Preferential delivery of the Sleeping Beauty transposon system to livers of mice by hydrodynamic injection

Nonviral, DNA-mediated gene transfer is an alternative to viral delivery systems for expressing new genes in cells and tissues. The Sleeping Beauty (SB) transposon system combines the advantages of viruses and naked DNA molecules for gene therapy purposes; however, efficacious delivery of DNA molecules to animal tissues can still be problematic. Here we describe the hydrodynamic delivery procedure for the SB transposon system that allows efficient delivery to the liver in the mouse. The procedure involves rapid, high-pressure injection of a DNA solution into the tail vein. The overall procedure takes <1 h although the delivery into one mouse requires only a few seconds. Successful injections result in expression of the transgene in 5-40% of hepatocytes 1 d after injection. Several weeks after injection, transgene expression stabilizes at approximately 1% of the level at 24 h, presumably owing to integration of the transposons into chromosomes.     

Temporal fossa bone grafts: a new technique in craniofacial surgery

The calvarium has become an increasingly popular bone-graft donor site. Previously described harvesting techniques are often difficult to perform and may produce unsatisfactory bone fragments. However, full-thickness bone grafts taken from the region of the temporal fossa, beneath the temporaiis muscle, have proven to be of high quality and technically easy to obtain. In our experience with eight patients, temporal fossa bone grafts were used primarily around the orbit, including reconstruction of the orbital floor, frontal bone, and zygoma. The procedure begins with a hemicoronal or bicoronal incision; the temporalis muscle is reflected, and an underlying bone plate up to 4 X 6 cm is removed. The resulting bone graft is consistently 3 to 4 mm in thickness. The cranial defect is packed with bone debris, and the muscle is replaced. This technique has proven to be safe, technically simple, consistently productive of high-quality bone grafts, and within discernible donor-site deformity.     

Placement Of A Coronary Sinus Pacing Lead From A Sub-occluded Left Subclavian Vein Using A Collateral Vein To The Right Subclavian Vein

Upgrading of a pacing system in the presence of a subclavian occlusion is technically challenging. We describe the case of a patient who underwent a successful upgrading procedure of an implantable cardioverter-defibrillator (ICD) to a biventricular defibrillator (ICD-CRT) in the presence of a suboccluded left subclavian vein, using a collateral vein that drained into the contralateral subclavian vein.     

Delivery of Therapeutic Agents Through Intracerebroventricular (ICV) and Intravenous (IV) Injection in Mice

Despite the protective role that blood brain barrier plays in shielding the brain, it limits the access to the central nervous system (CNS) which most often results in failure of potential therapeutics designed for neurodegenerative disorders ^1,2. Neurodegenerative diseases such as Spinal Muscular Atrophy (SMA), in which the lower motor neurons are affected, can benefit greatly from introducing the therapeutic agents into the CNS. The purpose of this video is to demonstrate two different injection paradigms to deliver therapeutic materials into neonatal mice soon after birth. One of these methods is injecting directly into cerebral lateral ventricles (Intracerebroventricular) which results in delivery of materials into the CNS through the cerebrospinal fluid ^3,4. The second method is a temporal vein injection (intravenous) that can introduce different therapeutics into the circulatory system, leading to systemic delivery including the CNS ⁵. Widespread transduction of the CNS is achievable if an appropriate viral vector and viral serotype is utilized. Visualization and utilization of the temporal vein for injection is feasible up to postnatal day 6. However, if the delivered material is intended to reach the CNS, these injections should take place while the blood brain barrier is more permeable due to its immature status, preferably prior to postnatal day 2. The fully developed blood brain barrier greatly limits the effectiveness of intravenous delivery. Both delivery systems are simple and effective once the surgical aptitude is achieved. They do not require any extensive surgical devices and can be performed by a single person. However, these techniques are not without challenges. The small size of postnatal day 2 pups and the subsequent small target areas can make the injections difficult to perform and initially challenging to replicate. Download video file.^{(37M, mov)}     

Delivery systems for gene therapy

Introduction of foreign genes into mammalian cellsin vitro has been accomplished previously by a variety of methods. The few techniques that have been developed for transfection of mammalian cellsin vivo, are technically difficult or lack cell specificity.We have developed a soluble, targetable DNA carrier system consisting of an asialoglycoprotein covalently coupled to a polycation. The strategy was based on: 1) the presence of unique receptors on hepatocytes which internalize galactose-terminal (asialo-)glycoproteins; 2) polycations can bind DNA in a non-covalent, non-damaging interaction. Using chloramphenicol acetyltransferase (CAT) as a marker gene, specific delivery and expression of CAT was demonstratedin vitro using asialoglycoprotein receptor ( +) and (-) cell lines.Intravenous injection of conjugate-DNA complexes in rats resulted in detection of CAT DNA sequences in liver 10 min later by dot blots with a CAT cDNA probe. CAT enzyme activity 24 hrs later was found specifically in liver but no other tissues or control livers. Targeted hepatic CAT expression was transient, maximal at 24 hrs but declined to barely detectable levels by 96 hrs. Persistent foreign gene expression was achieved by injection of DNA complex followed by 67% partial hepatectomy. High levels of hepatic CAT activity were detected through 11 weeks post-hepatectomy.The data indicate that a targetable gene delivery system can permitin vivo expression of an exogenous gene after simple intravenous injection. The foreign gene expression can be enhanced and made to persist by induction of hepatocyte replication.     

Specific Noradrenergic Neurones destroyed by 6-Hydroxydopamine Injection into Newborn Rats

SINCE 6-hydroxydopamine does not cross the blood-brain barrier in adult animals¹, it has to be given by intracerebral injection to produce specific degeneration of catecholamine-containing nerve endings in the central nervous system^2,3. The resulting permanent depletion of noradrenaline and dopamine in all regions of the brain makes it difficult to determine the neuronal tracts and brain regions involved in a physiological or behavioural disfunction after 6-hydroxydopamine treatment⁴. Localization of nerve destruction can be improved by direct intracerebral injection of 6-hydroxydopamine at specific neuronal sites⁵ by pretreatment with drugs that affect catecholamine uptake⁶ or by varying the dosage of 6-hydroxydopamine⁷. These procedures are, however, technically difficult.     

Surgical technique in the rat model of kidney transplantation

Today successful kidney transplantation procedures, techniques and immunosuppression protocols are a consequence of extensive research on animal models. During every transplantation surgery there are two crucial points for the success of the entire procedure: vascular (arterial end venous) and ureteral or ureterovesical anastomosis. Renal artery and vein of the donor kidney can be anastomosed end-to-side to the abdominal aorta and vena cava of the recipient (heterotopic transplantation), or end-to-end to the remains of renal artery and vain of the recipient (orthotopic transplantation) after nephrectomy. The ureter can be anastomosed also end-to-end or we can connect it directly to the urinary bladder (ureterocystoneostomy). The aim of this study was to elucidate which technique has better results according to: animal survival, reperfusion and perfusion of the transplanted kidney, elimination of the urine from the transplanted kidney and procedure costs. The study included 240 (120 donors and 120 recipients) male Wistar rats (3 months old; weight 250-300 g Our results are clearly showing that the end-to-end vascular anastomosis, and Paquins ureterovesical anastomosis have better results in transplanted rat kidneys survival and urine drainage compared to end-to-side vascular anastomosis and end-to-end ureteral anastomosis. Based on our experience we can conclude that described methods of end-to-end vascular anastomosis and Paquins ureterovesical anastomosis are less technically demanding and have a shorter learning curve. Therefore, we can recommend the use of described methods in kidney transplantation related researches.     

ANTI-TUMOR EFFECT OF INTRAVENOUS TNFα GENE DELIVERY NAKED PLASMID DNA USING A HYDRODYNAMICS-BASED PROCEDURE

High levels of foreign gene expression in mouse hepatocytes can be achieved by the rapid injection of a large volume of naked plasmid (pDNA) into animals via the tail vein, the so-called hydrodynamics-based procedure. In this study, we evaluated the efficacy of hydrodynamics-based tumor necrosis factor alpha (TNFα) transfer for tumor treatment, in which the naked pDNA encoding TNFα was administered into the tail vein following an intravenous injection of B16 melanoma cells. The mice treated with TNFα-expressing pDNA displayed a profound reduction in lung metastasis. These results suggest that the hydrodynamics-based transfer of naked pDNA is a convenient and efficient method of TNFα gene therapy against metastatic tumors.     

Anti-tumor effect of intravenous TNFalpha gene delivery naked plasmid DNA using a hydrodynamics-based procedure

High levels of foreign gene expression in mouse hepatocytes can be achieved by the rapid injection of a large volume of naked plasmid (pDNA) into animals via the tail vein, the so-called hydrodynamics-based procedure. In this study, we evaluated the efficacy of hydrodynamics-based tumor necrosis factor alpha (TNFalpha) transfer for tumor treatment, in which the naked pDNA encoding TNFalpha was administered into the tail vein following an intravenous injection of B16 melanoma cells. The mice treated with TNFalpha-expressing pDNA displayed a profound reduction in lung metastasis. These results suggest that the hydrodynamics-based transfer of naked pDNA is a convenient and efficient method of TNFalpha gene therapy against metastatic tumors.     

Retro-orbital injections in mice

Intravenous vascular access is technically challenging in the adult mouse and even more challenging in neonatal mice. The authors describe the technique of retro-orbital injection of the venous sinus in the adult and neonatal mouse. This technique is a useful alternative to tail vein injection for the administration of non-tumorigenic compounds. The authors report that they have routinely used this technique in the adult mouse to administer volumes up to 150?μl without incident. Administration of retro-orbital injections is more challenging in neonatal mice but can reliably deliver volumes up to 10?μl.     

Trichinella spiralis: vascular recirculation and organ retention of newborn larvae in rats

The recirculation of Trichinella spiralis newborn larvae was studied in inbred AO rats. Newborn larvae collected after in vitro incubation of adult T. spiralis worms for 2 or 24 hr were injected into rats through the tail vein or hepatic portal vein. Blood samples from the femoral vein, hepatic portal vein, and abdominal aorta were collected at intervals from 1 min to 24 hr after larval injection. Newborn larvae of both ages (24 hr or 2 hr old) persisted in femoral vein blood for less than or equal to 5 hr after injection, but they could be detected in portal vein blood by 24 hr after injection. The injection of larvae into a tail vein or the portal vein did not influence the pattern of larval circulation, although there was a 1-5 min delay in newborn larval appearance time after injection into the portal vein. Transcapillary migration through tissue and back to the circulation was evident in the appearance of newborn larvae in the thoracic duct lymph up to 24 (occasionally 48) hr after tail vein injection of newborn larvae. During the course of a natural primary infection, no evidence for trapping of larvae in the mesenteric lymph node could be found despite direct larval migration through this organ. Injected newborn larvae were retained in the lungs, and small numbers could be recovered 24 hr after intravenous injection. We conclude that a proportion of newborn larvae recirculates within the vasculature for several hours; a smaller population extravasates but can reenter the circulatory system via the lymphatics. Furthermore, some newborn larvae are found in organs rich in capillaries up to 24 hr after their entry into the blood.     

Birth of mice after nuclear transfer by electrofusion using tail tip cells

Mice have been successfully cloned from cumulus cells, fibroblast cells, embryonic stem cells, and immature Sertoli cells only after direct injection of their nuclei into enucleated oocytes. This technical feature of mouse nuclear transfer differentiates it from that used in domestic species, where electrofusion is routinely used for nuclear transfer. To examine whether nuclear transfer by electrofusion can be applied to somatic cell cloning in the mouse, we electrofused tail tip fibroblast cells with enucleated oocytes, and then assessed the subsequent in vitro and in vivo development of the reconstructed embryos. The rate of successful nuclear transfer (fusion and nuclear formation) was 68.8% (753/1094) and the rate of development into morulae/blastocysts was 40.8% (260/637). After embryo transfer, seven (six males and one female; 2.5% per transfer) normal fetuses were obtained at 17.5-21.5 dpc. These rates of development in vitro and in vivo are not significantly different from those after cloning by injection (44.7% to morulae/blastocysts and 4.8% to term). These results indicate that nuclear transfer by electrofusion is practical for mouse somatic cell cloning and provide an alternative method when injection of donor nuclei into recipient oocytes is technically difficult.     

A Novel Murine Model of Arteriovenous Fistula Failure: The Surgical Procedure in Detail

The arteriovenous fistula (AVF) still suffers from a high number of failures caused by insufficient remodeling and intimal hyperplasia from which the exact pathophysiology remains unknown. In order to unravel the pathophysiology a murine model of AVF-failure was developed in which the configuration of the anastomosis resembles the preferred situation in the clinical setting. A model was described in which an AVF is created by connecting the venous end of the branch of the external jugular vein to the side of the common carotid artery using interrupted sutures. At a histological level, we observed progressive stenotic intimal lesions in the venous outflow tract that is also seen in failed human AVFs. Although this procedure can be technically challenging due to the small dimensions of the animal, we were able to achieve a surgical success rate of 97% after sufficient training. The key advantage of a murine model is the availability of transgenic animals. In view of the different proposed mechanisms that are responsible for AVF failure, disabling genes that might play a role in vascular remodeling can help us to unravel the complex pathophysiology of AVF failure.     

Comparison of the lateral tail vein and the retro-orbital venous sinus as routes of intravenous drug delivery in a transgenic mouse model

In mice, intravenous injections are commonly administered in the lateral tail vein. This technique is sometimes difficult to carry out and may cause stress to mice. Though injection through the retro-orbital venous sinus can provide certain advantages over lateral tail vein injection, this method is poorly defined and infrequently used. To compare the efficacy of these two routes of drug delivery, the authors injected MAFIA transgenic mice with the depletion agent AP20187, which selectively induces apoptosis in macrophages. Each mouse received five consecutive daily injections through either the lateral tail vein or the retro-orbital venous sinus. The authors then compared macrophage depletion in different tissues (lung, spleen, bone marrow and peritoneal exudate cells). Both routes of injection were similarly effective. A separate experiment using BALB/c mice indicated that retro-orbital venous sinus injection was the less stressful of the two methods.     

Real-time dynamic imaging of virus distribution in vivo

The distribution of viruses and gene therapy vectors is difficult to assess in a living organism. For instance, trafficking in murine models can usually only be assessed after sacrificing the animal for tissue sectioning or extraction. These assays are laborious requiring whole animal sectioning to ascertain tissue localization. They also obviate the ability to perform longitudinal or kinetic studies in one animal. To track viruses after systemic infection, we have labeled adenoviruses with a near-infrared (NIR) fluorophore and imaged these after intravenous injection in mice. Imaging was able to track and quantitate virus particles entering the jugular vein simultaneous with injection, appearing in the heart within 500 milliseconds, distributing in the bloodstream and throughout the animal within 7 seconds, and that the bulk of virus distribution was essentially complete within 3 minutes. These data provide the first in vivo real-time tracking of the rapid initial events of systemic virus infection.     

The distally based lesser saphenous venofasciocutaneous flap for ankle and heel reconstruction

Finding an appropriate soft-tissue grafting material to close a wound located over the ankle and heel can be a difficult task. The distally based lesser saphenous venofasciocutaneous flap mobilized from the posterior aspect of the upper leg, used as an island pedicle skin flap, can be useful for this purpose. The vascular supply to the flap is derived from the retrograde perfusion of the accompanying arteries of the lesser saphenous vein. These arteries descend along both sides of the lesser saphenous vein to the distal third of the leg, either terminating or anastomosing with the septocutaneous perforators of the peroneal artery. Between February of 1999 and March of 2001, four variants of this flap were applied in 21 individuals, including 11 fasciocutaneous, five fascial, three sensory, and two fasciomyocutaneous flaps. Skin defects among all patients were combined with bone, joint, and/or tendon exposure. The authors found that the flap was reliable and technically simple to design and execute. This one-stage procedure not only preserves the major arteries and the sural nerve of the injured leg, but it also has proved valuable for covering a weight-bearing heel and filling a deep defect, because it potentially provides protective sensation and a well-vascularized muscle fragment. When conventional local flaps are inadequate, this flap should be considered for its reliability and low associated morbidity.     

Transhepatic Cholangiography

Percutaneous transhepatic cholangiography is a method of visualizing the biliary tree by the injection of radio-opaque medium through the abdominal wall and liver into an intrahepatic bile duct. The procedure is indicated in the immediate preoperative evaluation of patients with obstructive jaundice of unknown etiology and is usually diagnostic in these cases. It may also be of value in avoiding operation in poor-risk patients with obstructive jaundice. Biliary leak resulting in chemical peritonitis is a complication in about 5% of these procedures. Intraperitoneal hemorrhage is a complication in less than 1%. Death results from the procedure in less than 0.5% of cases. Transhepatic cholangiography during surgical operation is of value in demonstrating obstructive lesions of the bile ducts. However, preoperative percutaneous transhepatic cholangiography is preferred, since it makes possible adequate preparation for technically difficult repairs and resections.     

Quantitation and pharmacokinetic modeling of therapeutic antibody quality attributes in human studies

A thorough understanding of drug metabolism and disposition can aid in the assessment of efficacy and safety. However, analytical methods used in pharmacokinetics (PK) studies of protein therapeutics are usually based on ELISA, and therefore can provide a limited perspective on the quality of the drug in concentration measurements. Individual post-translational modifications (PTMs) of protein therapeutics are rarely considered for PK analysis, partly because it is technically difficult to recover and quantify individual protein variants from biological fluids. Meanwhile, PTMs may be directly linked to variations in drug efficacy and safety, and therefore understanding of clearance and metabolism of biopharmaceutical protein variants during clinical studies is an important consideration. To address such challenges, we developed an affinity-purification procedure followed by peptide mapping with mass spectrometric detection, which can profile multiple quality attributes of therapeutic antibodies recovered from patient sera. The obtained data enable quantitative modeling, which allows for simulation of the PK of different individual PTMs or attribute levels in vivo and thus facilitate the assessment of quality attributes impact in vivo. Such information can contribute to the product quality attribute risk assessment during manufacturing process development and inform appropriate process control strategy.     

Acute Variations in Retinal Vascular Oxygen Content in a Rabbit Model of Retinal Venous Occlusion

Purpose

To study the variation in intravascular oxygen saturation (oximetry) during an acute retinal vein occlusion (RVO) using hyperspectral computed tomographic spectroscopy based oximetry measurements.

Methods

Thirty rabbits were dilated and anesthetized for experiments. Baseline oximetry measurements were made using a custom-made hyperspectral computed tomographic imaging spectrometer coupled to a fundus camera. RVO were induced using argon green laser following an intravenous injection of Rose Bengal. RVO induction was confirmed by fluorescein angiography. Retinal oximetry measurements were repeated in arterial and venous branches one hour after RVO induction and up to 4 weeks afterwards. Comparison of retinal oximetry before and after vein occlusion was made using the Student T-test.

Results

One hour after RVO induction, we observed statistically significant reductions in the intravascular oxygen saturation in temporal retinal arteries (85.1±6.1% vs. 80.6±6.6%; p<0.0001) and veins (71.4±5.5% vs. 64.0±4.7%; p<0.0001). This decrease was reversible in animals that spontaneously recannulated the vein occlusion. There were no statistically significant differences in oxygen saturation in the nasal control arteries and veins before and after temporal vein RVO induction.

Conclusions

We demonstrate, for the first time, acute changes in the intravascular oxygen content of retinal vessels 1 hour after RVO. These changes are reversible upon spontaneous recannulation of retinal vessels. This study demonstrates that hyperspectral computer tomographic spectroscopy based oximetry can detect physiological variations in intravascular retinal oxygen saturation. The study also provides the first qualitative and quantitative evidence of the variation in retinal vascular oxygen content directly attributable to an acute retinal vein occlusion.