Live Animal Models Research Articles

Low copy expression vectors for use in Yersinia sp. and related organisms

In Yersinia, the most commonly used expression vectors for genetic studies such as gene complementation do not effectively allow for both induction and repression of gene expression. Additionally, there is no expression system available that can be induced in bacteria growing in vitro as well as in vivo, e.g. in eukaryotic cell lines or in living animal models. Here, we present a series of novel inducible low copy expression vectors that are well suited for use in the Yersinia species. Their tet operator/promoter/repressor system makes them distinct from other vectors, and gene transcription in bacteria can easily be induced by addition of anhydrotetracyline (ATc) either to the growth medium, to tissue culture medium during bacterial infections of cell lines or by injection into animals infected with bacteria. Researchers can choose between two different antibiotic resistances (kanamycin or spectinomycin), between two copy numbers (5 or 12–22) as well as between two different versions for expression from either the native RBS and ATG or RBS and ATG encoded in the plasmid. The whole vector series contains the same multi-cloning site from pBluescript II KS+ that allows for easy subcloning. Moreover, these vectors are built in a modular fashion that makes it simple to adapt them for other purposes. Finally, in addition to their use in Yersinia they are suitable for use in many other Enterobacteriaceae.

Endoscopic Full-Thickness Resection of Submucosal Gastric Tumors

Endoscopic full-thickness resection (EFTR) is a minimally invasive method for en bloc resection of gastrointestinal lesions, such as early cancer or submucosal tumor. The aim of this pilot study was to evaluate a novel EFTR prototype device for full-thickness resection of the gastric wall containing artificial submucosal lesions. Six artificial submucosal tumors were surgically created in the gastric submucosa by implanting 8-mm cork beads in anesthetized pigs. EFTR of the lesions was attempted using a prototype device which consists of a large transparent plastic cap, loaded onto the tip of the endoscope, into which the submucosal lesion and the surrounding gastrointestinal wall can be pulled by using suction, a grasping forceps, or a dedicated anchoring device. An over-the-scope clip (OTSC) can be deployed underneath the submucosal lesion and a pre-loaded snare is used for EFTR above the OTSC. The median procedure time was 15min (interquartile range 11-22). Successful resection of the artificial submucosal lesion was achieved in 4/6 (67%) cases. Successful EFTR of the gastric wall was achieved in 3/6 (50%) cases. In all cases, the OTSC closed the EFTR site completely. Gastric EFTR using the novel EFTR prototype device is feasible in a live animal model. The technique can achieve a full-thickness gastric wall and submucosal tumor resection with reliable closure of the gastric wall, but further refinements of the technique and device are necessary in order to reliably resect submucosal lesions, especially larger ones.

Fluorescent Peptide–PNA Chimeras for Imaging Monoamine Oxidase A mRNA in Neuronal Cells

Monoamine oxidases (MAO) catalyze the oxidative deamination of many biogenic amines and are integral proteins found in the mitochondrial outer membrane. Changes in MAO-A levels are associated with depression, trait aggression, and addiction. Here we report the synthesis, characterization, and in vitro evaluation of novel fluorescent peptide-peptide nucleic acid (PNA) chimeras for MAOA mRNA imaging in live neuronal cells. The probes were designed to include MAOA-specific PNA dodecamers, separated by an N-terminal spacer to a μ-opioid receptor targeting peptide (DAMGO), with a spacer and a fluorophore on the C-terminus. The probe was successfully delivered into human SH-SY5Y neuroblastoma cells through μ-opioid receptor-mediated endocytosis. The K(d) by flow cytometry was 11.6 ± 0.8 nM. Uptake studies by fluorescence microscopy showed ∼5-fold higher signal in human SH-SY5Y neuroblastoma cells than in negative control CHO-K1 cells that lack μ-opioid receptors. Moreover, a peptide-mismatch control sequence showed no significant uptake in SH-SY5Y cells. Such mRNA imaging agents with near-infrared fluorophores might enable real time imaging and quantitation of neuronal mRNAs in live animal models.

Vaginal distribution and retention of a multiparticulate drug delivery system, assessed by gamma scintigraphy and magnetic resonance imaging

BackgroundFor any new vaginal dosage form, the distribution and retention in the vagina has to be assessed by in vivo evaluation. We evaluated the vaginal distribution and retention of starch-based pellets in sheep as live animal model by gamma scintigraphy (using Indium-111 DTPA as radiolabel) and in women via magnetic resonance imaging (MRI, using a gadolinium chelate as contrast agent). A conventional cream formulation was used as reference in both studies. MethodCream and pellets were administered to sheep (n=6) in a two period-two treatment study and to healthy female volunteers (n=6) via a randomized crossover trial. Pellets (filled into hard gelatin capsule) and cetomacrogol cream, both labeled with Indium-111 DTPA (for gamma scintigraphy) or with gadolinium chelate (for MRI) were evaluated for their intravaginal distribution and retention over a 24h period. Spreading in the vagina was assessed based on the part of the vagina covered with formulation (expressed in relation to the total vaginal length). Vaginal retention of the formulation was quantified based on the radioactivity remaining in the vaginal area (sheep study), or qualitatively evaluated (women study). ResultsBoth trials indicated a rapid distribution of the cream within the vagina as complete coverage of the vaginal mucosa was seen 1h after dose administration. Clearance of the cream was rapid: about 10% activity remained in the vaginal area of the sheep 12h post-administration, while after 8h only a thin layer of cream was detected on the vaginal mucosa of women. After disintegration of the hard gelatin capsule, the pellet formulation gradually distributed over the entire vaginal mucosa. Residence time of the pellets in the vagina was longer compared to the semi-solid formulation: after 24h 23±7% radioactivity was detected in the vaginal area of the sheep, while in women the pellet formulation was still detected throughout the vagina. ConclusionA multi-particulate system containing starch-based pellets was identified as a promising novel vaginal drug delivery system, resulting in complete coverage of the vaginal mucosa and long retention time.

Anticancer Activity of an Imageable Curcuminoid 1‐[2‐Aminoethyl‐(6‐hydrazinopyridine‐3‐carbamidyl)‐3,5‐bis‐(2‐fluorobenzylidene)‐4‐piperidone (EFAH)

3,5-Bis(2-fluorobenzylidine)-4-piperidone or EF24 is a potent anticancer derivative of curcumin. Using an amine derivative of EF24, we synthesized a hydrazinonicotinic acid conjugate, EFAH, for Tc-99m radiolabelling and single photon emission tomography imaging. The aqueous solubility of EFAH (3.5 mg/mL) was significantly more than that of EF24 (1.2 mg/mL); the octanol/water partition coefficient of EFAH was estimated at log P = 0.33. As an antiproliferative agent, EFAH was as effective as EF24 in suppressing the proliferation of H441, MiaPaCa-2 and Panc-1 cells. Daily intraperitoneal injection of EFAH (5 μg) for 3 weeks in mice carrying xenografts of Panc-1 pancreatic cancer showed a mean tumour volume reduction of 79%; the tumour weight decreased by 82% in the treated group. For imaging and biodistribution, EFAH was labelled with Tc-99m (98% RCY) and intravenously administered in rats. Approximately 23.7% and 14.3% of injected dose accumulated in liver and intestine, respectively, suggesting that EFAH is mostly eliminated by hepatobiliary route. The results indicate that HYNIC modification of EF24 for Tc-99m radiolabelling does not affect its antiproliferative efficacy. For the first time, a visual biodisposition of EF24 in a live animal model has been demonstrated. Such knowledge could be of benefit in developing therapeutic curcuminoids, such as EF24.

Focused Ultrasound to Expel Calculi From the Kidney

A persistent stone burden after renal stone treatment may result in future patient morbidity and potentially lead to additional surgery. This problem is particularly common after treatment of lower pole stones. We describe a potential noninvasive therapeutic option using ultrasound waves to create a force sufficient to aid in stone fragment expulsion. Human stones were implanted by retrograde ureteroscopy or antegrade percutaneous access in a live porcine model. The calibrated probe of a system containing ultrasound imaging and focused ultrasound was used to target stones and attempt displacement. To assess for injury an additional 6 kidneys were exposed for 2 minutes each directly to the output used for stone movement. Another 6 kidneys were exposed to more than twice the maximum output used to move stones. Renal tissue was analyzed histologically with hematoxylin and eosin, and nicotinamide adenine dinucleotide staining. Stones were moved to the renal pelvis or ureteropelvic junction by less than 2 minutes of exposure. Stone velocity was approximately 1 cm per second. There was no tissue injury when tissue was exposed to the power level used to move stones. Localized thermal coagulation less than 1 cm long was observed in 6 of 7 renal units exposed to the level above that used for ultrasonic propulsion. Transcutaneous ultrasonic propulsion was used to expel calculi effectively and safely from the kidney using a live animal model. This study is the first step toward an office based system to clear residual fragments and toward use as a primary treatment modality in conjunction with medical expulsive therapy for small renal stones.

Molecular Imaging of TGFβ-Induced Smad2/3 Phosphorylation Reveals a Role for Receptor Tyrosine Kinases in Modulating TGFβ Signaling

The dual modality of TGFβ, both as a potent tumor suppressor and a stimulator of tumor progression, invasion, and metastasis, make it a critical target for therapeutic intervention in human cancers. The ability to carry out real-time, noninvasive imaging of TGFβ-activated Smad signaling in live cells and animal models would significantly improve our understanding of the regulation of this unique signaling cascade. To advance these efforts, we developed a highly sensitive molecular imaging tool that repetitively, noninvasively, and dynamically reports on TGFBR1 kinase activity. The bioluminescent TGFβR1 reporter construct was developed using a split firefly luciferase gene containing a functional sensor of Smad2 phosphorylation, wherein inhibition of TGFβ receptor1 kinase activity leads to an increase in reporter signaling. The reporter was stably transfected into mammalian cells and used to image in vivo and in vitro bioluminescent activity as a surrogate for monitoring TGFBR1 kinase activity. The reporter was successfully used to monitor direct and indirect inhibition of TGFβ-induced Smad2 and SMAD3 phosphorylation in live cells and tumor xenografts and adapted for high-throughput screening, to identify a role for receptor tyrosine kinase inhibitors as modulators of TGFβ signaling. The reporter is a dynamic, noninvasive imaging modality for monitoring TGFβ-induced Smad2 signaling in live cells and tumor xenografts. It has immense potential for identifying novel effectors of R-Smad phosphorylation, for validating drug-target interaction, and for studying TGFβ signaling in different metastasis models.

The Role of rap1b in Hoxb4 Transgenic Zebrafish Line,

Abstract 3413Self-renewal – the generation of daughter cells having the same regenerative properties as the parental cell – is the defining feature of hematopoietic stem cells (HSCs). Homeobox b4 (Hoxb4) is the key gene shown capable of expanding hematopoietic stem cells (HSCs) in vivo and in vitro. It is imperative to establish a stable Hoxb4 overexpressing transgenic live animal model. Here, using a Cre-loxP system, we established a transgenic zebrafish line that stably overexpressing enhanced green fluorescent protein (EGFP) – tagged hoxb4 protein under the control of hemangioblast-specific lmo2 promoter. The results indicate that Hoxb4 favors hematopoietic stem cell development of hemangioblasts in vivo. The mechanisms of Hoxb4-mediated HSC proliferation and the reason why Hoxb4 overexpression can cause retardation of specific and terminal blood cells in vivo are still unknown.Rap1 belongs to the Ras subfamily of small GTP-binding proteins. Rap1 can regulate cell proliferation, differentiation, and adhesion through distinct mechanisms. So we want to know whether Hoxb4 overexpression can influence Rap1b expression. The expression difference of Rap1b in blank control group(Tubegin Wild type), experimental control group (Tg(zLmo2:LDL-EGFP)×Tg(zLmo2:Cre)), experimental group (Tg(zLmo2:LDL-Hoxb4-EGFP) ×Tg(zLmo2:Cre)) zebrafish embryo were detected by whole embryo in situ hybridization (WISH) with Rap1b antisense mRNA probe. It was found that Rap1b expression was extended from 30 to 36 hour postfertilization(hpf) when Hoxb4 was overexpressed. In the Hoxb4 expressed lines, rap1b was upregulated and strongly expressed in the caudal hematopoietic tissue (CHT) at 36 hpf. Then, the EGFP labeled hematopoietic cell in transgenic line were collected with the fluorescence activated cell sorter (FACS). The Rap1b mRNA expression of sorting cell were compared by SqRT-PCR (Semi-quantitative Reverse Transcription and Polymerase Chain Reaction). The gene expressions of Rap1b was upregulated in Hoxb4 experimental group. These results indicate that Rap1b gene maybe is downstream target gene of Hoxb4 gene in hematopoietic system of zebrafish. Disclosures:No relevant conflicts of interest to declare.

Laparoscopic Animal Surgery for Training Without Sacrificing Animals; Introducing the Rabbit as a Model for Infantile Laparoscopy

Improvement in laparoscopic skills requires practicing, and it is mostly beneficial when live animal models are considered for use. Apart from pelvic trainer, dogs and rabbits are used as the animal models for training laparoscopic surgeries at our center. Every effort is made to keep the animals alive after surgery. From January 2007 to January 2010, German shepherd dogs and Angora rabbits were selected as the animal models for laparoscopic skill training. Under general anesthesia, trainees performed several laparoscopic surgeries under the supervision of experienced surgeons. A total number of 72 animals including 54 dogs and 18 rabbits were used for training laparoscopy. In total, some 107 different laparoscopic procedures were performed by trainees including nephrectomy, nephropexy, vesicotomy and vesicorrhaphy, vasectomies, spermatic cord ligation, and unilateral oophrectomy. There were one vascular and two visceral injuries in the rabbit model that were laparoscopically controlled, and conversion to open surgery happened in one case due to the failure in extracting the specimen from the abdominal cavity. Three visceral and six vascular injuries occurred in the canine model. Total mortality was five including three rabbits and two dogs. The sacrifice of the animal is important to be avoided from both ethical and technical stand points. Dogs and rabbits are good models for laparoscopic training in urology, and it is possible to keep the animals alive after surgery by close monitoring. We also found the rabbit to be a good model for practicing infantile laparoscopic surgery, as it simulates the real surgery in this difficult age group.

Research of the effect of persisters in Candida albicans biofilm on antifungal therapy using a Caenorhabditis elegans infection model

Objective To illuminate the effect of persisters on antifungal therapy by infecting Caenorhabditis elegans as a live-animal model with Candida albicans isolates of different persister levels, treating them with amphotericin B and comparing the survival rate. Methods Glp-4 (bn2ts); sek-1 (km4) worms were synchronized and grown to sterile to L4-stage, put on different Candida albicans strains lawns separately for 2 h. Dispensed 15-20 worms per well of the 96-well plate, and added serial dilutions of amphotericin B for each strain group. Wells filled without any amphotericin B were used as negative controls intra-group. Incubated the plate at 25C for 5 days, counted the survival rate of each well. Results Compared with negative controls, survival rate of drug wells in each group increased. In the same drug concentration, the increase for high-persister group was significantly lower than that for low-persister group (P＜0.01). Conclusion Caenorhabditis elegans provides a model for the study of persisters and antifungal pharmacodynamics.The drug tolerance of persisters may be a critical component responsible for antifungal drug failure and relapsing infections. Key words: Candida albicans; Biofilm; Persisters; Antifungal; Caenorhabditis elegans

Ex vivo evaluation of femtosecond pulse laser incision of urinary tract tissue in a liquid environment: Implications for endoscopic treatment of benign ureteral strictures

The femtosecond (FS) pulse laser incises soft tissues with minimal peripheral damage and is a promising cutting tool for ureteroscopic endoureterotomy of benign ureteral strictures. To evaluate the feasibility of applying the FS laser to ureteroscopic endoureterotomy. A commercial Ti:Sapphire regenerative amplifier system (Coherent, RegA 9050, USA) was used in this study. Normal saline, 5% glucose solution, 4% mannitol solution, distilled water, and a 1% (v/v) suspension of whole blood with each of these solutions were tested for their attenuation rate (AR) of the FS laser's power. Bladder specimens from Sprague-Dawley (SD) rats were used as a surrogate model. The laser incised slots of 2 mm in length at bladder samples using three power grades (5×, 10×, and 20× the threshold power) combined with five effective pulse rates (40, 20, 10, 5, and 2.5 kHz), both in air and in normal saline. After samples were processed with standard hematoxylin-eosin staining procedures, the incision depth and collateral damage range were determined microscopically. The ARs of blood suspensions with each of the three isosmotic solutions were significantly higher than the other five solutions (P < 0.001). The FS laser's cutting depth and the collateral damage were increased with the laser power or power density but the collateral damages were less than 100 µm. Microbubble formation was detected in the liquid environments tested and influenced the effective laser power. Endoscopic application of the FS laser is feasible. Microbubble formation with the laser incision, however, may influence cutting effects. Proposed methods to address these issues include increasing the irrigation rate, using distilled water as irrigation or using gas insufflation instead of irrigation. It is necessary to evaluate these methods, as well as the long-term biologic response to laser incision, on living animal models in endoscopic settings before use on humans.

A Novel Xenograft Model in Zebrafish for High-Resolution Investigating Dynamics of Neovascularization in Tumors

Tumor neovascularization is a highly complex process including multiple steps. Understanding this process, especially the initial stage, has been limited by the difficulties of real-time visualizing the neovascularization embedded in tumor tissues in living animal models. In the present study, we have established a xenograft model in zebrafish by implanting mammalian tumor cells into the perivitelline space of 48 hours old Tg(Flk1:EGFP) transgenic zebrafish embryos. With this model, we dynamically visualized the process of tumor neovascularization, with unprecedented high-resolution, including new sprouts from the host vessels and the origination from VEGFR2+ individual endothelial cells. Moreover, we quantified their contributions during the formation of vascular network in tumor. Real-time observations revealed that angiogenic sprouts in tumors preferred to connect each other to form endothelial loops, and more and more endothelial loops accumulated into the irregular and chaotic vascular network. The over-expression of VEGF165 in tumor cells significantly affected the vascularization in xenografts, not only the number and size of neo-vessels but the abnormalities of tumor vascular architecture. The specific inhibitor of VEGFR2, SU5416, significantly inhibited the vascularization and the growth of melanoma xenografts, but had little affects to normal vessels in zebrafish. Thus, this zebrafish/tumor xenograft model not only provides a unique window to investigate the earliest events of tumoral neoangiogenesis, but is sensitive to be used as an experimental platform to rapidly and visually evaluate functions of angiogenic-related genes. Finally, it also offers an efficient and cost-effective means for the rapid evaluation of anti-angiogenic chemicals.

Randomized Objective Comparison of Live Tissue Training versus Simulators for Emergency Procedures

There is a lack of objective analysis comparing live tissue and simulator training. This article aims to objectively determine the difference in outcomes. Twenty-four Air Force volunteers without prior experience performing emergency procedures were randomly assigned to receive training in tube thoracostomy (chest tube) and cricothyroidotomy training on either a pig model (Sus scrofa domestica) or on the TraumaMan simulator. One week posttraining, students were tested on human cadavers with objective and subjective results recorded. Average completion time for tube thoracostomy in the animal model group was 2 minutes 4 seconds and 1 minute 51 seconds in the simulator group with a mean difference of 12 seconds (P = 0.74). Average completion time for cricothyroidotomy in the animal model group was 2 minutes 35 seconds and 3 minutes 29 seconds in the simulator group with a mean difference of 53 seconds (P = 0.32). Overall confidence was 9 per cent higher in the animal trained group (P = 0.42). Success rate of cricothyroidotomy was 75 per cent in the animal model group and 58 per cent in the simulator-trained group (P = 0.67). Success rate of chest tube placement was 92 per cent in the animal group and 83 per cent in the simulator group (P = 1.00). There was no statistically significant difference in chest tube and cricothyroidotomy outcomes or confidence in the groups trained with live animal models or simulators at the 95 per cent confidence interval. Trends suggest a possible difference, but the number of cadavers required to reach greater than 95 per cent statistical confidence prohibited continuation of the study.

Simulation in surgery: a review

The ability to acquire surgical skills requires consistent practice, and evidence suggests that many of these technical skills can be learnt away from the operating theatre. The aim of this review article is to discuss the importance of surgical simulation today and its various types, exploring the effectiveness of simulation in the clinical setting and its challenges for the future. Surgical simulation offers the opportunity for trainees to practise their surgical skills prior to entering the operating theatre, allowing detailed feedback and objective assessment of their performance. This enables better patient safety and standards of care. Surgical simulators can be divided into organic or inorganic simulators. Organic simulators, consisting of live animal and fresh human cadaver models, are considered to be of high-fidelity. Inorganic simulators comprise virtual reality simulators and synthetic bench models. Current evidence suggests that skills acquired through training with simulators, positively transfers to the clinical setting and improves operative outcome. The major challenge for the future revolves around understanding the value of this new technology and developing an educational curriculum that can incorporate surgical simulators.

Tu1403 A Live Porcine Model of Bleeding Ulcer

Upper gastrointestinal bleeding (UGIB) is a frequent life-threatening condition. Endoscopic haemostasis and proton-pump inhibitors are the first-line treatments of hemorrhagic ulcers. Simulators, ex vivo animal models are currently used for training in endoscopic haemostasis, but they have important limitations. We aimed to validate a reproducible, realistic, quick-and-easy live animal model of acute bleeding ulcer for endoscopy training.

A New Technological Fusion of PET and MRI for Brain Imaging

Non-invasive imaging modalities for living animal models are a powerful research tool for the evaluation of potential drugs and treatments for human diseases. Parkinson’s disease is a neurodegenerative disorder associated with aging, and patients with this disease have a deficiency of dopaminergic neurons. In the study, we evaluated fusion imaging with positron emission tomography (PET) and magnetic resonance imaging (MRI) in the rat brain with [ 18 F]FP-CIT which binds with high affinity to dopamine transporters. PET and MRI fusion images were well registered with conventional Inveon Research Workplace software, even though they are different modalities. In PET/MRI fusion images, we showed that the uptake of [ 18 F]FP-CIT was clearly increased in the normal rat striatum and these images accurately matched the morphology in the rat brain atlas. PET/MRI images easily and accurately identified the regions of interest in the target tissue compared to PET/CT images and enabled us to calculate the uptake of the PET tracer. Therefore, combined PET/MRI analysis provides functional and anatomical information for studying biology and pathology in preclinical research.

Laparoendoscopic Single Site Nephrectomy With the SPIDER Surgical System: Engineering Advancements Tested in a Porcine Model

Abstract Background and Purpose: The Single Port Instrument Delivery Extended Reach (SPIDER) surgical system was developed for true continuous instrument triangulation during laparoendoscopic single site (LESS) surgery. We present our initial preclinical experience with the SPIDER surgical system during renal surgery. Bilateral laparoscopic nephrectomies were performed in a live adult porcine animal model using the SPIDER device. A standard surgical approach was used via direct video guidance. The procedure was successfully performed without surgical error or complication. The SPIDER system proved easy to use with only a minimal learning curve. Intracorporeal surgical knots were tied without difficulty using this single site system. Our initial experience with the SPIDER surgical system during renal surgery is promising. SPIDER allows for true single port instrument triangulation offering a superior operative experience to currently available LESS surgical systems.

Materials for multifunctional balloon catheters with capabilities in cardiac electrophysiological mapping and ablation therapy.

Developing advanced surgical tools for minimally invasive procedures represents an activity of central importance to improving human health. A key challenge is in establishing biocompatible interfaces between the classes of semiconductor device and sensor technologies that might be most useful in this context and the soft, curvilinear surfaces of the body. This paper describes a solution based on materials that integrate directly with the thin elastic membranes of otherwise conventional balloon catheters, to provide diverse, multimodal functionality suitable for clinical use. As examples, we present sensors for measuring temperature, flow, tactile, optical and electrophysiological data, together with radiofrequency electrodes for controlled, local ablation of tissue. Use of such 'instrumented' balloon catheters in live animal models illustrates their operation, as well as their specific utility in cardiac ablation therapy. The same concepts can be applied to other substrates of interest, such as surgical gloves.

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load. This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Akt Suppresses Retrograde Degeneration of Dopaminergic Axons by Inhibition of Macroautophagy

Axon degeneration is a hallmark of neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. Such degeneration is not a passive event but rather an active process mediated by mechanisms that are distinct from the canonical pathways of programmed cell death that mediate destruction of the cell soma. Little is known of the diverse mechanisms involved, particularly those of retrograde axon degeneration. We have previously observed in living animal models of degeneration in the nigrostriatal projection that a constitutively active form of the kinase, myristoylated Akt (Myr-Akt), demonstrates an ability to suppress programmed cell death and preserve the soma of dopamine neurons. Here, we show in both neurotoxin and physical injury (axotomy) models that Myr-Akt is also able to preserve dopaminergic axons due to suppression of acute retrograde axon degeneration. This cellular phenotype is associated with increased mammalian target of rapamycin (mTor) activity and can be recapitulated by a constitutively active form of the small GTPase Rheb, an upstream activator of mTor. Axon degeneration in these models is accompanied by the occurrence of macroautophagy, which is suppressed by Myr-Akt. Conditional deletion of the essential autophagy mediator Atg7 in adult mice also achieves striking axon protection in these acute models of retrograde degeneration. The protection afforded by both Myr-Akt and Atg7 deletion is robust and lasting, because it is still observed as protection of both axons and dopaminergic striatal innervation weeks after injury. We conclude that acute retrograde axon degeneration is regulated by Akt/Rheb/mTor signaling pathways.