Retro-orbital Sinus Research Articles

Safety of a Medicinal Product Based on Human Glial Progenitor Cells: A Pilot Study of Retrobulbar Administration in C57BL/6J Mice

INTRODUCTION. Stem cell therapy is a promising treatment method for various diseases and injuries, but its safety has yet to be determined. Therefore, studying the safety of administering a xenogeneic cell-based medicinal product (CBMP) into the retro-orbital venous sinus is essential for developing protocols for further studies of potential medicinal products for neurological conditions.AIM. The aim of the study was to determine the optimal dose of a CBMP derived from glial progenitor cells (GPCs) and to evaluate its safety during retrobulbar administration in C57BL/6J mice.MATERIALS AND METHODS. GPCs were derived from human induced pluripotent stem cells by stepwise differentiation and cultured in DMEM/F12 supplemented with epidermal growth factor and ciliary neurotrophic factor. Matrigel was used as a substrate. GPCs were injected into the retro-orbital venous sinus of male C57BL/6J mice under isoflurane anaesthesia once a week for two months. The study analysed changes in biochemical blood parameters and behaviour. The quantities of activated astrocytes and glial cells were determined by postmortem immunohistochemical staining.RESULTS. The administration of GPCs at a dose of 500×103 cells/mouse, which was selected using literature data, induced an increase in the plasma levels of ala nine aminotransferase and aspartate aminotransferase. This could indicate cell damage and the development of inflammatory reactions. At doses reduced to one-third the initial GPC concentration or lower, the biochemical blood parameters of the treatment groups did not differ significantly from those of the control group. There were no significant differences in neuroinflammatory markers between the groups receiving GPCs at different doses, except for an increase in astrocyte activation at a dose of 150×103 cells/mouse, which could potentially indicate inflammatory processes in the brain. The study detected no pathological changes in the brain or cell damage markers in the blood of mice after retrobulbar GPC injections of 15×103 or 50×103 cells/mouse.CONCLUSIONS. The study results indicate that long-term therapy with GPCs is potentially safe for mice if the dose is optimal. The authors suggest using the optimal doses and the administration route established in this study for further research into the safety of intravenous administration of CBMPs for neurological conditions.

The Effect of Cinnamomum Burmanii Ethanol Extract on Isoniazid-induced Serum Levels of Serum Glutamate Piruvate Transaminase (SGPT) Wistar Strain Male Rats

Hepatotoxicity can be caused by excessive drug use, triggered by increased oxidative stress which is considered as the initial mechanism of Drug Induced Liver Injury (DILI). One of the causes of DILI is Isoniazid. One of the plants acting as a hepatoprotector in protecting liver cells is cinnamon (Cinamommum burmanii). This study aims to determine the effect of 96% ethanolic cinnamon extract as a hepatoprotector against an increase in Alanine Transaminase (ALT) levels induced by isoniazid. The parameters used to assess liver damage were rat plasma ALT. The sample used in this study was rat plasma, which was taken through the retro orbital sinus. This research is an experimental study with a sample of 25 experimental animals divided into five groups, namely negative control (K1), positive control l (K2), and experimental group with cinnamon ethanolic extract at a dose of 100 mg/kgbw (K3), 200 mg/kgbw(K4), and 400 mg/kgbw(K5). The research used a post-test-only control group design. The results were analyzed using the One-way ANOVA test followed by the post hoc Tukey test. The results proved that cinnamon ethanolic extract at doses of 100 mg/kgbw (P = 0.029), 200 mg/kgbw (P = 0.001), and 400 mg/kgbw (P = 0.000) was effective in reducing plasma SGPT levels in isoniazid-induced rats when compared with the positive control group (K2). The most effective dose was at 200 mg/kgbw (K4). Thus, this proves that all of the doses in experimental groups have a hepatoprotective effect against isoniazid-induced liver damage. Keywords: Alanine Transaminase (ALT), cinnamon ethanol extract, Drug Induced Liver Injury (DILI), isoniazid

Biodistribution of PET radiotracers in tumor-bearing TRAMP mice administered by retroorbital or jugular vein injections

Nuclear medicine is an important tool for use in molecular imaging of important biological processes. Methods for intravenous delivery of radiotracers remains a challenge, with tail vein injections demonstrated to be technically difficult and lacking in reproducibility. Other intravenous methods include jugular vein (JV) injection, which requires a more invasive and precise microsurgical technique. Although the retroorbital (RO) sinus drains directly into the JV, and RO injections are minimally invasive and simpler to perform, they remain underutilized, perhaps due to a lack of studies demonstrating their performance. This study provides a comprehensive comparison of dynamic tissue biodistribution of three categories of commonly utilized radiopharmaceuticals between JV and RO injection methods in prostate tumor-bearing mice using PET-CT imaging. Results show that JV and RO injections have equivalent dynamic tissue biodistributions across the three categories of radiopharmaceuticals used: (1) small molecule measuring tumor metabolism (18F-flurodeoxyglucose [FDG]); (2) peptide-based probe measuring angiogenesis (64Cu-NOTA-PEG4-cRGD2); and (3) dextran-based nanocarrier (64Cu-NOTA-D20). Although RO injections present with some limitations such as type of injectate and difficulty for measuring acute, dynamic pharmacokinetics, this study demonstrates that RO injections are a viable, minimally invasive or stressful, and efficient alternative intravenous delivery technique for molecular imaging.

Cytotoxicity and cellular uptake capacity of a berberine-loaded nanogold/collagen drug delivery system in lung cancer

Lung cancer is the leading cause of cancer-related deaths worldwide. As an emerging technology, nanomedicine offers a more effective and biocompatible approach to treating lung cancer by targeting and killing uncontrolled cancer cells. This study fabricated a nanocarrier system comprising gold nanoparticles, type I collagen, and alkaline berberine (Au-Col-BB). It then thoroughly evaluated the physical and biological properties of this novel drug delivery system. The material properties were characterized using UV-Vis spectroscopy, Fourier-transform infrared spectroscopy, scanning electron microscopy, dynamic light scattering, energy-dispersive X-ray spectroscopy, and X-ray photoelectron spectroscopy. MTT and Calcein AM staining of A549 lung cancer cells demonstrated that treatment with Au-Col-BB significantly decreased cell viability. Annexin V/propidium iodide double staining and cell cycle progression revealed that treatment with Au-Col-BB increased apoptosis 1.82-fold in A549 cells but decreased apoptosis 0.94-cold in BEAS-2B normal lung cells. The percentage of A549 cells in the sub-G1 phase increased from 4.0 % to 11.6 % (p < 0.001), while the percentage of those in the S phase decreased from 14.5 % to 9.6 % (p < 0.010), indicating enhanced apoptosis and reduced proliferation. Treatment with Au-Col-BB significantly reduced the migration distance of A549 cells by 51 %. Au-Col-BB was found to primarily enter cells via clathrin-mediated endocytosis and autophagy, which were inhibited by chlorpromazine and bafilomycin A1, respectively. Retroorbital sinus injection of Au-Col-BB into BALB/c mice demonstrated its integrity and safety in vivo. Overall, this study suggests that the Au-Col-BB nanocarrier system is a promising alternative for antineoplastic drugs, offering effective cancer cell targeting and apoptosis induction while minimizing damage to surrounding healthy tissues.

Efficient retinal ganglion cells transduction by retro-orbital venous sinus injection of AAV-PHP.eB in mature mice

Gene therapy is one of the strategies that may reduce or reverse progressive neurodegeneration in retinal neurodegenerative diseases. However, efficiently delivering transgenes to retinal ganglion cells (RGCs) remains hard to achieve. In this study, we innovatively investigated transduction efficiency of adeno-associated virus (AAV)-PHP.eB in murine RGCs by retro-orbital venous sinus injection. Five doses of AAV-PHP.eB-EGFP were retro-orbitally injected in venous sinus in adult C57/BL6J mice. Two weeks after administration, RGCs transduction efficiency was quantified by retinal flat-mounts and frozen section co-labeling with RGCs marker Rbpms. In addition, safety of this method was evaluated by RGCs survival rate and retinal morphology. To conform efficacy of this new method, AAV-PHP.eB-CNTF was administrated into mature mice through single retro-orbital venous injection after optic nerve crush injury to evaluate axonal elongation. Results indicated that AAV- PHP.eB readily crossed the blood-retina barrier and was able to transduce more than 90% of RGCs when total dose of virus reached 5 × 1010 vector genomes (vg). Moreover, this technique did not affect RGCs survival rate and retinal morphology. Furthermore, retro-orbital venous delivery of AAV-PHP.eB-CNTF effectively transduced RGCs, robustly promoted axonal regeneration after optic nerve crush injury. Thus, novel AAV-PHP.eB retro-orbital injection provides a minimally invasive and efficient route for transgene delivery in treatment of retinal neurodegenerative diseases.

Focused ultrasound on the substantia nigra enables safe neurotensin-polyplex nanoparticle-mediated gene delivery to dopaminergic neurons intranasally and by blood circulation

Neurotensin-polyplex nanoparticles provide efficient gene transfection of nigral dopaminergic neurons when intracerebrally injected in preclinical trials of Parkinson’s disease because they do not cross the blood–brain barrier (BBB). Therefore, this study aimed to open BBB with focused ultrasound (FUS) on the substantia nigra to attain systemic and intranasal transfections and evaluate its detrimental effect in rats. Systemically injected Evans Blue showed that a two-pulse FUS opened the nigral BBB. Accordingly, 35 μL of neurotensin-polyplex nanoparticles encompassing the green fluorescent protein plasmid (79.6 nm mean size and + 1.3 mV Zeta-potential) caused its expression in tyrosine hydroxylase(+) cells (dopaminergic neurons) of both substantiae nigrae upon delivery via internal carotid artery, retro-orbital venous sinus, or nasal mucosa 30 min after FUS. The intracarotid delivery yielded the highest transgene expression, followed by intranasal and venous administration. However, FUS caused neuroinflammation displayed by infiltrated lymphocytes (positive to cluster of differentiation 45), activated microglia (positive to ionized calcium-binding adaptor molecule 1), neurotoxic A1 astrocytes (positive to glial fibrillary acidic protein and complement component 3), and neurotrophic A2 astrocytes (positive to glial fibrillary acidic protein and S100 calcium-binding protein A10), that ended 15 days after FUS. Dopaminergic neurons and axonal projections decreased but recuperated basal values on day 15 after transfection, correlating with a decrease and recovery of locomotor behavior. In conclusion, FUS caused transient neuroinflammation and reversible neuronal affection but allowed systemic and intranasal transfection of dopaminergic neurons in both substantiae nigrae. Therefore, FUS could advance neurotensin-polyplex nanotechnology to clinical trials for Parkinson’s disease.

Haematological Profile of Various Inbred Strains of Mice Maintained at IMTech Centre for Animal Resources and Experimentation (iCARE)

Animal models are frequently employed in scientific studies to collect data from an entire set of experiments. Haematological tests on these laboratory mice are essential for confirming scientific findings because diverse mouse strains have been used to examine various diseases and novel therapeutic methods. This work is aimed to establish haematological values in mouse strains used in biomedical research. Blood samples were taken from the retro-orbital sinus of mice from various strains and haematological parameters including total White Blood Cells (WBC), Red Blood Cells (RBC), Haemoglobin (HGB), Haematocrit (HCT), Mean Corpuscular Volume (MCV), Mean Corpuscular Haemoglobin (MCH), Mean Corpuscular Haemoglobin Concentration (MCHC), Red Blood Cell Dimension Width ( RDW), platelets, Mean Platelet Volume (MPV) and Platelet Dimension Width (PDW) were assessed. Compared to other strains (BALB/c, M:9.61, F:9.51; C57BL6/N, M:10.13, F:10.73; DBA/2, M:11.96, F:10.88), the mean values of WBCs (*103/μL) in DBA/1 mice were higher (M:14.02; F:14.65) and lower in NOD (M:7.14; F:6.54). The mean RBCs in different strains of mice used in this study ranged from 8.78-11.67*106/μL. Male mean haemoglobin valves in BALB/c, C57BL/6N, DBA/1, DBA/2 and NOD were 14.60, 13.32, 13.00, 14.35 and 13.48 (g/dL), respectively, while in females were 14.3, 14.09, 13.11, 12.65 and 13.24 (g/dL), respectively. The mean platelet counts in NOD mice (M:1826.60 and F:1557.80*103/μL) were significantly higher than other strains. Haematological profiles of the mice used in the study were consistent as reported previously for mice maintained in different animal facilities. The haematological values presented in this study can be used as reference values in biomedical research.

Smallpox vaccination in a mouse model.

The monkeypox epidemic, which became unusually widespread among humans in 2022, has brought awareness about the necessity of smallpox vaccination of patients in the risk groups. The modern smallpox vaccine variants are introduced either intramuscularly or by skin scarification. Intramuscular vaccination cannot elicit an active immune response, since tissues at the vaccination site are immunologically poor. Skin has evolved into an immunologically important organ in mammals; therefore, intradermal delivery of a vaccine can ensure reliable protective immunity. Historically, vaccine inoculation into scarified skin (the s.s. route) was the first immunization method. However, it does not allow accurate vaccine dosing, and high-dose vaccines need to be used to successfully complete this procedure. Intradermal (i.d.) vaccine injection, especially low-dose one, can be an alternative to the s.s. route. This study aimed to compare the s.s. and i.d. smallpox immunization routes in a mouse model when using prototypic second- and fourth-generation low-dose vaccines (104 pfu). Experiments were conducted using BALB/c mice; the LIVP or LIVP-GFP strains of the vaccinia virus (VACV) were administered into the tail skin via the s.s. or i.d. routes. After vaccination (7, 14, 21, 28, 42, and 56 days post inoculation (dpi)), blood samples were collected from the retro-orbital venous sinus; titers of VACV-specific IgM and IgG in the resulting sera were determined by ELISA. Both VACV strains caused more profound antibody production when injected via the i.d. route compared to s.s. inoculation. In order to assess the level of the elicited protective immunity, mice were intranasally infected with a highly lethal dose of the cowpox virus on 62 dpi. The results demonstrated that i.d. injection ensures a stronger protective immunity in mice compared to s.s. inoculation for both VACV variants.

Comparison of the Transduction Capacity of AAV5 and AAV PHP.eB Serotypes in Hippocampus Astroglia

In the present study, we compared the astrocyte-transducing potential of the relatively novel engineered AAV PHP.eB serotype and the well-examined conventional AAV5 serotype. We generated the AAV-based genetic constructs with membrane-bound fluorescent markers under the control of the astroglial promoter GfaABC1D to target astrocytes in vivo, either via local injection into the hippocampus (AAV5, AAV PHP.eB) or via systemic injection in the retro-orbital venous sinus (AAV PHP.eB). We collected new data on the transduction properties of locally injected PHP.eB and AAV5 viruses. A morphological examination and immunostainings of mouse brain slices revealed a dose-dependent shift of cellular tropism for locally injected PHP.eB from astroglial to astroglial-neuronal as the concentration increased. When the high doses of PHP.eB viruses were administered systemically, we observed strong astrocyte transduction throughout the brain, as confirmed by the morphological examination and GFAP immunostaining. AAV5 exhibited consistent astrocytic expression in all tested concentrations. The obtained results suggest that AAV5 is more suitable for astrocyte targeting in routine stereotaxic viral injection experiments. The widely used engineered PHP.eB capsid was originally designed for the transduction of both neurons and glia. Dual cellular tropism of PHP.eB viruses, observed using different doses and different delivery protocols (local vs. systemic), suggests that the usage of AAV5 is more reliable for astrocyte labeling and that intrahippocampal injection is more suitable than systemic injection for the preferential labeling of hippocampal astroglia.

Molecular detection of Leptospira and Bartonella in Mastomys natalensis and its ectoparasites in Morogoro, Tanzania

Abstract Rodents play an important role in the transmission of zoonotic diseases. This study investigated the prevalence of Leptospira spp. and Bartonella spp. in Mastomys natalensis and its ectoparasites (fleas and mites) in selected villages of Morogoro, Tanzania. Mastomys natalensis were captured live in fallow habitats using Sherman® traps and anesthetized using Halothane. Blood samples were obtained from the retroorbital sinus Ectoparasites were removed from the fur using a hard brush and preserved in 70 % ethanol. Real time–qPCR was used to detect Leptospira spp. and Bartonella spp. from Mastomys natalensis blood and ectoparasites respectively. The study revealed a relatively larger number of males than females captures. Leptospira spp. was demonstrated in one out of 100 Mastomys natalensis. For Bartonella spp., prevalence of (14 %) was recorded in mites with a higher proportion in mites from adult male Mastomys natalensis than females. Upon Sanger sequencing, four positive samples showed a complete sequence of the ITS gene. Indicating that all samples belonged to Uncultured Bartonella. Low prevalence of Leptospira spp. and a high prevalence of Bartonella spp. was observed in Mastomys natalensis. Further exploration of rodent pathogens is recommended to raise awareness of the role of commensal rodents in disease transmission via their ectoparasites.

Differentiation Induction of Mesenchymal Stem Cells by a Au Delivery Platform.

Au decorated with type I collagen (Col) was used as a core material to cross-link with stromal cell-derived factor 1α (SDF1α) in order to investigate biological performance. The Au-based nanoparticles were subjected to physicochemical determination using scanning electron microscopy (SEM), dynamic light scattering (DLS) and ultraviolet-visible (UV-Vis) and Fourier-transform infrared spectroscopy (FTIR). Mesenchymal stem cells (MSCs) were used to evaluate the biocompatibility of this nanoparticle using the MTT assay and measuring reactive oxygen species (ROS) production. Also, the biological effects of the SDF-1α-conjugated nanoparticles (Au-Col-SDF1α) were assessed and the mechanisms were explored. Furthermore, we investigated the cell differentiation-inducing potential of these conjugated nanoparticles on MSCs toward endothelial cells, neurons, osteoblasts and adipocytes. We then ultimately explored the process of cell entry and transportation of the nanoparticles. Using a mouse animal model and retro-orbital sinus injection, we traced in vivo biodistribution to determine the biosafety of the Au-Col-SDF1α nanoparticles. In summary, our results indicate that Au-Col is a promising drug delivery system; it can be used to carry SDF1α to improve MSC therapeutic efficiency.

Experimental application results of mesenchymal stem cell microvesicles in the mouse model of acute renal failure

An important role in restoration of damaged organs and tissues is played by mesenchymal stem cells (MSCs) and microvesicular particles (MV) produced by them. They can be a source of cytokines, anti- apoptotic and growth stimulating factors. In addition, MVs carry out transport of mRNA, miRNA, and signal proteins into damaged tissues. This increases the ability of cells to regenerate and to inhibit apoptosis, promote to angiogenesis and stimulate cell proliferation. The aim of our research was to study the immunoregulatory and pro-regenerative properties of mesenchymal stem cell microvesicles (MSC-MV) in a model of glycerol- induced acute renal failure (ARF) in mice. The experiments were carried out on CBA mice aged 3-4 months. AKI was induced by a single intramuscular injection of 50% glycerol. MSCs were obtained from the bone marrow of healthy animals and cultivated under standard conditions. Microvesicles were obtained by centrifugation at 12000g of MSC supernatant after induction of their apoptosis by culturing under oxygen deprivation conditions and in serum-free medium. MSC-MV was injected intravenously into the retroorbital sinus one day after induction of ARF. The MV dose was calculated as equivalent to (derived from) 1 million MSCs, which was 100 mL per mouse. Animals were taken out of the experiment on days 4 and 11 after MSC-MV injection. Blood plasma was taken to determine the level of creatinine, urine – for albumin analysis, kidneys – for histological examination. It has been shown that MVs induced by MSCs dose-dependently stimulated splenocyte proliferation in both spontaneous and Con-A induced tests. The addition of MV caused a decrease in doxorubicin-induced apoptosis of splenic lymphocytes in mice. Probably, in this case, MV produced by MSCs had an immunostimulatory and antiapoptotic effect. Also, MVs had a positive impact on the restoration of structure and function kidneys in a model of ARF in mice. The use of MSC-MV in treatment of acute renal failure induced by a single injection of 50% glycerol contributed to decrease albumin level urine and restoration of creatinine level in blood serum of animals. Morphological studies have shown decrease in the height cell and collecting duct diameter in the medulla and a decrease in the largest transverse diameter of superficial glomeruli in the renal cortex of sick mice. Thus, the obtained results indicate significant therapeutic and pro-regenerative properties of MSC-MV, which require further study.

High fidelity sensory-evoked responses in neocortex after intravenous injection of genetically encoded calcium sensors.

Two-photon imaging of genetically-encoded calcium indicators (GECIs) has traditionally relied on intracranial injections of adeno-associated virus (AAV) or transgenic animals to achieve expression. Intracranial injections require an invasive surgery and result in a relatively small volume of tissue labeling. Transgenic animals, although they can have brain-wide GECI expression, often express GECIs in only a small subset of neurons, may have abnormal behavioral phenotypes, and are currently limited to older generations of GECIs. Inspired by recent developments in the synthesis of AAVs that readily cross the blood brain barrier, we tested whether an alternative strategy of intravenously injecting AAV-PHP.eB is suitable for two-photon calcium imaging of neurons over many months after injection. We injected C57BL/6 J mice with AAV-PHP.eB-Synapsin-jGCaMP7s via the retro-orbital sinus. After allowing 5 to 34 weeks for expression, we performed conventional and widefield two-photon imaging of layers 2/3, 4 and 5 of the primary visual cortex. We found reproducible trial-by-trial neural responses and tuning properties consistent with known feature selectivity in the visual cortex. Thus, intravenous injection of AAV-PHP.eB does not interfere with the normal processing in neural circuits. In vivo and histological images show no nuclear expression of jGCaMP7s for at least 34 weeks post-injection.

A Western Diet Modifies the Activation Profile of Circulating Platelets

Background: While it is known that obesity increases the risk for cardiovascular disease (CVD), there has been no clear investigation into the effect an obesogenic diet exerts onto circulating platelets. As platelets are the critical actors in the formation of occlusive clots that manifest as a myocardial infarction or stroke, it is pertinent to establish any potential link between obesity and platelet activity. Methods: To examine the effect of a Western diet on baseline platelet activity, male C57BL6/J mice were fed either a control (10% fat, 7% sucrose, 0.3% salt Research Diets Inc. D12450J) or Western (40% fat, 30% sucrose, 8% salt Research Diets Inc. D06111701) diet ad libitum for 20 weeks beginning at the time of weaning. At 12, 16 and 20 weeks after beginning diet administration whole blood was collected via the retro-orbital sinus. Blood was used to generate platelet rich plasma (PRP) which was stimulated with collagen (20mg/ml) prior to staining for flow cytometry analysis. Flow cytometry staining utilized antibodies to recognize the platelet receptors GPIbα, P-Selectin and the activated form of integrin α IIb β 3 (JON/A). Additionally, body composition was likewise determined at these time points using Echo-MRI while bodyweight was measured weekly. Results: Western diet fed mice gained slightly more weight than control mice and had a higher percentage of body fat (measured by EchoMRI). Surprisingly, the two groups differed in GPIbα and activated α IIb β 3 expression on circulating platelets at all three observed time points. Specifically, platelets from control-fed mice displayed significantly higher GPIbα expression throughout the study while Western diet fed animals had higher levels of activated α IIb β 3 . There was no difference in platelet expression of P-Selectin between the groups at any time point. Conclusions: Platelet activation is not a binary process where platelets display a definitive “active” or “inactive” phenotype. Though terminal platelet activation elicits de-granulation and an increase in P-Selectin expression, the spectrum of platelet stimulation preceding this is marked by variable incidences of integrin activation and GPIbα shedding. Here we demonstrate proof of principle that dietary factors, such as the consumption of a Western diet, can facilitate early changes to the stimulation pattern of circulating platelets which is then maintained long-term. While the consequence of this altered platelet stimulation profile is unknown with regards to the pathways of hemostasis and thrombosis, further investigations of downstream signaling and amplification pathways are needed. Funding: Arkansas Children's Research Institute/Arkansas Bioscience Institute Postgraduate Fellowship: AWD-037176 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Implementing a Novel Orthopedic Polytraumatic Injury Model in Laboratory Mice

Background/Objective: Traumatic injury is consistently the leading cause of death up to middle age and is the largest factor in reducing the United States economic productivity. Currently, there are few murine models that emulate orthopedic polytraumatic injury. Existing models typically require sophisticated vascular access resulting in higher costs. We have identified the need for a simple and easily reproducible model. Therefore, we developed a new polytrauma mouse model which included femoral fracture, quadriceps muscle crush, andhemorrhagic shock.&#x0D; Methods: Sixty 12-week-old, C57BL/6 female mice were used in this study. Briefly, a mid-shaft femur fracture was surgically induced on the right leg of each mouse and was stabilized by an intramedullary wire. Next, the ipsilateral quadriceps muscle underwent an interval of crushing injury. Then, to create hemorrhagic shock, mice had approximately 400μL removed through the retroorbital sinus. Lasty, mice were resuscitated with intraperitoneal 1.6 ml of saline 90 minutes later. Other mice served as uninjured controls. Splenocytes were harvested in the control group and at 0-, 1-, 4-, and 24-hours post-trauma (n=12/group). Flow cytometric analyses and animal survival were examined.&#x0D; Results: Of the 48 C57BL/6 mice undergoing the polytrauma model, all survived to their respective time point. There was a significant increase (p&lt;0.05) in neutrophils from the 4-hour cohort compared to the control. There were also significant increases in the 24-hour group in natural killer (NK) cells compared to control (p&lt;0.05), 0-hour (p&lt;0.0001),1-hour (p&lt;0.0001), and 4-hour groups (p&lt;0.05).&#x0D; Conclusion: The survival of all of the mice shows the effectiveness of this model to emulate non-fatal, poly-traumatic injuries. Additionally, increased levels of neutrophils, and NK cells, along with steady levels of B cells are consistent with the known immunologic traumatic response, further validating this model’s ability to create survivable polytrauma conditions.

Gene Silencing in the Brain with siRNA to Promote Long-Term Post-Stroke Recovery.

RNA interference is a promising strategy to degrade target RNAs of interest after stroke using small interfering RNA (siRNA). An optimized targeting such as combining a siRNA with a nontoxic transfection reagent that facilitates the effective delivery of siRNAs to the brain and subsequent cellular uptake after stroke is needed. Furthermore, an appropriate route of administration such as intravenous (tail vein or retro-orbital sinus) or cerebroventricular injection has to be used. Using siRNAs tagged with fluorescent probes shows the cellular uptake of siRNA. Efficacy and window of opportunity for a siRNA needs to be determined by testing multiple doses and time frame that alters the long-term functional outcomes. Real-time PCR/western blots can be used to determine the siRNA efficiency by evaluating the knockdown of the RNA/protein of interest. In siRNA studies, it is also essential to identify a proper dose (efficacious, but not toxic) by histopathologic testing to identify any toxicity in the peripheral organs and CNS. This chapter describes the strategies to deliver siRNAs to treat stroke and to facilitate post-stroke long-term recovery.

Метод взятия крови у мышей из периферической вены хвоста для исследования специфической активности эритропоэтина

One of the main quality indicators showing the effectiveness and safety of recombinant human erythropoietin drugs is their specific activity. The determination of this specific activity is recommended to carry out by the biological method in vivo on polycythemic and normocythemic mice. The main method of blood collection was the method of taking blood from the retroorbital sinus of the eye. The disadvantages of this method are the high risk of complications and the inability to take blood again after a short period of time. The next blood sampling is possible only 10 days after the procedure. This article describes a simple method of taking blood that allows us to select a sufficient amount of blood to study the specific activity of erythropoietin, minimize the traumatism of mouse tail tissues, reduce contamination of the obtained blood with foreign particles, minimize the number of complications, the possibility of repeated blood sampling after a short period of time.

DDEL-18. PEG-AU-BP NANOCARRIERS FOR DBTRG HUMAN GLIOMA CELLS

Abstract n-butylidenephthalide (BP) has been verified to have the superior ability of cancer cell toxicity. Furthermore, gold nanoparticles (Au) are biocompatible materials as well as the effective carrier to deliver bio-active molecular for cancer therapeutics. In present research, Au nanoparticles were firstly conjugated with polyethylene glycol (PEG), and cross-linked with BP to obtain PEG-Au-BP nanocarriers. The physicochemical properties were characterized through Ultraviolet-visible spectroscopy (UV-Vis), Fourier-transform infrared spectroscopy (FTIR) and Dynamic light scattering (DLS) to confirm the combination of PEG,Au,and BP. Through iv vitro, normal BAEC cells and DBTRG human glioma cells were treated with PEG-Au-BP nanocarriers to investigate the cytotoxicity, cell uptake efficiency, and mechanism of endocytic routes. According to the results of MTT assay, PEG-Au-BP could significantly inhibit DBTRG cells proliferation. Cell uptake efficiency and potential cellular transportation in both BAEC and DBTRG cell lines were observed to be significantly higher at the time point of 2hr and 24 hr. Moreover, the mechanisms of endocytosis, clathrin-mediated endocytosis and cell autophagy, were explored to routes for BAEC and DBTRG cells to absorb PEG-Au-BP nanocarriers. Next, the cell progression and apoptosis of DBTRG cells after PEG-Au-BP treatment was investigated by flow cytometry. The results figured out that PEG-Au-BP could remarkably regulate DBTRG cell cycle at Sub-G1 phase as well as induced more apoptotic cells. The expression of apoptotic-related proteins in DBTRG cells was determined. After the treatment of PEG-Au-BP, the apoptotic cascade proteins, p21, BAX, Act-caspase-3 were significantly expressed in DBTRG cells. Through in vivo, the tissue morphology and particle distribution in mice were examined by retro-orbital sinus injection with PEG-Au-BP nanocarriers. The results demonstrated the tissue integrity in brain (forebrain, midbrain, cerebellum), and other organs did not show significant destruction. The above finding elucidated PEG-Au-BP to be a potential nanodrug for brain tumor therapies.

Rift Valley fever MP-12 vaccine elicits an early protective immune response in mice

Rift Valley fever virus (RVFV) is an important mosquito-borne pathogen that causes outbreaks of severe disease in people and livestock throughout Africa and the Arabian Peninsula. The development of an effective veterinary and human vaccine to protect against Rift Valley fever (RVF) disease remains a high priority. The live attenuated RVFV MP-12 is a promising vaccine candidate for the prevention of RVF in both human and domestic ruminants. The aim of this study was to determine the onset of protective immunity elicted in mice by a single dose of this vaccine. Groups of CD-1 mice were vaccinated intraperitoneally with RVFV MP-12 vaccine and challenged on days 2, 5, 6 and 7 post-vaccination (PV) with a lethal dose of virulent RVFV. The mice were observed once daily for terminal morbidity and blood samples were obtained from the retro-orbital sinus complex on days 23 and 28 PV of surviving mice to determine RVFV neutralizing antibody titers. In one test, 2 of 3 mice challenged on day 2 PV survived and all 3 mice challenged at days 5 and 7 PV also survived. A second test of 10 mice per group was performed, and half (5) of those challenged at day 2 PV survived while all (10) survived challenge at day 4 and 6 PV. All surviving animals develop antibody that ranged from 1:80 to 1:1,280 PV. In a separate experiment, RVFV MP-12 vaccinated CD-1 mice, but not challenged developed a low viremia for the first 3 days PV and neutralzing antibody was detected on days 5 through day 28 PV. These findings demonstrated that the RVFV MP-12 vaccine elicited a rapid protective immune response in mice as early as 2 days PV, thus further supporting the effectiveness of this vaccine candidate for preventing RVF among humans and domestic ruminants.

Effect of Badri cow urine distillate on hemogram in Wistar rats

Present study was designed to observe the effect of Badri cow urine distillate on hematological parameters in Wistar rats. A total of 35 rats for this experiment were divided randomly into two groups viz. Group I (GI) and Group II (GII). Group I contain 20 rats and were kept as control. Group II contain 15 rats which were administered Badri cow urine distillate at dose of 0.25ml/day/rat with drinking water from 0 day post treatment till 90th day post treatment. The blood samples were collected from retro orbital sinus of rats at 0th, 30th, 60th, 90th day post treatment in EDTA vials for assessment of hematological parameters. There was significant increase in Hemoglobin (22.03%), Packed cell volume (21.1%), Total erythrocyte count (25.68%), Total leucocyte (33.02%), Absolute neutrophil count (20.61%) and Absolute lymphocyte count (61.63%) in treated rats as compare to control group. However, there was no significant difference observed in MCH, MCV and MCHC values in treated rats as compared to controls. The findings of this study suggest that the Badri cow urine distillate boosts the hematopoiesis in Wistar rats. This constitutes to be the first report of its kind on effect of Badri cow urine distillate on hemogram of Wistar rats. Keywords: Badri cow urine distillate, Hematology, Wistar rats.