued at 7 mmHg with the same volume of green tracer, followed by a fixative. In two control groups, the eyes were constantly perfused at either 30 mmHg （n=6） or 7 mmHg （n=6） using the same methods. The outflow facility （C, pJ.min.-lmmHg-1）, was continuously recorded. Confocal images were taken along the inner wall （IW） of the aqueous plexus （AP） in frontal sections. The percent of the effective filtration length （PEFL, PEFL=IW length exhibiting tracer labeling/total length of IW） was measured. Sections with AP were processed and examined by light microscopy. The total length of IW and the length exhibiting separation （SL） in the juxtacanalicular connective tissue （JCT） were measured. A minimum of eight collector channel （CC） ostia per eye were analyzed for herniations. Results In the experimental （30-7 mmHg） group, the outflow facility was significantly higher at 7 mmHg （（4.81±1.33） #lmin-1 mmHg-1） than that at 30 mmHg （（0.99±0.15） μl.min-1 mmHg-1, P=-0.002）, corresponding to a significant increase in the PEFL （P=-0.0003）. The percent of CC ostia exhibiting herniations in the experimental group （（67.40±8.90） μl.min·-1mmHg-1） decreased significantly compared to that in the control at 30 mmHg （（94.44±3.33） μl.min-lmmHg-1, P=-0.03）, but higher than that in the control at 7 mmHg （（29.43±4.60） μl.min-1mmHg-1, P=0.01）. Washout-associated separation between the IW and JCT was found by light microscopy and percent separation length （PSL, PSL=SL/total length of IW） was decreased in the control at 30 mmHg compared to that in the experimental group and control at 7 mmHg. Conclusions The pressure-induced morphological and hydrodynamic changes were reversible. Changes （collapse of AP, separation between the JCT and IW, and herniation into CC ostia） influence the effective filtration area that regulates outflow facility.

Development of a novel two color tracer perfusion technique for the hydrodynamic study of aqueous outflow in bovine eyes

Background Elevation of intraocular pressure is usually associated with primary open angle glaucoma and caused by increased outflow resistance. A two-color fluorescent tracer technique was developed to investigate the hydrodynamics of aqueous humor outflow with changing intraocular pressure within the same eye, to better understand the relationship between outflow facility and effective filtration area. Methods Eighteen enucleated bovine eyes were first perfused at 30 mmHg with Dulbecco＇s phosphate-buffered saline containing 5.5 mmol/L D-glucose. After a stable baseline facility, red fluorescent microspheres （0.5 um, 0.002% v/v） were exchanged and perfused. Eyes in the one-color control group （n=6） were immediately perfused with fixative. In the experimental group （n=6）, eyes were perfused with green tracer after intraocular pressure reduced to 7 mmHg, while in the two-color control group （n=6）, eyes were perfused with green tracer with intraocular pressure remaining at 30 mmHg. All 12 eyes were then per＇fusion-fixed. Outflow facility was continuously recorded in all eyes. Confocal images were taken along the inner wall of the aqueous plexus and the percent of the effective filtration length （PEFL; length of inner wall exhibiting tracer labeling/total length of inner wall） was measured. The relationships between outflow facility and PEFL were analyzed statistically. Results No significant differences were found in baseline facilities （ul.min-1.mmHg-1） among the three groups （the experimental group： 0.93±0.12; the two-color control group： 0.90±0.19; the one-color control group： 0.98±0.13）. In the experimental group, the outflow facility was significantly higher at 7 mmHg （4.29±1.01） than that at 30 mmHg （1.90±0.67, P 〈0.001）, which corresponded to a significant increase in the PEFL at 7 mmHg （54.70±8.42） from that at 30 mmHg （（11.76±4.56）%, P 〈0.001）. The PEFL labeled by red fluorescent microspheres in the experimental group （（11.76±4.56）%） showed no significant difference from that of the one-color control group （（13.39±2.19）%, P=-0.473） or the two-color control group （（11.49±4.95）%, P=-0.930）. The PEFL labeled by green fluorescent microspheres in the experimental group （（54.70±8.42）%） was significantly higher than that of the two color control group （（37.34±8.17）%, P=0.010）. A positive correlation was found between outflow facility and PEFL（r=0.897, R2=0.804） in the experimental group. Conclusions Changes in aqueous humor outflow patterns before and after a change in intraocular pressure can be successfully distinguished within the same eye using our newly developed two-color tracer perfusion technique. The PEFL showed positive correlation with the outflow facility.