Lumbar Intrathecal Injection in Adult and Neonatal Mice.

This article describes a step-by-step process of lumbar intrathecal injection of Evans blue dye and AAV9-EGFP in adult (2-month-old) and neonatal (postnatal day 10) mice. Intrathecal injection is a clinically translatable technique that has already been extensively applied in humans. In mice, intrathecal injection is considered a challenging procedure that requires a trained and experienced researcher. For both adult and neonatal mice, lumbar intrathecal injection is directed into the L5-L6 intervertebral space. Intrathecally injected material enters the cerebrospinal fluid (CSF) within the intrathecal space from where it can directly access the central nervous system (CNS) parenchyma. Simultaneously, intrathecally injected material exits the CSF with pressure gradient and enters the endoneurial fluid and ultimately the peripheral nerves. While in the CSF, the injectable material also enters the bloodstream and systemic circulation through the arachnoid villi. A successful lumbar intrathecal injection results in adequate biodistribution of the injectable material in the CNS, PNS, and peripheral organs. When correctly applied, this technique is considered as minimally invasive and non-disruptive and can be used for the lumbar delivery of any solute. © 2024 Wiley Periodicals LLC. Basic Protocol 1: C57BL/6 adult and P10 mice lumbar intrathecal injection Basic Protocol 2: Tissue collection and preparation for evaluating Evans blue dye diffusion Basic Protocol 3: Tissue collection and preparation for immunohistochemistry staining Basic Protocol 4: Tissue collection and vector genome copy number analysis.

[Restraint stress induces blood-brain barrier injury in rat amygdala by activating the Rho/ROCK signaling pathway].

OBJECTIVE: To investigate the role of Rho/ROCK signaling pathway in mediating restraint stress-induced blood-brain barrier (BBB) injury in the amygdala of rats. METHODS: Sixty male SD rats were randomized equally into control group (with food and water deprivation for 6 h per day), restraint stress group (with restraint for 6 h per day), stress + fasudil treatment (administered by intraperitoneal injection at 1 mg/100 g 30 min before the 6-h restraint) group, and fasudil treatment alone group. The elevated plus-maze test was used to detect behavioral changes of the rats, serum corticosterone and S100B levels were determined with ELISA, and Evans Blue leakage in the brain tissue was examined to evaluate the changes in BBB permeability. The changes in expression levels of tight junction proteins in the amygdala were detected using immunofluorescence assay and Western blotting, and Rho/ROCK pathway activation was detected by Pull-down test and Western blotting. Ultrastructural changes of the cerebral microvascular endothelial cells were observed using transmission electron microscopy. RESULTS: Compared with those in the control group, the rats in restrain stress group and stress+fasudil group showed obvious anxiety-like behavior with significantly increased serum corticosterone level (P<0.001). Compared with those in the control group and stress+fasudil group, the rat models of restrain stress showed more obvious Evans Blue leakage and higher S100B expression (P<0.01) but lower expressions of tight junction proteins in the amygdala. Pull-down test and Western blotting confirmed that the expression levels of RhoA-GTP, ROCK2 and P-MLC 2 were significantly higher in stress group than in the control group and stress + fasudil group (P<0.05). Transmission electron microscopy revealed obvious ultrastructural changes in the cerebral microvascular endothelial cells in the rat models of restrain stress. CONCLUSION: Restraint stress induces BBB injury in the amygdala of rats by activating the Rho/ROCK signaling pathway.

Enhanced capillary delivery with nanobubble-mediated blood-brain barrier opening and advanced high resolution vascular segmentation.

Overcoming the blood-brain barrier (BBB) is essential to enhance brain therapy. Here, we utilized nanobubbles with focused ultrasound for targeted and improved BBB opening in mice. A microscopy technique method assessed BBB opening at a single blood vessel resolution employing a dual-dye labeling technique using green fluorescent molecules to label blood vessels and Evans blue brain-impermeable dye for quantifying BBB extravasation. A deep learning architecture enabled blood vessels segmentation, delivering comparable accuracy to manual segmentation with a significant time reduction. Segmentation outcomes were applied to the Evans blue channel to quantify extravasation of each blood vessel. Results were compared to microbubble-mediated BBB opening, where reduced extravasation was observed in capillaries with a diameter of 2-6 µm. In comparison, nanobubbles yield an improved opening in these capillaries, and equivalent efficacy to that of microbubbles in larger vessels. These results indicate the potential of nanobubbles to serve as enhanced agents for BBB opening, amplifying bioeffects in capillaries while preserving comparable opening in larger vessels.

Efficacy of [67Cu]Cu-EB-TATE Theranostic Against Somatostatin Receptor Subtype-2-Positive Neuroendocrine Tumors.

ß--emitting 177Lu-octreotate is an approved somatostatin receptor subtype 2 (SSTR2)-directed peptide receptor radionuclide therapy for the treatment of gastroenteropancreatic neuroendocrine tumors (NETs). However,177Lu-octreotate has fast pharmacokinetics, requiring up to 4 treatment doses. Moreover, 177Lu is less than ideal for theranostics because of the low branching ratio of its γ-emissions, which limits its SPECT imaging capability. Compared with 177Lu, 67Cu has better decay properties for use as a theranostic. Here, we report the preclinical evaluation of a long-lived somatostatin analog, [67Cu]Cu-DOTA-Evans blue-TATE (EB-TATE), against SSTR2-positive NETs. Methods: The in vitro cytotoxicity of [67Cu]Cu-EB-TATE was investigated on 2-dimensional cells and 3-dimensional spheroids. In vivo pharmacokinetics and dosimetry were studied in healthy BALB/c mice, whereas ex vivo biodistribution, micro-SPECT/CT imaging, and therapy studies were done on athymic nude mice bearing QGP1.SSTR2 and BON1.SSTR2 xenografts. Therapeutic efficacy was compared with [177Lu]Lu-EB-TATE. Results: Projected human effective doses of [67Cu]Cu-EB-TATE for male (0.066 mSv/MBq) and female (0.085 mSv/MBq) patients are tolerable. In vivo micro-SPECT/CT imaging of SSTR2-positive xenografts with [67Cu]Cu-EB-TATE showed tumor-specific uptake and prolonged accumulation. Biodistribution showed tumor accumulation, with concurrent clearance from major organs over a period of 72 h. [67Cu]Cu-EB-TATE was more effective (60%) at eliminating tumors that were smaller than 50 mm3 within the first 15 d of therapy than was [177Lu]Lu-EB-TATE (20%) after treatment with 2 doses of 15 MBq administered 10 d apart. Mean survival of [67Cu]Cu-EB-TATE-treated groups was 90 d and more than 90 d, whereas that of [177Lu]Lu-EB-TATE was more than 90 d and 89 d against vehicle control groups (26 d and 53 d), for QGP1.SSTR2 and BON1.SSTR2 xenografts, respectively. Conclusion: [67Cu]Cu-EB-TATE exhibited high SSTR2-positive NET uptake and retention, with favorable dosimetry and SPECT/CT imaging capabilities. The antitumor efficacy of [67Cu]Cu-EB-TATE is comparable to that of [177Lu]Lu-EB-TATE, with [67Cu]Cu-EB-TATE being slightly more effective than [177Lu]Lu-EB-TATE for complete remission of small tumors. [67Cu]Cu-EB-TATE therefore warrants clinical development.

MiR-221-5p regulates blood-brain barrier dysfunction through the angiopoietin-1/-2/Tie-2 signaling axis after subarachnoid hemorrhage.

AIM: To explore the potential role of microRNA miR-221-5p on the angiopoietin-1 (Ang-1)/Ang-2/Tie-2 signaling axis after subarachnoid hemorrhage (SAH) in a rat model. METHODS: Aspects of the rat's behavior were measured using the Kaoutzanis scoring system to test neurological responses. This included feeding behavior, body contraction, motor, and eye-opening responses. Brain sections were studied using transmission electron microscopy and Evans blue extravasation. Levels of Ang-1, Ang-2, and Tie-2 were determined by Western blot, while miR-221-5p was quantified using stem-loop real-time quantitative PCR (RT-qPCR). RESULTS: The SAH group responded worse to the neurological response test than the sham-operated group. The intercellular space was widened in the SAH group, but not in the sham-operated group. Evans blue dye leaked significantly more into brain tissue cells of the SAH group. Stem-loop qRT-PCR showed elevated miR-221-5p levels. Additionally, Ang-1 and Tie-2 were reduced but Ang-2 expression was increased after SAH. This led to a significant reduction of the Ang-1/Ang-2 ratio in the brain tissue, which was associated with the destruction of the blood-brain barrier. CONCLUSION: The data indicate that miR-221-5p might regulate blood-brain barrier dysfunction through the Ang-1/Ang-2/Tie-2 signaling axis, suggesting that it should be further investigated as a potential novel biomarker.

Characteristics of the New Mast Cell-Rich Nodal Structure in the Rat Skin Surface.

Background: : Acupuncture, practiced for millennia, lacks a clear anatomical definition for acupoints. A prevailing theory suggests that acupoints overlap with skin areas with higher mast cell density. Skin spots stained with intravenously infused Evans blue (EB), indicative of neurogenic inflammation, have recently been posited as acupoints in rats. Objectives: : To demonstrate the concordance between EB-reactive skin spots and mast cell-enriched acupoints. Methods: : We employed staining and RNA-seq analysis to delineate the morphological characteristics and gene expression profiles of EB-reactive skin spots in rats. Results: : EB infusion revealed a novel nodal structure on the rat skin surface, visible to the naked eye, with dimensions of approximately 1 mm in both diameter and height. Around 30 such nodes were identified on one side of the abdominal area, spaced roughly 3 mm apart, excluding the linea alba. RNA-seq analysis indicated that the gene expression patterns within these nodes markedly differed from both non-nodal skin areas and lymph nodes. Histological examination using toluidine blue revealed a significantly greater mast cell count in the nodes than in non-nodal skin regions. Additionally, the nodes stained positively with Alcian blue and Hemacolor, reagents known to mark primo vascular tissues. Conclusion: : Our findings suggest that EB-reactive nodes are indeed rich in mast cells. Further research is warranted to establish these skin nodes as surface primo nodes.

Topical Skin Application of Small-Molecule Antiplatelet Agent against Pressure Injury in Rat Models.

Due to prolonged forced positioning, the incidence of intraoperative pressure injuries is high. This study aimed to explore the impact of small-molecule antiplatelet drugs on pressure injuries by locally applying them before an injury occurs. In the first part of this study, water-soluble tracers with different molecular weights were applied to normal and early-stage pressure-injured skin. Through digital cameras, spectrophotometers, and histological observations, the penetration of tracers into the epidermis was clarified. In the second part of this study, a water-soluble antiplatelet drug called Trapidil (molecular weight = 205 Da) was applied to the left side of the back of a rat before, during, and after compression, and the contralateral side served as a non-intervention control group. The differences in pressure injuries between the two groups were observed through a digital camera, an ultraviolet camera, and temperature measurement, and skin circulation and perfusion were assessed via an intravenous injection of Evans Blue. The first part of this study found that water-soluble tracers did not easily penetrate normal skin but could more easily penetrate pressure-damaged skin. The smaller the molecular weight of the tracer, the easier it penetrated the skin. Therefore, in the next step of research, water-soluble drugs with smaller molecular weights should be selected. The second part of this study found that, compared with the control group, the occurrence rates and areas of ulcers were lower, the gray value was higher, and the skin temperature was lower in the Trapidil group (p < 0.05). After the intravenous Evans Blue injection, skin circulation and perfusion in the Trapidil group were found to be better. In conclusion, this study found that the topical skin application of a small-molecule antiplatelet agent may have significant effects against pressure injuries by improving post-decompression ischemia, providing new insights into the prevention and treatment of intraoperative pressure injuries.

Neuroprotection of rhubarb extract against cerebral ischaemia-reperfusion injury via the gut-brain axis pathway.

BACKGROUND: The gut-brain axis (GBA) plays a central role in cerebral ischaemia-reperfusion injury (CIRI). Rhubarb, known for its purgative properties, has demonstrated protective effects against CIRI. However, it remains unclear whether this protective effect is achieved through the regulation of the GBA. AIM: This study aims to investigate the mechanism by which rhubarb extract improves CIRI by modulating the GBA pathway. METHODS: We identified the active components of rhubarb extract using LC-MS/MS. The model of middle cerebral artery occlusion (MCAO) was established to evaluate the effect of rhubarb extract. We conducted 16S rDNA sequencing and untargeted metabolomics to analyze intestinal contents. Additionally, we employed HE staining, TUNEL staining, western blot, and ELISA to assess intestinal barrier integrity. We measured the levels of inflammatory cytokines in serum via ELISA. We also examined blood-brain barrier (BBB) integrity using Evans blue (EB) penetration, transmission electron microscopy (TEM), western blot, and ELISA. Neurological function scores and TTC staining were utilized to evaluate neurological outcomes. RESULTS: We identified twenty-six active components in rhubarb. Rhubarb extract enhanced α-diversity, reduced the abundance of Enterobacteriaceae, and partially rectified metabolic disorders in CIRI rats. It also ameliorated pathological changes, increased the expressions of ZO-1, Occludin, and Claudin 1 in the colon, and reduced levels of LPS and d-lac in serum. Furthermore, it lowered the levels of IL-1ß, IL-6, IL-10, IL-17, and TNF-α in serum. Rhubarb extract mitigated BBB dysfunction, as evidenced by reduced EB penetration and improved hippocampal microstructure. It upregulated the expressions of ZO-1, Occludin, Claudin 1, while downregulating the expressions of TLR4, MyD88, and NF-κB. Similarly, rhubarb extract decreased the levels of IL-1ß, IL-6, and TNF-α in the hippocampus. Ultimately, it reduced neurological function scores and cerebral infarct volume. CONCLUSION: Rhubarb effectively treats CIRI, potentially by inhibiting harmful bacteria, correcting metabolic disorders, repairing intestinal barrier function, alleviating BBB dysfunction, and ultimately improving neurological outcomes.

Non-invasive evaluation of vascular permeability in formalin-induced orofacial pain model using infrared thermography.

Enhanced vascular permeability at the site of injury is a prominent feature in acute inflammatory pain models, commonly assessed through the Evans Blue test. However, this invasive test requires euthanasia, thereby precluding further investigations on the same animal. Due to these limitations, the integration of non-invasive tools such as IRT has been sought. Here, we aimed to evaluate the use of thermography in a common orofacial pain model that employs formalin as a chemical irritant to induce local orofacial inflammation. Male Hannover rats (290-300 g, N = 43) were used. In the first approach, radiometric images were taken before and after formalin administration, assessing temperature changes and extravasated Evans Blue. The second approach included capturing pre- and post-formalin test radiometric images, followed by cytokine measurements in excised vibrissae tissue. Rats were anesthetized for vibrissae tissue collection, allowing correlations between thermographic patterns, nocifensive behavior duration, and cytokine levels in this area. Our findings revealed a positive correlation between local temperature, measured via thermography, and vascular permeability in the contralateral (r2 = 0.3483) and ipsilateral (r2 = 0.4502) side, measured using spectrophotometry. The obtained data supports the notion that thermography-based temperature assessment can effectively evaluate vascular permeability in the orofacial region.

Intravenous administration of human chorionic membrane mesenchymal stem cells promotes functional recovery in a rat traumatic brain injury model.

Human chorionic membrane mesenchymal stem cells (hCM-MSCs) have increasingly emerged as an excellent source of transplanted cells for regenerative therapy as they can be isolated via a non-invasive and simple method with high proliferative capabilities. However, the roles and mechanisms of hCM-MSCs on traumatic brain injury (TBI) animal models have not been investigated yet. The aim of this study was to investigate the therapeutic potential and mechanism of hCM-MSCs transplantation in a rat model of TBI. Adult male Sprague-Dawley rats were subjected to moderate lateral fluid percussion-induced TBI. At 2 h after TBI, hCM-MSCs, or PBS were administered intravenously via the tail vein. Neurological function, brain water content, Evans blue dye extravasation, immunofluorescence staining, and enzyme-linked immunosorbent were evaluated. The results showed that transplanted hCM-MSCs were observed in the injured brain. Compared with the PBS group, hCM-MSCs treatment significantly decreased the numbers of M1 macrophages/microglia, MPO + neutrophils and caspase-3 + cells ( P  < 0.01). Meanwhile, hCM-MSCs treatment significantly reduced the expression levels of the pro-inflammatory cytokines (TNF-α, interleukin-(IL)6 and IL-1ß) while increasing the numbers of M2 macrophages/microglia and the expression of the anti-inflammatory cytokines IL-10 ( P  < 0.01). In addition, hCM-MSCs treatment significantly reduced brain water content and Evans blue extravasation. Lastly, hCM-MSCs treatment significantly promoted neurogenesis and angiogenesis, and attenuated neurological deficits. Collectively, these findings indicate that hCM-MSCs exhibited effective therapeutic efficacy in a rat TBI model, and its mechanism may be by reducing inflammation, apoptosis and the blood-brain barrier disruption, promoting angiogenesis and neurogenesis.

The role of beta-adrenoreceptors in postoperative ileus in rats.

The aim of the research was to evaluate the influence of antagonists of specific beta-adrenergic receptor subtypes on bowel motility following abdominal surgery in rat model of postoperative ileus. Bowel motility was measured by the intestinal transit of Evans blue introduced via orogastric tube after surgical procedures of skin incision, laparotomy and laparotomy with gut manipulation. Male rats were given individual adrenergic receptor subtypes antagonists intraperitoneally, and the influence of administered agents on intestinal transit of Evans blue was then evaluated. No statistically significant differences in the length of intestine in tested rats were observed. Propranolol administered prior to surgical procedure has shown protective effect on Evans blue migration in rats undergoing laparotomy and gut manipulation. Intestinal dye transit for propranolol doses of 10, 30 and 45 mg/kg was 18.00 ± 1.88c m, 23.75 ± 1.71 cm and 22.5 ± 2.43 cm, respectively, and for last two doses, statistically significant increase of dye passage was noted, compared to Evans blue transit of 11.00 ± 2.43 cm in the control group. No acceleration of dye migration was seen following administration of beta1-, beta2- and beta3-selective adrenergic receptor antagonist metoprolol, ICI 118.551 and SR58894A, respectively. Our research confirmed that propranolol at high doses, as seen by other researchers, improved bowel motility in early phase of postoperative ileus. That slight acceleration of intestinal dye transit after surgery with gut manipulation is rather connected with membrane-stabilizing action, than the receptor blocking effect, as this effect was not observed after the application of selective antagonists of respective subtypes of beta-adrenergic receptor.

Effect of different suspension moxibustion methods on syndrome characteristics of rats with rheumatoid arthritis of heat bi syndrome based on "moxibustion can be used for heat syndrome" / 中国针灸

OBJECTIVE@#To observe the effects of different suspension moxibustion methods on the syndrome characteristics and inflammatory factors of rats with rheumatoid arthritis (RA) of heat bi syndrome and to prove the concept of "moxibustion can be used for heat syndrome".@*METHODS@#Among seventy Wistar rats, 12 rats were randomly selected as a normal group, and the remaining rats were induced by collagen combined with wind, dampness, and heat environmental stimulation to establish the RA model of heat bi syndrome. Forty-eight rats with successful model establishment were further randomly divided into a model group and three moxibustion groups (mild moxibustion group, rotating moxibustion group and sparrow-pecking moxibustion group), with 12 rats in each group. The acupoints "Quchi" (LI 11), "Dazhui" (GV 14) and ashi point were used in all moxibustion groups, with mild moxibustion, rotating moxibustion, and sparrow-pecking moxibustion intervention given respectively, each acupoint was treated with moxibustion for 10 min a day, and 6 days were considered one course of treatment, with a total of three courses. After the intervention, the arthritis index (AI), the Evans blue (EB) extravasated volume in the soft tissue of the right hind paw, and the levels of tumor necrosis factor (TNF)-α and interleukin (IL)-10 in the serum were measured by ELISA in each group. The volume of the bilateral hind paw was measured; the infrared thermal imaging was collected to analyze the temperature of the plantar area of the bilateral foot pads, and the reaction time of plantar heat pain was calculated before and after modeling, as well as after the 1st, 2nd and 3rd courses of interrention. The ankle dorsiflexion angle of the right hind foot was also measured before and after modeling, as well as after the intervention.@*RESULTS@#After modeling, compared with the normal group, the rats in the model group had more high-temperature areas in the bilateral hind limbs, abnormal AI score, abnormal bilateral hind paw volume, abnormal temperature of the plantar area of the bilateral foot pads, abnormal foot pain response time, abnormal right hind ankle dorsiflexion angle, abnormal right hind paw soft tissue EB extravasation, and abnormal serum TNF-α and IL-10 levels (P<0.01, P<0.05). After the intervention, compared with the model group, the rats in each moxibustion group had decreased or disappeared high-temperature areas in the bilateral hind limbs, EB extravasated volume in the soft tissue of the right hind paw was reduced (P<0.05), and the right ankle dorsiflexion angle was increased (P<0.05), serum level of TNF-α was reduced, and level of IL-10 increased (P<0.05); the AI scores in the mild moxibustion group and the sparrow-pecking moxibustion group was decreased (P<0.01, P<0.05). After the 1st, 2nd and 3rd courses of intervention, compared with the model group, the bilateral hind paw volume of rats in each moxibustion group was decreased (P<0.05, P<0.01), and plantar heat pain reaction time was increased (P<0.05). After the 2nd course and the 3rd course of intervention, the temperature of the right hind paw pad area was decreased in each moribustion group (P<0.05); after the 3rd courses of intervention, the temperature of the left hind paw pad area was decreased in the mild moxibustion group (P<0.05).@*CONCLUSION@#Suspension moxibustion could adjust the serum levels of TNF-α and IL-10 to improve the syndrome characteristics of RA rats of heat bi syndrome, such as joint redness, swelling, heat, pain and activity restriction. The effect of mild moxibustion is the most prominent. The findings could provide scientific basis for "moxibustion can be used for heat syndrome".

Tanshinone ⅡA activates PI3K/AKT signaling pathway to inhibit the apoptosis of mice cochlear pericytes induced by high glucose / 中华耳鼻咽喉头颈外科杂志

Objective: To investigate whether tanshinone ⅡA can protect the apoptosis of mice cochlear pericytes induced by high glucose and its specific protective mechanism, so as to provide experimental evidence for the prevention and treatment of diabetic hearing loss. Methods: C57BL/6J male mice were used to prepare type 2 diabetes model, which were divided into normal (NG) group, diabetic (DM) group, diabetic+tanshinone ⅡA (HG+tanshinone ⅡA) group and tanshinone ⅡA group. Each group had 10 animals. Primary cochlear pericytes were divided into NG group, HG group (high glucose 35 mmol/L), HG+tanshinone ⅡA (1, 3, 5 μmol/L) group, HG+Tanshinone ⅡA+LY294002 (PI3K/AKT pathway inhibitor) group, LY294002 group, tanshinone ⅡA group and DMSO group. Auditory brainstem response (ABR) was used to measure hearing threshold. Evans blue was used to detect the permeability of blood labyrinth barrier in each group. TBA methods were used to detect oxidative stress levels in various organs of mice. Morphological changes of stria vascularis were observed by hematoxylin-eosin staining (HE). Evans blue was used to detect the vascular labyrinth barrier permeability in cochlea. The expression of apoptosis protein in stria vascularis pericytes was observed by immunofluorescence. Pericytes apoptosis rate was observed by flow cytometry. DCFH-DA was combined with flow cytometry to detect intracellular ROS content, and Western blot was used to detect the expression of apoptotic proteins (Cleaved-caspase3, Bax), anti-apoptotic proteins (BCL-2) and pathway proteins (PI3K, p-PI3K, AKT, p-AKT). SPSS software was used for statistical analysis. Independent sample t test was performed, and P<0.05 was considered statistically significant. Results: Animal experiments: Tanshinone ⅡA decreased the hearing threshold of DM group [(35.0±3.5) dB SPL vs. (55.3±8.1) dB SPL] (t=4.899, P<0.01), decreased the oxidative stress level in cochlea (t=4.384, P<0.05), improved the structure disorder, atrophy of cochlea vascular lines, vacuole increased phenomenon. Tanshinone ⅡA alleviated the increased permeability of the blood labyrinth barrier [Evans blue leakage (6.84±0.27) AU vs. (8.59±0.85) AU] in the cochlea of DM mice (t=2.770, P<0.05), reversed the apoptotic protein: Caspase3 (t=4.956, P<0.01) and Bax (t=4.388, P<0.05) in cochlear vascularis. Cell experiments: Tanshinone ⅡA decreased intracellular ROS content in a concentration-dependent way (t=3.569, P<0.05; t=4.772, P<0.01; t=7.494, P<0.01); Tanshinone ⅡA decreased apoptosis rate and apoptotic protein, and increased the expression of anti-apoptotic protein, p-PI3K/PI3K and p-AKT/AKT in concentration-dependent manner (all P values<0.05); LY294002 reversed the protective effect of tanshinone ⅡA on pericytes apoptosis (all P values<0.05). Conclusion: Tanshinone ⅡA can inhibit the apoptosis of cochlear pericytes induced by high glucose by reducing oxidative stress level and activating PI3K/AKT signaling pathway under high glucose environment, thus playing a protective role in diabetic hearing loss.

Schisandrin B Inhibits NLRP3 Inflammasome Pathway and Attenuates Early Brain Injury in Rats of Subarachnoid Hemorrhage / 中国结合医学杂志

OBJECTIVE@#To determine whether Schisandrin B (Sch B) attenuates early brain injury (EBI) in rats with subarachnoid hemorrhage (SAH).@*METHODS@#Sprague-Dawley rats were divided into sham (sham operation), SAH, SAH+vehicle, and SAH+Sch B groups using a random number table. Rats underwent SAH by endovascular perforation and received Sch B (100 mg/kg) or normal saline after 2 and 12 h of SAH. SAH grading, neurological scores, brain water content, Evan's blue extravasation, and terminal transferase-mediated dUTP nick end-labeling (TUNEL) staining were carried out 24 h after SAH. Immunofluorescent staining was performed to detect the expressions of ionized calcium binding adapter molecule 1 (Iba-1) and myeloperoxidase (MPO) in the rat brain, while the expressions of B-cell lymphoma 2 (Bcl-2), Bax, Caspase-3, nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3), apoptosis-associated specklike protein containing the caspase-1 activator domain (ASC), Caspase-1, interleukin (IL)-1β, and IL-18 in the rat brains were detected by Western blot.@*RESULTS@#Compared with the SAH group, Sch B significantly improved the neurological function, reduced brain water content, Evan's blue content, and apoptotic cells number in the brain of rats (P<0.05 or P<0.01). Moreover, Sch B decreased SAH-induced expressions of Iba-1 and MPO (P<0.01). SAH caused the elevated expressions of Bax, Caspase-3, NLRP3, ASC, Caspase-1, IL-1β, and IL-18 in the rat brain (P<0.01), all of which were inhibited by Sch B (P<0.01). In addition, Sch B increased the Bcl-2 expression (P<0.01).@*CONCLUSION@#Sch B attenuated SAH-induced EBI, which might be associated with the inhibition of neuroinflammation, neuronal apoptosis, and the NLRP3 inflammatory signaling pathway.

Verification with the utility of an established rapid assessment of brain safety for newly developed vaccines / 한국실험동물학회지

In the twenty-first century, high contagious infectious diseases such as SARS (Severe Acute Respiratory Syndrome), MERS (Middle East Respiratory Syndrome), FMD (Foot-and-Mouth Disease) and AI (Avian Influenza) have become very prevalent, causing treat harm to humans and animals in aspect of public health, and economical issues. The critical problem is that newly-reported infectious diseases that humans firstly experience are expected to continue to emerge, and these diseases will be spreading out rapidly. Therefore, rapid and safe supplies of effective vaccines are most pivotal to prevent the rapid prevalent of new infection, but international standards or assessing protocol the safety of urgent vaccines are not established well. In our previous study, since we established a module to assess the brain safety of urgent vaccines, therefore, it is necessary to verify that this established module for assessing brain safety could work effectively in commercially available two vaccines (one killed- and on live-vaccines). We compared the results of Evans blue (EB) assay and qPCR analysis by injection of two kinds of vaccines, PBS and Lipopolysaccharide (LPS) under the condition of the module previously reported. We confirmed that the brain safety test module for urgent vaccine we established is very reproducible. Therefore, it is believed that this vaccine safety testing method can be used to validate brain safety when prompt supply of a newly developed vaccines is needed.

MiR-590 Inhibits Endothelial Cell Apoptosis by Inactivating the TLR4/NF-κB Pathway in Atherosclerosis

PURPOSE: Previous study has well documented the anti-apoptotic effects of miR-590 on oxidized low-density lipoprotein (ox-LDL)-treated endothelial cells (ECs). However, the mechanism underlying the anti-apoptotic effects of miR-590 in ox-LDL-treated ECs remains to be further addressed. MATERIALS AND METHODS: ApoE(−/−) mice fed with a high-fat diet (HFD) and human aortic endothelial cells (HAECs) treated with ox-LDL were used as in vivo and in vitro models of atherosclerosis. The expressions of miR-590 and toll-like receptor 4 (TLR4) were detected by quantitative real-time PCR and Western blot, respectively. Atherosclerotic lesion analysis was performed using Evans blue and hematoxylin-eosin staining. Cell proliferation was assessed by MTT assay. Apoptosis was examined using flow cytometry analysis and Western blot analysis of Cleaved poly (ADP-ribose) polymerase (PARP) and Cleaved Caspase-3 levels. The effect of miR-590 on TLR4/nuclear factor kappa B (NF-κB) pathway was evaluated by Western blot. Binding between miR-590 and TLR4 was confirmed by luciferase reporter assay and Western blot. RESULTS: miR-590 was downregulated in the aorta tissues from HFD-fed apoE(−/−) mice and ox-LDL-treated HAECs. miR-590 overexpression inhibited atherosclerotic lesion in HFD-induced apoE(−/−) mice and promoted proliferation and inhibited apoptosis of ox-LDL-treated HAECs. Additionally, TLR4 was identified as a direct target of miR-590 in ox-LDL-treated HAECs. Moreover, anti-miR-590 reversed TLR4 knockdown-mediated promotion of cell proliferation and suppression of apoptosis in ox-LDL-treated HAECs. miR-590 overexpression suppressed the TLR4/NF-κB pathway, and inhibition of the TLR4/NF-κB pathway promoted cell proliferation and impeded apoptosis in ox-LDL-treated HAECs. CONCLUSION: miR-590 promoted proliferation and blocked ox-LDL-induced apoptosis in HAECs through inhibition of the TLR4/NF-κB pathway.

β₂-Adrenoceptor Blockade Deteriorates Systemic Anaphylaxis by Enhancing Hyperpermeability in Anesthetized Mice

PURPOSE: Patients treated with propranolol, a nonselective β-adrenoceptor antagonist, develop severe anaphylaxis, but the mechanism remains unknown. We determined effects of β₁- and β₂-adrenoceptor antagonists on the anaphylaxis-induced increase in vascular permeability in mice. METHODS: In anesthetized ovalbumin-sensitized C57BL mice, mean arterial blood pressure (MBP) was measured, and Evans blue dye extravasation and hematocrit (Hct) were assessed at 20 minutes after antigen injection. The following pretreatment groups (n=7/group) were studied: (1) sensitized control (non-pretreatment), (2) propranolol, (3) the selective β₂-adrenoceptor antagonist ICI 118,551, (4) the selective β₁-adrenoceptor antagonist atenolol, (5) adrenalectomy, (6) the selective β₂-adrenoceptor agonist terbutaline, and (7) non-sensitized groups. RESULTS: The antigen injection decreased MBP, and increased Hct and vascular permeability in the kidney, lung, mesentery, and intestine, but not in the liver or spleen. Pretreatment with ICI 118,551, propranolol and adrenalectomy, but not atenolol, reduced the survival rate and augmented the increases in Hct and vascular permeability in the kidney, intestine, and lung as compared with the sensitized control group. Pretreatment with terbutaline abolished the antigen-induced alterations. Plasma epinephrine levels were increased significantly in the sensitize control mice. CONCLUSIONS: Blockade of β₂-adrenoceptor can deteriorate systemic anaphylaxis by augmenting hyperpermeability-induced increase in plasma extravasation by inhibiting beneficial effects of epinephrine released from the adrenal glands in anesthetized mice.

Angiopoietin-1 and Angiopoietin-2 Expression Imbalance Influence in Early Period After Subarachnoid Hemorrhage / 대한배뇨장애요실금학회지

PURPOSE: Microvascular endothelial integrity is important for maintaining the blood-brain barrier (BBB). However, subarachnoid hemorrhage (SAH) disrupts this integrity, making the BBB dysfunctional—an important pathophysiological change after SAH. Angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) regulate microvascular permeability by balancing each other’s expression. METHODS: This study investigated the dynamics of Ang-1 and Ang-2 expression after SAH and the protective effect of Ang-1 on BBB functioning using an endovascular puncture model of rat SAH. The Ang-1 and Ang-2 expression in brain tissue was determined by immunohistochemistry. In addition, Western blotting was used to estimate Ang-1 and Ang-2 concentration and to compare them at 6–72 hours post-SAH cortex and hippocampus. Evans blue viability assay was used to evaluate BBB permeability, and neurological testing was implemented to evaluate neurological impairment during SAH. RESULTS: It was found that following SAH, Ang-1 expression decreases and Ang-2 expression increases in the cortex, hippocampus, and microvessels. The Ang-1/Ang-2 ratio decreased as quickly as 6 hours after SAH and reached its lowest 1 day after SAH. Finally, it was found that exogenous Ang-1 reduces SAH-associated BBB leakage and improves neurological function in post-SAH rats. CONCLUSIONS: Our findings suggest that the equilibrium between Ang-1 and Ang-2 is broken in a period shortly after SAH, and the treatment of exogenous Ang-1 injection alleviates neurological dysfunctions through decreasing BBB destruction.

Efficacy of fungal decolorization of a mixture of dyes belonging to different classes

Dyes are the most difficult constituents to remove by conventional biological wastewater treatment. Colored wastewater is mainly eliminated by physical and chemical procedures, which are very expensive and have drawbacks. Therefore, the advantage of using biological processes, such as the biotransformation of dyes, is that they may lead to complete mineralization or formation of less toxic products. To prove the possibility of using fungal processes for decolorization and other applications, the analysis of the toxicity of the processes' products is required. The decolorization of the mixture of two dyes from different classes - triphenylmethane brilliant green and azo Evans blue (GB - total concentration 0.08 g/L, proportion 1:1 w/w) - by Pleurotus ostreatus (BWPH and MB), Gloeophyllum odoratum (DCa), RWP17 (Polyporus picipes) and Fusarium oxysporum (G1) was studied. Zootoxicity (Daphnia magna) and phytotoxicity (Lemna minor) changes were estimated at the end of the experiment. The mixture of dyes was significantly removed by all the strains that were tested with 96 h of experimental time. However, differences among strains from the same species (P. ostreatus) were noted. Shaking improved the efficacy and rate of the dye removal. In static samples, the removal of the mixture reached more than 51.9% and in shaken samples, more than 79.2%. Tests using the dead biomass of the fungi only adsorbed up to 37% of the dye mixture (strain BWPH), which suggests that the process with the living biomass involves the biotransformation of the dyes. The best results were reached for the MB strain, which removed 90% of the tested mixture under shaking conditions. Regardless of the efficacy of the dye removal, toxicity decreased from class V to class III in tests with D. magna. Tests with L. minor control samples were classified as class IV, and samples with certain strains were non-toxic. The highest phytotoxicity decrease was noted in shaken samples where the elimination of dye mixture was the best.

Permeability Parameters Measured with Dynamic Contrast-Enhanced MRI: Correlation with the Extravasation of Evans Blue in a Rat Model of Transient Cerebral Ischemia

OBJECTIVE: The purpose of this study was to correlate permeability parameters measured with dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) using a clinical 3-tesla scanner with extravasation of Evans blue in a rat model with transient cerebral ischemia. MATERIALS AND METHODS: Sprague-Dawley rats (n = 13) with transient middle cerebral artery occlusion were imaged using a 3-tesla MRI with an 8-channel wrist coil. DCE-MRI was performed 12 hours, 18 hours, and 36 hours after reperfusion. Permeability parameters (K(trans), v(e), and v(p)) from DCE-MRI were calculated. Evans blue was injected after DCE-MRI and extravasation of Evans blue was correlated as a reference with the integrity of the blood-brain barrier. Correlation analysis was performed between permeability parameters and the extravasation of Evans blue. RESULTS: All permeability parameters (K(trans), v(e), and v(p)) showed a linear correlation with extravasation of Evans blue. Among them, K(trans) showed highest values of both the correlation coefficient and the coefficient of determination (0.687 and 0.473 respectively, p < 0.001). CONCLUSION: Permeability parameters obtained by DCE-MRI at 3-T are well-correlated with Evans blue extravasation, and K(trans) shows the strongest correlation among the tested parameters.