Glibenclamide Administration Attenuates Infarct Volume, Hemispheric Swelling, and Functional Impairments following Permanent Focal Cerebral Ischemia in Rats

Studies from a single laboratory have shown that in rodent models of permanent stroke, administration of the sulfonylurea glibenclamide (Glib) is highly effective in reducing edema, mortality, and lesion volume. The Stroke Therapy Academic Industry Roundtable (STAIR) recommends that new acute treatments for ischemic stroke to be replicated across different laboratories. Accordingly, we examined the effect of low-dose Glib in a permanent suture occlusion model of stroke. Male Sprague-Dawley rats underwent permanent middle cerebral artery occlusion (pMCAO) followed by an initial intraperitoneal injection of Glib (10 μg/kg) and the start of a constant infusion (200 ng/h) via miniosmotic pump at the onset of ischemia. Functional deficits were assessed by Neurological Severity Score (NSS) and grip-strength meter at 24 and 48 h after pMCAO. Glib-treated rats showed a significant reduction in infarct volume, lower NSS, and less hemispheric swelling compared to vehicle. Grip strength was decreased significantly in pMCAO rats compared to shams and significantly improved by treatment with Glib. Taken together, these data indicate that Glib has strong neuroprotective effects following ischemic stroke and may warrant further testing in future clinical trials for human stroke

The Effect of Gold Nanoparticles and Apricot Kernel Extract on the Osseointegration of Dental Implants - A Rabbit Model

Introduction The osseointegration of dental implants is critical to their longevity and success. Various factors such as surface modifications, both additive and subtractive, systemic drug administration, growth factors, and plasma derivatives can influence osseointegration. Aims and Objectives This study aimed to study the effects of gold nanoparticles and aqueous extract of apricot kernels on the osseointegration of titanium implants in the tibia of rabbits. Materials and Methods Apricot kernels were dried, grounded, and dissolved in water to obtain an aqueous extract. It was filtered and stored in the refrigerator at 4oC. Gold nanoparticles (AuNPs) were prepared by mixing 3 ml of 0.02 mM hydrogen tetrachloroaurate with 1 ml of olive leaf extract. The mixture was stirred vigorously for 15 minutes on a heater stirrer at 50oC. Titanium screw implants were sterilized in an autoclave at 120oC with 15 pounds per square inch for 30 minutes. Sterilized implants were stored in sealed containers until use. The study was carried out at the College of Veterinary Medicine, University of Mosul, Iraq. The ethical approval for the study was obtained from the institutional animal care and use committee, College of Veterinary Medicine, University of Mosul, Mosul, Iraq, on 15/04/2023 on reference no UM.VET.2023.013. Nine healthy male adult white New Zealand rabbits aged between 10-12 months were utilized for the study. Three pure titanium implants, each of a type, were placed in each tibia. The implants were categorized as follows: control group (18 uncoated implants) and experimental group (36 coated implants). The implant placement was divided into three groups: controls with uncoated implants (n=18), AuNPs-coated implants (n=18), and apricot kernel extract-coated implants (n=18). General anesthesia was induced by intramuscular injection of ketamine hydrochloride 50mg (1ml /kg of body weight) plus xylazine 2% (0.2 ml /kg of body weight). The AuNPs-coated implants were implanted in the superior osteotomy site in the tibia. The apricot kernel extract-coated implants were implanted in the inferior osteotomy site and the uncoated implants were implanted in the middle osteotomy sites. Histological and histomorphometric tests were performed on all bone implant blocks at one-week, two-week, and three-week intervals after implant placement. The results were evaluated based on the amount of bone formation, cellular components, and inflammation. Tabulated data were statistically analyzed using SPSS v23.0 (IBM, Armonk, NY) using ANOVA with a statistical significance of p <0.05 Results We observed a significant amount of osteogenesis with reduced inflammation in AuNPs-coated implants and apricot kernel extract-coated implants compared to untreated implants. The osteoclastic activity was significantly higher in untreated implants than the treated implant sites. A statistically significant association was observed between coated implants and untreated implants. Conclusion The application of gold nanoparticles and aqueous extract of apricot kernels appears to be promising in reducing inflammation and improving bone apposition on the titanium implant surface. However, this preliminary investigation warrants further research

Vitamin D prevents hypoxia/reoxygenation-induced blood-brain barrier disruption via vitamin D receptor-mediated NF-kB signaling pathways.

Maintaining blood-brain barrier integrity and minimizing neuronal injury are critical components of any therapeutic intervention following ischemic stroke. However, a low level of vitamin D hormone is a risk factor for many vascular diseases including stroke. The neuroprotective effects of 1,25(OH)2D3 (vitamin D) after ischemic stroke have been studied, but it is not known whether it prevents ischemic injury to brain endothelial cells, a key component of the neurovascular unit. We analyzed the effect of 1,25(OH)2D3 on brain endothelial cell barrier integrity and tight junction proteins after hypoxia/reoxygenation in a mouse brain endothelial cell culture model that closely mimics many of the features of the blood-brain barrier in vitro. Following hypoxic injury in bEnd.3 cells, 1,25(OH)2D3 treatment prevented the decrease in barrier function as measured by transendothelial electrical resistance and permeability of FITC-dextran (40 kDa), the decrease in the expression of the tight junction proteins zonula occludin-1, claudin-5, and occludin, the activation of NF-kB, and the increase in matrix metalloproteinase-9 expression. These responses were blocked when the interaction of 1,25(OH) )2D3 with the vitamin D receptor (VDR) was inhibited by pyridoxal 5'-phosphate treatment. Our findings show a direct, VDR-mediated, protective effect of 1,25(OH) )2D3 against ischemic injury-induced blood-brain barrier dysfunction in cerebral endothelial cells

1,25(OH)₂D₃ effects on tight junction protein expression and cell viability after hypoxia/reoxygenation in bEnd.3 cells.

<p><b>(A)</b> Representative double immunofluorescence staining of ZO-1 (green), claudin-5 (green), occludin (red), and 4’,6-Diamidino-2-phenylindole (DAPI, blue) under hypoxia-reoxygenation (H/R) in the presence or absence of 100 nmol/L 1,25(OH)₂D₃ with or without vitamin D receptor antagonist pyridoxal 5 phosphate pyridoxal 5’-phosphate (P5P) in brain endothelial cells. <b>(B)</b> Immunoblot images and quantitative data showing the expression of occludin and claudin-5 after H/R in bEnd.3 cell monolayers. H/R-induced loss of occludin and claudin-5 compared with normoxia was prevented by pretreatment with 1,25(OH)₂D₃. These effects were blocked by P5P treatment. The density of proteins in normoxia was used as a standard (arbitrary unit) to compare the relative density of the other groups. Values shown are mean ± s.e.m. (n = 3). Similar results were obtained from three independent experiments. <b>(C)</b> Representative data from MTT assay showing cell viability after 16 h hypoxia/10 min reperfusion in b.End3 cells. The reduced cell viability after hypoxia was prevented by pretreatment with 1,25(OH)₂D₃. These effects were blocked by P5P treatment. Values shown are mean ± s.e.m. (n = 16). * <i>p</i> < 0.05, ** <i>p</i> < 0.01, and *** <i>p</i>< 0.001</p

Schematic presentation of a possible mechanism of 1,25(OH)₂D₃ action on hypoxia-induced blood-brain barrier (BBB) disruption.

<p>Following hypoxic injury, an increase in intracellular reactive oxygen species (ROS) causes mitochondrial dysfunction, which further leads to intracellular oxidative stress (1). As a result, damaged mitochondria activate nuclear factor kappa B (NF-κB) signaling pathways, causing IkB proteosomal degradation <i>via</i> phosphorylation of IkBα (2), which leads to proteolysis and the translocation of NF-κB to the nucleus (3). The attachment of NF-κB subunits to its DNA binding site modulates MMP-9 transcription, which leads to increased MMP-9 expression (4). Increased expression of MMP-9 mediates BBB disruption (low TEER values) through degradation of tight junction proteins such as ZO-1, occludin, and claudin-5 (5). 1,25(OH)₂D₃ blocks the signaling cascade by binding to the vitamin D receptor (VDR) (6) and preventing the translocation of p65 into the nucleus and the phosphorylation of I<i>k</i>B (7). The VDR antagonist pyridoxal 5’-phosphate blocks the effect of 1,25(OH)₂D₃ on BBB markers (8).</p

Effect of 1,25(OH)2D3 on NF-kB activation in bEnd.3 cells after hypoxia.

<p><b>(A)</b> Representative immunofluorescence images show the translocation of the p65 subunit of NF-κB (green) with DAPI (blue) nuclear counterstaining. bEnd.3 cells were treated with 1,25(OH)₂D₃ (100 nmol/L), or 1,25(OH)₂D₃ plus vitamin D receptor antagonist pyridoxal 5’-phosphate (P5P; 1 mM) under hypoxia/reoxygenation. Scale bar 50 μm. 1,25(OH)₂D₃ prevented the hypoxia-induced translocation of the p65 subunit of NF-kB into the nucleus, which was blocked by P5P treatment. <b>(B)</b> Protein levels of phosphorylated IkBα in bEnd.3 cells as determined using Western blotting and imaging analysis. Representative Western blots for IkBα. The density of proteins in normoxia was used as a standard (arbitrary unit) to compare the relative density of the other groups. Values shown are mean ± s.e.m. (n = 3) of a representative experiment. Similar results were obtained from three independent experiments; *<i>p</i> < 0.05, **<i>p</i> < 0.01, and ***<i>p</i> < 0.001.</p

Effects of 1,25(OH)₂D₃ on hypoxia/reoxygenation-induced mitochondrial superoxide production and hydrogen peroxide in bEnd.3 cells.

<p><b>(A)</b> Mitochondrial superoxide production in live cells was measured by fluorescence microscopy using MitoSOX Red dye. Representative fluorescence images show localization of MitoSOX Red fluorescence and DAPI fluorescence. Scale bar 50 μm. <b>(B)</b> MitoSOX Red fluorescence per cell was quantified using ImageJ software. Image data from 51–60 cells per treatment condition were averaged (n = 3). <b>(C)</b> Extracellular H₂O₂ production with the use of Amplex Red (n = 4). Data are expressed as mean ± s.e.m; *<i>p</i> < 0.05, **<i>p</i> < 0.01, and ***<i>p</i> < 0.001.</p

Effects of 1,25(OH)₂D₃ on MMP-2/9 in bEnd.3 cells.

<p>Cells were pretreated with 1,25(OH)₂D₃ at 100 nM or 1,25(OH)₂D₃ with vitamin D receptor antagonist pyridoxal-5-phosphate (1mM) for 24 h. <b>(A)</b> Representative Western blots for MMP-2/9. <b>(B-C)</b> The density of proteins in normoxia was used as a standard (arbitrary unit) to compare the relative density of the other groups. Values shown are mean ± s.e.m. Similar results were obtained from three independent experiments; a, different from normoxia; *<i>p</i> < 0.05, **<i>p</i> < 0.01, and ***<i>p</i> < 0.001.</p