Small Experimental Animals Research Articles

Chelating the valley of death: Deferoxamine's path from bench to wound clinic.

There is undisputable benefit in translating basic science research concretely into clinical practice, and yet, the vast majority of therapies and treatments fail to achieve approval. The rift between basic research and approved treatment continues to grow, and in cases where a drug is granted approval, the average time from initiation of human trials to regulatory marketing authorization spans almost a decade. Albeit with these hurdles, recent research with deferoxamine (DFO) bodes significant promise as a potential treatment for chronic, radiation-induced soft tissue injury. DFO was originally approved by the Food and Drug Administration (FDA) in 1968 for the treatment of iron overload. However, investigators more recently have posited that its angiogenic and antioxidant properties could be beneficial in treating the hypovascular and reactive-oxygen species-rich tissues seen in chronic wounds and radiation-induced fibrosis (RIF). Small animal experiments of various chronic wound and RIF models confirmed that treatment with DFO improved blood flow and collagen ultrastructure. With a well-established safety profile, and now a strong foundation of basic scientific research that supports its potential use in chronic wounds and RIF, we believe that the next steps required for DFO to achieve FDA marketing approval will include large animal studies and, if those prove successful, human clinical trials. Though these milestones remain, the extensive research thus far leaves hope for DFO to bridge the gap between bench and wound clinic in the near future.

Orthovoltage X-ray Minibeam Radiation Therapy for the Treatment of Ocular Tumours-An In Silico Evaluation.

(1) Background: Radiotherapeutic treatments of ocular tumors are often challenging because of nearby radiosensitive structures and the high doses required to treat radioresistant cancers such as uveal melanomas. Although increased local control rates can be obtained with advanced techniques such as proton therapy and stereotactic radiosurgery, these modalities are not always accessible to patients (due to high costs or low availability) and side effects in structures such as the lens, eyelids or anterior chamber remain an issue. Minibeam radiation therapy (MBRT) could represent a promising alternative in this regard. MBRT is an innovative new treatment approach where the irradiation field is composed of multiple sub-millimetric beamlets, spaced apart by a few millimetres. This creates a so-called spatial fractionation of the dose which, in small animal experiments, has been shown to increase normal tissue sparing while simultaneously providing high tumour control rates. Moreover, MBRT with orthovoltage X-rays could be easily implemented in widely available and comparably inexpensive irradiation platforms. (2) Methods: Monte Carlo simulations were performed using the TOPAS toolkit to evaluate orthovoltage X-ray MBRT as a potential alternative for treating ocular tumours. Dose distributions were simulated in CT images of a human head, considering six different irradiation configurations. (3) Results: The mean, peak and valley doses were assessed in a generic target region and in different organs at risk. The obtained doses were comparable to those reported in previous X-ray MBRT animal studies where good normal tissue sparing and tumour control (rat glioma models) were found. (4) Conclusions: A proof-of-concept study for the application of orthovoltage X-ray MBRT to ocular tumours was performed. The simulation results encourage the realisation of dedicated animal studies considering minibeam irradiations of the eye to specifically assess ocular and orbital toxicities as well as tumour response. If proven successful, orthovoltage X-ray minibeams could become a cost-effective treatment alternative, in particular for developing countries.

Acidic Exo-Polysaccharide Obtained from Bacillus sp. NRC5 Attenuates Testosterone-DMBA-Induced Prostate Cancer in Rats via Inhibition of 5 α-Reductase and Na+/K+ ATPase Activity Mechanisms

Bacillus sp. NRC5 is a new strain that grows in Egyptian beaches. This strain produces acidic exo-polysaccharide that have excellent antioxidant, anti-inflammatory and anti-tumor properties. The current study aimed to introduce a new natural product feasible for prostate cancer therapies. The anti-prostate cancer of acidic exo-polysaccharide produced from marine Bacillus sp. NRC5 (EBPS) was determined using 7,12-dimethylbenz-(a)-anthracene; DMBA-induced prostate cancer in male Sprague Dawley rats. Rats were subcutaneously injected with testosterone (3 mg/kg/day for 3 months) and a single dose of DMBA (65 mg/kg) for induction of prostate cancer. EBPS was administrated orally at dose 200 mg/kg/day for 3 months. To study protective effect of EBPS, animals received EBPS before cancer induction, meanwhile in therapeutic effect animals received EBPS after cancer induction. EBPS debug oxidative stress and inflammatory conditions associated with prostate cancer. EBPS either protective or therapeutic material considerably reduced cancer growth rate-limiting enzyme—i.e., 5-α-reductase (46.89 ± 1.72 and 44.86 ± 2.56 µg Eq/mL) and Na+/K+ ATPase (0.44 ± 0.03 and 0.42 ± 0.02 µg Eq/mL), compared to cancer control (69.68 ± 3.46 µg Eq/mL). In addition, both cancer biomarkers—i.e., prostate-specific antigen and carcinoembryonic antigen were significantly lowered as evidence of the ability of EBPS to protect and treat prostate cancer in chemically induced rats. EBPS showed protective and therapeutic efficacy on testosterone–DMBA-induced prostate cancer rats with a good safety margin. This study may go to clinical trials after a repeated study on another type of small experimental animal, their offspring, and one big experimental animal.

A Sparse Orthogonal Collimators System for Experiments on Small-Animal Scale

<h3>Purpose/Objective(s)</h3> Intensity-modulated radiotherapy (IMRT) is the clinical gold standard for concentrated dose delivery. However, IMRT for small animal experiments has faced paramount technical challenges, preventing effective clinical translation of preclinical experiments. Sparse orthogonal collimator (SOC) based on the Rectangular Aperture Optimization (RAO) framework is the first general-purpose small animal IMRT platform, but its original planning algorithm was inefficient for complex cases such as total marrow irradiation (TMI) of mice. Here we introduce a significantly improved optimization framework for efficient SOC-based treatment planning and demonstrate TMI efficacy. <h3>Materials/Methods</h3> The SOC system is designed and fabricated with four orthogonal, double-focused tungsten leaf pairs that are capable of forming two rectangles simultaneously. The rectangles were optimized using RAO. Since the Kronecker product representation of the rectangular aperture is the main limitation of the computational performance, we reformulated matrix multiplication in the data fidelity term using multiplication with small matrices instead of the Kronecker product of the matrices. The optimization problem can then be efficiently solved using the Fast Iterative Shrinkage-Thresholding Algorithm (FISTA). Five mice CTs with manually delineated bone marrow and OARs, including the kidneys, lungs, heart, spleen, liver and bowel, were used in the planning study. 12 Gy was prescribed to the bone marrow target while expecting the bones to receive 2.5X higher doses due to kV photoelectric interactions. RAO plans were compared with manual 3D plans with custom lead blocks. <h3>Results</h3> SOC plans markedly improved TMI PTV hot spots and significantly reduced OAR doses compared with other experimental data. The PTV D5, indicating the maximum dose, was reduced by 21.6%. The average doses to the kidneys, lungs, heart, liver, and bowel were reduced by 98.4%, 95.7%, 79.1%, 97.7%, and 82.8%. The average plan optimization total time was 40 minutes. <h3>Conclusion</h3> This study shows the feasibility of creating SOC-based RAO plans for complex and large targets such as TMI. The marked improvement in OAR sparing allows mouse experiments to more closely simulate human treatments, which is critical for TMI, whose treatment response and outcome heavily depend on both the target and off-target doses and toxicity.

An efficient rectangular optimization method for sparse orthogonal collimator based small animal irradiation

Objective. Intensity-modulated radiotherapy (IMRT) is widely used in clinical radiotherapy, treating varying malignancies with conformal doses. As the test field for clinical translation, preclinical small animal experiments need to mimic the human radiotherapy condition, including IMRT. However, small animal IMRT is a systematic challenge due to the lack of corresponding hardware and software for miniaturized targets. Approach. The sparse orthogonal collimators (SOC) based on the direct rectangular aperture optimization (RAO) substantially simplified the hardware for miniaturization. This study investigates and evaluates a significantly improved RAO algorithm for complex mouse irradiation using SOC. Because the Kronecker product representation of the rectangular aperture is the main limitation of the computational performance, we reformulated matrix multiplication in the data fidelity term using multiplication with small matrices instead of the Kronecker product of the dose loading matrices. Solving the optimization problem was further accelerated using the Fast Iterative Shrinkage-Thresholding Algorithm (FISTA). Main results. Four mouse cases, including a liver, a brain tumor, a concave U-target, and a complex total marrow irradiation (TMI) case, were included in this study with manually delineated targets and OARs. Seven coplanar-field SOC IMRT (sIMRT) plans were compared with idealistic fluence map based IMRT (iIMRT) plans. For the first three cases with simpler and smaller targets, the differences between sIMRT plans and iIMRT plans in the planning target volumes (PTV) statistics are within 1%. For the TMI case, the sIMRT plans are superior in reducing hot spots (also termed D max) of PTV, kidneys, lungs, heart, and bowel by 20.5%, 31.5%, 24.67%, 20.13%, and 17.78%, respectively. On average, in four cases in this study, the sIMRT plan conformity is comparable to that of the iIMRT’s with lightly increased R50 and Integral Dose by 2.23% and 2.78%. Significance. The significantly improved sIMRT optimization method allows fast plan creation in under 1 min for smaller targets and makes complex TMI planning feasible while achieving comparable dosimetry to idealistic IMRT with fluence map optimization.

Feasibility of epidural temporal interference stimulation for minimally invasive electrical deep brain stimulation: simulation and phantom experimental studies

Objective. Temporal interference stimulation (TIS) has shown the potential as a new method for selective stimulation of deep brain structures in small animal experiments. However, it is challenging to deliver a sufficient temporal interference (TI) current to directly induce an action potential in the deep area of the human brain when electrodes are attached to the scalp because the amount of injection current is generally limited due to safety issues. Thus, we propose a novel method called epidural TIS (eTIS) to address this issue; in this method, the electrodes are attached to the epidural surface under the skull. Approach. We employed finite element method (FEM)-based electric field simulations to demonstrate the feasibility of eTIS. We first optimized the electrode conditions to deliver maximum TI currents to each of the three different targets (anterior hippocampus, subthalamic nucleus, and ventral intermediate nucleus) based on FEM, and compared the stimulation focality between eTIS and transcranial TIS (tTIS). Moreover, we conducted realistic skull-phantom experiments for validating the accuracy of the computational simulation for eTIS. Main results. Our simulation results showed that eTIS has the advantage of avoiding the delivery of TI currents over unwanted neocortical regions compared with tTIS for all three targets. It was shown that the optimized eTIS could induce neural action potentials at each of the three targets when a sufficiently large current equivalent to that for epidural cortical stimulation is injected. Additionally, the simulated results and measured results via the phantom experiments were in good agreement. Significance. We demonstrated the feasibility of eTIS, facilitating more focalized and stronger electrical stimulation of deep brain regions than tTIS, with the relatively less invasive placement of electrodes than conventional deep brain stimulation via computational simulation and realistic skull phantom experiments.

Preparation of 3-O-, 5-O- and N-palmitoyl derivatives of fumonisin B1 toxin and their characterisation with NMR and LC-HRMS methods

We have previously published six esterified O-acyl (EFB1) and three N-acyl fumonisin B1 derivatives extracted from rice cultures inoculated with Fusarium verticillioides, amongst these the identification of N-palmitoyl-FB1 has been clearly established in a spiking experiment. At that time, it was assumed that as in the case of O-acyl-FB1 derivatives, linoleic-, oleic- or palmitic acid esterify through the OH group on the 3C or 5C atom of the carbon chain of the fumonisins. In our most recent experiments, we have synthetically acylated the FB1 toxin and subsequently purified 3-O-palmitoyl- and 5-O-palmitoyl-FB1 toxins in addition to the N-palmitoyl-FB1 toxin. They were identified and characterised using 1H and 13C NMR as well as LC-HRMS. Our aim was the identification of the previously detected O-acyl-FB1 derivatives over the course of a spiking experiment, which were obtained through the solid-phase fermentation of Fusarium verticillioides. By spiking the three synthesized and identified components one-by-one into the fungal culture extract and analysing these cultures using LC-MS, it was clearly demonstrated that the F. verticillioides strain produced both the 5-O-palmitoyl-FB1 and N-palmitoyl-FB1 toxins, but did not produce 3-O-palmitoyl-FB1. Thus, it is highly probable that the components thought to be 3-O-acyl-(linoleoyl-, oleoyl-, palmitoyl-) FB1 derivatives in our previous communication are presumably 10-O-acyl-FB1 derivatives. Since these acylated FB1 derivatives can occur naturally in e.g. maize, the use of these synthesized components as reference materials is of great importance in order to obtain accurate qualitative and quantitative data on the occurrence of acylated fumonisins in different matrices including maize based feed samples. The production of these substances has also made it possible to test their toxicity in cell culture and small animal experiments.

Development of portable image-guidance system (PIGS) for preclinical small animal experiments with the cone-beam irradiation of X-RAD

Development of portable image-guidance system (PIGS) for preclinical small animal experiments with the cone-beam irradiation of X-RAD

Estimating the Neovascularity of Human Finger Tendon Through High-Frequency Ultrasound Micro-Doppler Imaging.

Neovascularization of injured tendons prolongs the proliferative phase of healing, but prolonged neovascularization may cause improper healing and pain. Currently, ultrasound Doppler imaging is used for measuring the neovascularization of injured tendons (e.g., Achilles tendon). However, the resolution of state-of-the-art clinical ultrasound machines is insufficient for visualizing the neovascularization in finger tendons. In this study, a high-frequency micro-Doppler imaging (HFμDI) based on 40-MHz ultrafast ultrasound imaging was proposed for visualizing the neovascularization in injured finger tendons during multiple rehabilitation phases. The vessel visibility was enhanced through a block-wise singular value decomposition filter and several curvilinear structure enhancement strategies, including the bowler-hat transform and Hessian-based vessel enhancement filtering. HFμDI was verified through small animal kidney and spleen imaging because the related vessel structure patterns of mice are well studied. Five patients with finger tendon injuries underwent HFμDI examination at various rehabilitation phases after surgery (weeks 11-56), and finger function evaluations were performed for comparisons. The results of small animal experiments revealed that the proposed HFμDI provides excellent microvasculature imaging performance; the contrast-to-noise ratio of HFμDI was approximately 15 dB higher than that of the conventional singular value decomposition filter, and the minimum detectable vessel size for mouse kidney was 35 μm without the use of contrast agent. In the human study, neovascularization was clearly observed in injured finger tendons during the early phase of healing (weeks 11-21), but it regressed from week 52 to 56. Finger rehabilitation appears to help reduce neovascularization; neovascular density decreased by approximately 1.8%-8.0% in participants after 4 weeks of rehabilitation. The experimental results verified the performance of HFμDI for microvasculature imaging and its potential for injured finger tendon evaluations.

In vivo non-invasive monitoring of tissue development in 3D printed subcutaneous bone scaffolds using fibre-optic Raman spectroscopy

The development of novel biomaterials for regenerative therapy relies on the ability to assess tissue development, quality, and similarity with native tissue types in in vivo experiments. Non-invasive imaging modalities such as X-ray computed tomography offer high spatial resolution but limited biochemical information while histology and biochemical assays are destructive. Raman spectroscopy is a non-invasive, label-free and non-destructive technique widely applied for biochemical characterization. Here we demonstrate the use of fibre-optic Raman spectroscopy for in vivo quantitative monitoring of tissue development in subcutaneous calcium phosphate scaffolds in mice over 16 weeks. Raman spectroscopy was able to quantify the time dependency of different tissue components related to the presence, absence, and quantity of mesenchymal stem cells. Scaffolds seeded with stem cells produced 3–5 times higher amount of collagen-rich extracellular matrix after 16 weeks implantation compared to scaffolds without. These however, showed a 2.5 times higher amount of lipid-rich tissue compared to implants with stem cells. Ex vivo micro-computed tomography and histology showed stem cell mediated collagen and bone development. Histological measures of collagen correlated well with Raman derived quantifications (correlation coefficient in vivo 0.74, ex vivo 0.93). In the absence of stem cells, the scaffolds were largely occupied by adipocytes. The technique developed here could potentially be adapted for a range of small animal experiments for assessing tissue engineering strategies at the biochemical level.

In vitro Adaptation, Molecular Characterization and Tissue Tropism of Foot-and-mouth Disease Virus in Guinea Pigs

Background: Foot-and-mouth disease is a highly contagious disease in cloven-footed animals. It is reported to be endemic in Pakistan. Methods: Molecular characterization of FMD virus field isolate was carried out, followed by an investigation of the tissue tropism in guinea pigs’ heart and respiratory organs. After calculating the biological titer of the virus and guinea pigs infectious dose50, the virus was injected into the hind metatarsal feet pads of 30 guinea pigs to study the pathogenesis of the disease at different day intervals from day 1 to 28. Result: Phylogenetic analysis showed that the isolate belonged to ME-SA topotype of serotype “O” with lineage Pan-Asia II. In pathological findings, hemorrhages and congestion in the heart and trachea and edema in the lungs were observed. Histopathological examination showed myocardial necrosis and tracheal epithelium sloughing along with bronchial edema. In Immunohistochemical studies, antigen load was detected in the heart, trachea and lungs up to 14 days post-inoculation. The study suggested that small experimental animals would be a better cost-effective replacement for large animals to study the pathogenesis of the disease as the virus did not change its behavior in these laboratory animals.

3D Printed Bioreactor Enabling the Pulsatile Culture of Native and Angioplastied Large Arteries.

Routine interventions such as balloon angioplasty, result in vascular activation and remodeling, often requiring re-intervention. 2D in vitro models and small animal experiments have enabled the discovery of important mechanisms involved in this process, however the clinical translation is often underwhelming. There is a critical need for an ex vivo model representative of the human vascular physiology and encompassing the complexity of the vascular wall and the physical forces regulating its function. Vascular bioreactors for ex vivo culture of large vessels are viable alternatives, but their custom-made design and insufficient characterization often hinders the reproducibility of the experiments. The objective of the study was to design and validate a novel 3D printed cost-efficient and versatile perfusion system, capable of sustaining the viability and functionality of large porcine arteries for 7 days and enabling early post-injury evaluations. MultiJet Fusion 3D printing was used to engineer the EasyFlow insert, converting a conventional 50 ml centrifuge tube into a mini bioreactor. Porcine carotid arteries either left untreated or injured with an angioplasty balloon, were cultured under pulsatile flow for up to 7 days. Pressure, heart rate, medium viscosity and shear conditions were adjusted to resemble arterial in vivo hemodynamics. Tissue viability, cell activation and matrix remodeling were analyzed by immunohistochemistry, and vascular function was monitored by duplex ultrasound. Culture conditions in the EasyFlow bioreactor preserved endothelial coverage and smooth muscle organization and extracellular matrix structure in the vessel wall, as compared to static culture. Injured arteries presented hallmarks of early remodeling, such as intimal denudation, smooth muscle cell disarray and media/adventitia activation in flow culture. Duplex ultrasound confirmed continuous pulsatile blood flow conditions, dose-dependent vasodilator response to nitroglycerin in untreated vessels and impaired dilator response in angioplastied vessels. The scope of this work is to validate a low-cost, robust and reproducible system to explore the culture of native and injured large arteries under pulsatile flow. While the study of vascular pathology is beyond the scope of the present paper, our system enables future investigations and provides a platform to test novel therapies and devices ex vivo, in a patient relevant system.

State Estimation of Time-Varying MRI with Radial Golden Angle Sampling

We propose a state estimation approach to time-varying magnetic resonance imaging utilizing a priori information. In state estimation, the time-dependent image reconstruction problem is modeled by separate state evolution and observation models. In our method, we compute the state estimates by using the Kalman filter and steady-state Kalman smoother utilizing a data-driven estimate for the process noise covariance matrix, constructed from conventional sliding window estimates. The proposed approach is evaluated using radially golden angle sampled simulated and experimental small animal data from a rat brain. In our method, the state estimates are updated after each new spoke of radial data becomes available, leading to faster frame rate compared with the conventional approaches. The results are compared with the estimates with the sliding window method. The results show that the state estimation approach with the data-driven process noise covariance can improve both spatial and temporal resolution.

A simple algorithm for diffuse optical tomography without Jacobian inversion

A computationally simpler algorithm to reconstruct the optical property distribution of turbid media using diffuse optical tomographic principles is presented. The proposed algorithm eliminates the requirement of large Jacobian matrix inversion which otherwise is essential for tomographic imaging. The most significant Jacobians are identified based on proper thresholding of the measurement and the intersection of these Jacobians gives the approximate spatial location of the inhomogeneity. The algorithm is tested and optimized using simulations and further validated using tissue-mimicking phantom-based experiments and in-vivo small-animal experiments.

Techniques for digital histological morphometry of the pineal gland

IntroductionThe pineal gland is a small photo-neuro-endocrine organ. This study used human post-mortem pineal glands to microscopically assess immunohistochemical marker intensity and percentage of positivity using known and novel digital techniques. Materials and methodsAn experimental non-inferiority study has been performed on 72 pineal glands harvested from post-mortem examinations. The glands have been stained with glial fibrillary acidic protein (GFAP), synaptophysin (SYN), neuron-specific enolase (NSE), and neurofilament (NF). Slides were digitally scanned. Morphometric data were obtained using optical analysis, CaseViewer, ImageJ, and MorphoRGB ResultsStrong and statistically significant correlations were found and plotted using Bland-Altman diagrams between the two image analysis software in the case of mean percentage and intensity of GFAP, NSE, NF, and SYN. DiscussionsSoftware such as SlideViewer and ImageJ, with our novel software MorphoRGB were used to perform histological morphometry of the pineal gland. Digital morphometry of a small organ such as the pineal gland is easy to do by using whole slide imaging (WSI) and digital image analysis software, with potential use in clinical settings. MorphoRGB provides slightly more accurate data than ImageJ and is more user-friendly regarding measurements of parenchyma percentage stained by immunohistochemistry. The results show that MorphoRGB is not inferior in functionality. ConclusionsThe described morphometric techniques have potential value in current practice, experimental small animal models and human pineal glands, or other small endocrine organs that can be fully included in a whole slide image. The software we used has applications in quantifying immunohistochemical stains.

PLGA Nanoparticle Rapamycin- or Necrostatin-1-Coated Sutures Inhibit Inflammatory Reactions after Arterial Closure in Rats.

Background: The inflammatory reaction of sutures and oozing after arterial closure depends on the suture material and the amount of oozing surrounding the sutures. Anti-inflammation coatings have been proven to be an effective strategy to decrease this reaction. The aim of this study was to establish an arterial closure oozing model in rats and to test the effect of poly (lactic-co-glycolic acid) (PLGA) nanoparticle (NP) rapamycin- or necrostatin-1(NEC-1)-coated sutures on the inflammatory reaction after arterial closure. Methods and Materials: A 10 mm arteriotomy was carried out on the carotid artery of Sprague-Dawley rats and closed using 11-0 sutures. PLGA NP-rapamycin and NEC-1 were made. The 11/0 nylon sutures were coated with PLGA NP-rapamycin and NEC-1. Sutures were examined by scanning electron microscopy, hemolysis test, and cumulative release. The carotid arteriotomy was closed using uncoated PLGA NP-rapamycin- and NP-NEC-1-coated sutures. The carotid artery was harvested on day 7. Tissues were examined by histology and immunohistochemistry. Results: There were severe inflammatory reactions in the oozing arteries compared to the normal healing arteries (P = 0.0192). PLGA NP-rapamycin- and NEC -1-coated sutures reduced foreign body reaction compared to the uncoated sutures. There were significantly smaller number of CD3 (P = 0.0068), CD45 (P = 0.0300), and CD68 (P = 0.0011) cells in the PLGA NP-rapamycin- and NP-NEC-1-coated groups compared to the uncoated group. There was a smaller number of p-mTOR (P = 0.0198)-positive cells in the PLGA NP-rapamycin-coated group compared to the uncoated group. There was a smaller number of TNFα (P = 0.0198)-positive cells in the PLGA NP-NEC-1-coated group compared to the uncoated group. Conclusions: In this rat carotid artery oozing model, PLGA NP-rapamycin- or NP-NEC-1-coated sutures can inhibit inflammatory reaction and foreign body reaction. Although this was a small rodent animal experiment, this coated suture may have a potential clinical application in the future.

To the possibility of detecting the "coronary theft" syndrome in small laboratory animals

The purpose of the study. There are investigation of the possibility of using the laser Doppler flowmetry method to assess blood microcirculation and analyze the mechanisms of its regulation in conditionally intact and ischemic myocardium in small laboratory animals (rats) to identify the "coronary theft" syndrome. Materials and methods. The experiments were carried out on white mongrel anesthetized (urethane 1300 mg/kg /b) male rats weighing 200-250 g. 28 days after the reproduction of an experimental myocardial infarction in an open chest and artificial lung ventilation, the microcirculation level was assessed in conditionally intact and ischemic areas of the myocardium by laser Doppler flowmetry. Using spectral wavelet analysis, the amplitudes of microcirculation oscillations normalized to the total perfusion associated with various regulation mechanisms were determined. Results. It was found that the microcirculation level is significantly lower in the ischemic zone of the myocardium compared with the conditionally intact one (17.3±2.8 and 30.3±1.3 per. units, respectively, p = 0.006, n = 8). Spectral wavelet analysis showed that in the ischemic injury zone, in comparison with the conditionally intact myocardium, the amplitudes of microcirculation oscillations normalized to the general level of microcirculation increase for all regulatory mechanisms. Conclusion. The laser Doppler flowmetry method can be used to assess the intensity of microcirculation in conditionally intact and ischemic myocardium. Using this method makes it possible to identify the "coronary theft" syndrome in experiments on small experimental animals.

Sperm quality and testicular histopathology of Wistar albino male rats treated with hydroethanolic extract of Cordia dichotoma fruits

Context Cordia dichotoma Forst. (Boraginaceae) has potent pharmacological impact. Meanwhile, its effect on fertility is unclear. Objective This study investigates the effect of Cordia fresh fruits hydroethanolic extract on fertility. Materials and methods 120 Wistar albino male rats were divided into four groups (n = 30). The first group was negative control, and the second, third, and fourth groups received 125, 250, and 500 mg extract/kg bodyweight for 56 days. After 56 days, Cordia force-feeding stopped, and all groups were kept under laboratory conditions for another month to study the recovering effect. Results After day 56, extract at 500 mg/kg significantly reduced sperm total count, motility%, and alive%, to 47.60 ± 2.27 × 106 sperm/mL, 43.33% ± 1.49, and 63.67% ± 1.19, respectively, abnormalities% increased considerably (26.67% ± 0.54), compared to the negative control. Also, significant depletion on follicle-stimulating hormone (2.66 ± 0.21 mIU/L), luteinizing hormone (1.07 ± 0.06 mIU/L), and testosterone (2.69 ± 0.13 nmol/L) level was recorded, compared to the negative control. Cordia negative effect showed on histopathological studies of testes, prostate, and seminal vesicles. Fortunately, these adverse effects of Cordia recovered remarkably after stopping administration for one month. Conclusions Cordia antifertility effect may be due to its hypocholesterolemic effect, where cholesterol, the steroid cycle precursor, was significantly reduced. This study can be incorporated in clinical research after being repeated on another small experimental animal, their offspring, and one large experimental animal, then going to a clinical study that we plan to do in the future.

AAV2-VEGF-B gene therapy failed to induce angiogenesis in ischemic porcine myocardium due to inflammatory responses

Therapeutic angiogenesis induced by gene therapy is a promising approach to treat patients suffering from severe coronary artery disease. In small experimental animals, adeno-associated viruses (AAVs) have shown good transduction efficacy and long-term transgene expression in heart muscle and other tissues. However, it has been difficult to achieve cardiac-specific angiogenic effects with AAV vectors. We tested the hypothesis whether AAV2 gene transfer (1 × 1013 vg) of vascular endothelial growth factor B (VEGF-B186) together with immunosuppressive corticosteroid treatment can induce long-term cardiac-specific therapeutic effects in the porcine ischemic heart. Gene transfers were delivered percutaneously using direct intramyocardial injections, improving targeting and avoiding direct contact with blood, thus reducing the likelihood of immediate immune reactions. After 1- and 6-month time points, the capillary area was analyzed, myocardial perfusion reserve (MPR) was measured with radiowater positron emission tomography ([15O]H2O-PET), and fluorodeoxyglucose ([18F]FDG) uptake was used to evaluate myocardial viability. Clinical chemistry and immune responses were analyzed using standard methods. After 1- and 6-month follow-up, AAV2-VEGF-B186 gene transfer failed to induce angiogenesis and improve myocardial perfusion and viability. Here, we show that inflammatory responses attenuated the therapeutic effect of AAV2 gene transfer by significantly reducing successful transduction and long-term gene expression despite the efforts to reduce the likelihood of immune reactions and the use of targeted local gene transfer methods.

Intravital microscopic observation of the microvasculature during hemodialysis in healthy rats

Hemodialysis (HD) provides life-saving treatment for kidney failure. Patient mortality is extremely high, with cardiovascular disease (CVD) being the leading cause of death. This results from both a high underlying burden of cardiovascular disease, as well as additional physiological stress from the HD procedure itself. Clinical observations indicate that HD is associated with microvascular dysfunction (MD), underlining the need for a fundamental pathophysiological assessment of the microcirculatory consequences of HD. We therefore successfully developed an experimental small animal model, that allows for a simultaneous real-time assessment of the microvasculature. Using in-house built ultra-low surface area dialyzers and miniaturized extracorporeal circuit, we successfully dialyzed male Wistar Kyoto rats and combined this with a simultaneous intravital microscopic observation of the EDL microvasculature. Our results show that even in healthy animals, a euvolemic HD procedure can induce a significant systemic hemodynamic disturbance and induce disruption of microvascular perfusion (as evidence by a reduction in the proportion of the observed microcirculation receiving blood flow). This study, using a new small animal hemodialysis model, has allowed direct demonstration that microvascular blood flow in tissue in skeletal muscle is acutely reduced during HD, potentially in concert with other microvascular beds. It shows that preclinical small animal models can be used to further investigate HD-induced ischemic organ injury and allow rapid throughput of putative interventions directed at reducing HD-induced multi-organ ischemic injury.