Testicular injection of busulfan for recipient preparation in transplantation of spermatogonial stem cells in mice

2016 ◽  
Vol 28 (12) ◽  
pp. 1916 ◽  
Author(s):  
Yusheng Qin ◽  
Ling Liu ◽  
Yanan He ◽  
Wenzhi Ma ◽  
Huabin Zhu ◽  
...  
Keyword(s):  
Germ Cells ◽  
Fluorescent Protein ◽  
Positive Control ◽  
Negative Control ◽  
Seminiferous Tubules ◽  
Red Fluorescent Protein ◽  
Efferent Duct ◽  
Testicular Injection ◽  
Transplantation Recipient

Intraperitoneal busulfan injections are used to prepare recipients for spermatogonial stem cell (SSC) transplantation but they are associated with haematopoietic toxicity. Testicular injections of busulfan have been proposed to overcome this limitation. To date, testicular injections have not been studied in the mouse model. Therefore, in the present study we used ICR mice as recipients for SSC transplantation and prepared these mice by testicular injection of busulfan on both sides (2, 3, 4 or 6 mg kg–1 per side). Following this, donor germ cells expressing red fluorescent protein (RFP) from transgenic C57BL/6J male mice were transplanted into recipients via the efferent duct on Days 16–17 after busulfan treatment. Positive control mice were prepared by intraperitoneal injection of 40 mg kg–1 busulfan and negative control mice were treated with bilateral testicular injection of 50% dimethyl sulfoxide. On Day 49 after transplantation, recipient mice that were RFP-positive by in vivo imaging were mated with ICR female mice. Donor-derived germ cell colonies with red fluorescence were observed on Day 60 after transplantation, and donor-derived offspring were obtained. The results demonstrated that endogenous germ cells were successfully eliminated in the seminiferous tubules via testicular busulfan administration, and that exogenous SSCs successfully undergo spermatogenesis in the testes of recipient mice prepared by testicular injections of busulfan. In addition to its effects on recipient preparation, this method was safe in rodents and could possibly be adapted for use in other species.

scholarly journals Intra-testicular injection of adenoviral constructs results in Sertoli cell-specific gene expression and disruption of the seminiferous epithelium

Reproduction ◽  
2009 ◽  
Vol 137 (2) ◽  
pp. 361-370 ◽  
Author(s):  
R P Hooley ◽  
M Paterson ◽  
P Brown ◽  
K Kerr ◽  
P T K Saunders
Keyword(s):  
Seminiferous Tubule ◽  
Fluorescent Protein ◽  
Immune Cell ◽  
Adenoviral Vectors ◽  
Seminiferous Tubules ◽  
Specific Gene ◽  
Viral Particles ◽  
Junctional Complexes ◽  
Testicular Injection

Spermatogenesis is a complex process that cannot be modelledin vitro. The somatic Sertoli cells (SCs) within the seminiferous tubules perform a key role in supporting maturation of germ cells (GCs). Progress has been made in determining what aspects of SC function are critical to maintenance of fertility by developing rodent models based on the Cre/LoxP system; however, this is time-consuming and is only applicable to mice. The aim of the present study was to establish methods for direct injection of adenoviral vectors containing shRNA constructs into the testis as a way of inducing target-selective knock-downin vivo. We describe here a series of experiments using adenovirus expressing a green fluorescent protein (GFP) transgene. Injection via the efferent ductules resulted in SC-specific expression of GFP; expression levels paralleled the amount of infective viral particles injected. At the highest doses of virus seminiferous tubule architecture were grossly disturbed and immune cell invasion noted. At lower concentrations, the expression of GFP was variable/negligible, the seminiferous tubule lumen was maintained but stage-dependent GC loss and development of numerous basal vacuoles was observed. These resembled intercellular dilations of SC junctional complexes previously described in rats and may be a consequence of disturbances in SC function due to interaction of the viral particles with the coxsackie/adenovirus receptor that is a component of the junctional complexes within the blood testis barrier. In conclusion, intra-testicular injection of adenoviral vectors disturbs SC functionin vivoand future work will therefore focus on the use of lentiviral delivery systems.

Fluorescent proteins for in vivo imaging, where's the biliverdin?

2020 ◽  
Vol 48 (6) ◽  
pp. 2657-2667
Author(s):  
Felipe Montecinos-Franjola ◽  
John Y. Lin ◽  
Erik A. Rodriguez
Keyword(s):  
Hydrogen Peroxide ◽  
In Vivo Imaging ◽  
Near Infrared ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Fluorescent Imaging ◽  
Infrared Light ◽  
Red Fluorescent Protein ◽  
Color Palette

Noninvasive fluorescent imaging requires far-red and near-infrared fluorescent proteins for deeper imaging. Near-infrared light penetrates biological tissue with blood vessels due to low absorbance, scattering, and reflection of light and has a greater signal-to-noise due to less autofluorescence. Far-red and near-infrared fluorescent proteins absorb light >600 nm to expand the color palette for imaging multiple biosensors and noninvasive in vivo imaging. The ideal fluorescent proteins are bright, photobleach minimally, express well in the desired cells, do not oligomerize, and generate or incorporate exogenous fluorophores efficiently. Coral-derived red fluorescent proteins require oxygen for fluorophore formation and release two hydrogen peroxide molecules. New fluorescent proteins based on phytochrome and phycobiliproteins use biliverdin IXα as fluorophores, do not require oxygen for maturation to image anaerobic organisms and tumor core, and do not generate hydrogen peroxide. The small Ultra-Red Fluorescent Protein (smURFP) was evolved from a cyanobacterial phycobiliprotein to covalently attach biliverdin as an exogenous fluorophore. The small Ultra-Red Fluorescent Protein is biophysically as bright as the enhanced green fluorescent protein, is exceptionally photostable, used for biosensor development, and visible in living mice. Novel applications of smURFP include in vitro protein diagnostics with attomolar (10−18 M) sensitivity, encapsulation in viral particles, and fluorescent protein nanoparticles. However, the availability of biliverdin limits the fluorescence of biliverdin-attaching fluorescent proteins; hence, extra biliverdin is needed to enhance brightness. New methods for improved biliverdin bioavailability are necessary to develop improved bright far-red and near-infrared fluorescent proteins for noninvasive imaging in vivo.

scholarly journals UJI AKTIVITAS ANTIBAKTERI ISOLAT SENYAWA GOLONGAN TERPENOID DARI BIJI PEPAYA (Carica papaya L.) TERHADAP Staphylococcus aureus SECARA IN VIVO PADA KELINCI JANTAN

2021 ◽  
Vol 10 (1) ◽  
Author(s):  
Keyword(s):  
Staphylococcus Aureus ◽  
Ethyl Acetate ◽  
Carica Papaya ◽  
Test Group ◽  
Positive Control ◽  
Negative Control ◽  
Test Substance ◽  
Color Test ◽  
Group 15

research on the isolation of terpenoid class of compounds from the seeds of papaya (Carica papaya L.) and test its activity against Staphylococcus aureus in vivo in male rabbits. This research aims to prove that the terpenoid compounds isolated from the seeds of papaya (Carica papaya L.) can inhibit the growth of Staphylococcus aureus in vivo. Separation of terpenoid compounds by column chromatography ((eluent n-hexane: ethyl acetate: ethanol)) resulted in 25 eluates, and then merged based on the results of identification by TLC ((nhexane: ethyl acetate (8: 2)) and the color test reagent Lieburmann -Burchard produce 5 fraction groups. fraction D showed positive terpenoids with Rf 0.75 and the color purple with Lieburmann-Burchard reagent. study using 15 rabbits were divided into 5 groups: P1 (negative control), P2 (positive control) , P3 (5% of the test group), P4 (10% of the test group), P5 (test group 15%). each group was given the intracutaneous Staphylococcus aureus as 0,2ml on the backs of rabbits. Having symptoms of infection each group was given the test substance 3 times a day topically, the observed parameter is the diameter of the wound, and histopathological observations performed on days 3,6 and 9 Analysis of the results of research conducted using ANSIRA showed highly significant differences between groups (p <0.05). Then proceed with the analysis of the results of the analysis HSD test showed highly significant differences in the test group 5% to 10% of the test group and the test group 15%. Isolates terpenoid class of compounds from the seeds of papaya (Carica Papaya L.) with a concentration of 10% and 15% can inhibit the growth of Staphylococcus aureus. Keywords: Antibacterial, Staphylococcus aureus, male rabbits

scholarly journals In-Vivo Effectiveness of 5% Azadirachta indica Oil Cream as Anti-Scabies

2019 ◽  
Vol 4 (1) ◽  
pp. 10
Author(s):  
Patihul Husni ◽  
Mayang K. Dewi ◽  
Norisca A. Putriana ◽  
Rini Hendriani
Keyword(s):  
Azadirachta Indica ◽  
Recovery Time ◽  
Sarcoptes Scabiei ◽  
Positive Control ◽  
Negative Control ◽  
Neem Oil ◽  
Once Daily ◽  
Irritation Test ◽  
Primary Irritation

Scabies is an infectious skin disease caused by mite Sarcoptes scabiei. Neem tree (Azadirachta indica) has the potential to be used as an anti-parasite due to the presence of azadirachtin compound that is commonly found in the seeds. The aim of this study was to evaluate in-vivo effectiveness of neem oil as an anti-scabies. This study used an experimental method.  The effectiveness of the cream as an anti-scabies was tested on New Zealand white rabbits which were infected with scabies. Permethrin cream was used as a positive control and cream base was used as a negative control.  Cream was applied once daily and left for 8 hours. The data were analyzed using Kruskal Wallis and Mann Whitney. Dermal acute irritation test was performed by applying  0.5 g cream on the rabbit dorsal. We found that 5% neem oil cream was effective as an anti-scabies with 20-21 days recovery time. The recovery time is longer than permethrin cream (7-8 days), but shorter compared to negative control with recovery time over 30 days. Primary irritation index for 5% neem oil creams was 0, indicating negligible irritation category. In conclusion, A. indica cream was effective for the treatment of scabies although its recovery time is shorter than permethrin cream.  Keywords: effectiveness test, irritation test, neem oil cream, scabies

scholarly journals In-vivo Antiplasmodial Effect of Dihydroartemisinin-Piperaquine-Nitrofurantoin on Plasmodium berghei- Infected Mice

2021 ◽  
pp. 12-20
Author(s):  
Udeme O. Georgewill ◽  
Festus Azibanigha Joseph ◽  
Elias Adikwu
Keyword(s):  
Plasmodium Berghei ◽  
Blood Cells ◽  
Hematological Parameters ◽  
White Blood Cells ◽  
Positive Control ◽  
Negative Control ◽  
Antiplasmodial Activity ◽  
Significant Difference ◽  
Tract Infections

Nitrofurantoin (NT) used for the treatment of urinary tract infections may have antiplasmodial activity. Dihydroartemisinin-piperaquine (DP) is an artemisinin based combination therapy used for the treatment of malaria. This study evaluated the antiplasmodial effect of dihydroartemisinin-piperaquine-nitrofurantoin (DP-NT) on mice infected with Plasmodium berghei. Adult Swiss albino mice (30-35 g) of both sexes were used. The mice were randomly grouped, inoculated with Plasmodium berghei, and treated orally with DP (1.7/13.7 mg/kg), NT (57.1 mg/kg) and DP-NT (1.71/13.7/ 57.1 mg/kg), respectively using curative, prophylactic and suppressive tests. The negative control was orally treated with normal saline (0.3 mL), while the positive control was orally treated with chloroquine CQ (10mg/kg). After treatment, blood samples were collected and evaluated for percentage parasitemia, inhibitions and hematological parameters. Liver samples were evaluated for histological changes. The mice were observed for mean survival time (MST). Treatment with DP-NT decreased parasitemia levels when compared to individual doses of DP and NT with significant difference observed at p<0.05. DP-NT prolonged MST when compared to individual doses of DP and NT with significant difference observed at p<0.05. The decrease in packed cell volume, red blood cells, hemoglobin and increase in white blood cells in parasitized mice were significantly restored by DP-NT  when compared to individual doses of DP and NT with difference observed at p<0.05. DP-NT eradicated liver Plasmodium parasite.  NT remarkably increased the antiplasmodial activity of DP. DP-NT may be used for the treatment of malaria.

scholarly journals Study the effect of the mixture alcoholic extract of Peganum harmala seeds and cones of Cupressus sempervirens and their effect on viability of protoscolices of Echinococcus granulosus in vivo

2011 ◽  
Vol 5 (2) ◽  
pp. 44-52
Author(s):  
Noor Nihad Baker ◽  
Fawzia Ahmed AL-Shanawi
Keyword(s):  
Hydatid Cyst ◽  
Positive Control ◽  
Negative Control ◽  
Control Group ◽  
Alcoholic Extract ◽  
Peganum Harmala ◽  
Cupressus Sempervirens ◽  
Positive Group ◽  
Plant Mixture

this study included the using of the mixture of alcoholic extract Peganum harmala seeds and cones of Cupressus sempervirens at concentrations (1+50) mgml. And then experimentation on the mice injected with protoscolices and its comparison with the mice injected with only protoscolices (as positive control group), and the mice injected with normal saline (as negative control) to investigate the effect of plant mixture in vivo, it appeared of getting the reduction of hydatid cyst with percentage 100% in processed group with the mixture compared with positive group as its absence of the hydatid cyst in processed group. The lowering significantly occurred in the averages of the weights of the liver and spleen and the averages of its distension in processed groups and about of the positive group and which was approach to the negative group. Also study the tissular changes occurred in the liver and spleen, in the liver it occurred of changes in the liver cell and increase in the number of the kupffer cell as a defensive in the processed group were less than what it appeared in the positive control, but the spleen, it appeared the dilation of the whit pulp and the appearance of the cell composing of the hemic platelets (megakaryocyte cells) in the mice processed in comparison with negative control. These changes were of less acuity in the group processed. Thus from the results of this study at appeared the possibility of using the mixture in vivo in successful and safe way by it a capability of initiating the immunity system to the inhibition of the protoscolices and prevent the development of the secondary hydatid cyst in vivo without causing the negative side effect.

scholarly journals In Vivo Evaluation of the Genotoxic Effects of Poly (Butylene adipate-co-terephthalate)/Polypyrrole with Nanohydroxyapatite Scaffolds for Bone Regeneration

Materials ◽  
2019 ◽  
Vol 12 (8) ◽  
pp. 1330 ◽  
Author(s):  
Conceição de Maria Vaz Elias ◽  
Antônio Luiz Martins Maia Filho ◽  
Laryssa Roque da Silva ◽  
Fabrício Pires de Moura do Amaral ◽  
Thomas J. Webster ◽  
...  
Keyword(s):  
Micronucleus Test ◽  
Infusion Pump ◽  
Tail Length ◽  
Positive Control ◽  
Negative Control ◽  
Electrospinning Process ◽  
Genotoxic Effects ◽  
In Vivo Evaluation ◽  
In Vivo Assays

Here, butylene adipate-co-terephthalate/polypyrrole with nanohydroxyapatite (PBAT/PPy/nHAp) scaffolds were fabricated and characterized. The electrospinning process was carried out using 12 kV, a needle of 23 G, an infusion pump set at 0.3 mL/h, and 10 cm of distance. Afterwards, nHAp was directly electrodeposited onto PBAT/PPy scaffolds using a classical three-electrode apparatus. For in vivo assays (comet assay, acute and chronic micronucleus), 60 male albino Wistar rats with 4 groups were used in each test (n = 5): PBAT/PPy; PBAT/PPy/nHAp; positive control (cyclophosphamide); and the negative control (distilled water). Peripheral blood samples were collected from the animals to perform the comet test after 4 h (for damage) and 24 h (for repair). In the comet test, it was shown that the scaffolds did not induce damage to the % DNA tail and neither for tail length. After the end of 48 h (for acute micronucleus) and 72 h (for chronic micronucleus), bone marrow was collected from each rat to perform the micronucleus test. All of the produced scaffolds did not present genotoxic effects, providing strong evidence for the biological application of PBAT/PPy/nHAp scaffolds.

scholarly journals Essential oils as tick repellents on clothing

2019 ◽  
Vol 79 (2) ◽  
pp. 209-219 ◽  
Author(s):  
Oliver Soutar ◽  
Freya Cohen ◽  
Richard Wall
Keyword(s):  
Essential Oils ◽  
No Reduction ◽  
Positive Control ◽  
Negative Control ◽  
Tick Abundance ◽  
Spearmint Oil ◽  
Oregano Oil ◽  
Negative Controls ◽  
Tick Repellents

Abstract Essential oils show promise as natural alternatives to synthetic tick repellents, but few studies have investigated their repellent efficacy in vivo or under field conditions. Here, blanket-drags and standardised walks were employed to evaluate tick acquisition by 1 m2 cotton blankets or cotton trousers, respectively, in woodland edge habitats of known high tick abundance. Blankets and trousers had been treated with one of 5% oregano, rosemary, spearmint or thyme oils, 20% DEET (N,N-diethyl-3-methylbenzamide) (positive control) or ethanol excipient-only (negative control). The number of ticks present on the blankets or trousers differed significantly between treatments: spearmint oil treatments resulted in significantly fewer ticks than the negative controls for both blankets and trousers and significantly fewer ticks were present on the oregano oil treated blankets. For ticks that did attach to the trousers, the rate of drop off within 3 min was significantly higher for trousers treated with spearmint oil or thyme oil than ethanol, oregano oil and rosemary oil. No reduction in repellence was detected over a 24 h period between treatment and testing. The results suggest that 5% oregano and spearmint oils exhibit potential as natural clothing repellents, with an effective equivalence to 20% DEET.

250 ALL REGIONS OF THE MOUSE EPIDIDYMIS ARE ABLE TO PHAGOCYTIZE IMMATURE SPERMATOGENIC CELLS

2013 ◽  
Vol 25 (1) ◽  
pp. 272
Author(s):  
P. Ramos-Ibeas ◽  
E. Pericuesta ◽  
R. Fernandez-Gonzalez ◽  
M. A. Ramirez ◽  
A. Gutierrez-Adan
Keyword(s):  
Quality Control ◽  
Green Fluorescent Protein ◽  
Germ Cells ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Sperm Quality ◽  
Seminiferous Tubules ◽  
Spermatogenic Cells ◽  
Green Fluorescent ◽  
Immature Cells

Successful mammalian fertilization requires gametes with an intact structure and functionality. Although it is well known that epididymal functions are sperm maturation, sustenance, transport, and storage, there is controversial information about its role in sperm quality control, and it has been suggested that some regions of the rat epididymis are able to phagocytize germ cells. Our objective was to analyse whether different segments of the mouse epididymal epithelium act as a selection barrier for abnormal spermatogenic cells by removing immature cells from the lumen by phagocytosis. To detect the presence of immature germ cells along the epididymis, transgenic mice expressing enhanced green fluorescent protein under a Deleted in Azoospermia-Like (mDazl) promoter were generated. The transgenic animals express specifically enhanced green fluorescent protein in spermatogonias, spermatocytes, and spermatids; thus, immature spermatogenic cells can be easily identified by fluorescence microscopy. Colchicine, a microtubule disruptor that leads to severe alterations in the architecture of the seminiferous tubules, was administered in the rete testis to induce the release of immature germ cells into the epididymis. Mice were killed daily, from Day 1 to 8 post-administration, and epididymides were collected and observed under a fluorescence stereoscope to determine the transit of immature germ cells along the epididymis. Epididymides from control mice without colchicine administration were also collected. Fluorescent immature germ cells were present in the caput epididymis 24 h after colchicine administration, and they progressed through the corpus and cauda, leaving the epididymis 7 days after colchicine administration. After fluorescence observation, epididymides were fixed, sectioned, and stained with hematoxylin solution. Immature germ cells and phagosomes were not observed in control epididymides. By contrast, the presence of phagosomes in the principal cells of the epididymal epithelium containing immature germ cells in different degrees of degradation was observed by light microscopy in mice injected with colchicine. Phagocytosis was observed along the epididymis following the main wave of fluorescent immature cells. Thus, when immature cells had reached the corpus epididymis, phagocytosis was detected in several segments of the caput epididymis. Later, once the immature cells had arrived to the cauda epididymis or had abandoned the epididymis, phagocytosis was observed in the corpus and cauda epididymis. The presence of phagosomes was observed in all epididymal tubules within a phagocytosis area. In conclusion, we demonstrated that the epididymal epithelium is engaged in sperm quality control by clearing immature germ cells after a massive shedding into the epididymal lumen, and that this phenomenon is not restricted to a specific segment of the epididymis.

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