Name: trans-tympanic drug delivery for the treatment of ototoxicity
Uploaded: 2018-03-16T12:30:00
Description: Watch this Scientific Journal Video about Trans-Tympanic Drug Delivery for the Treatment of Ototoxicity at JoVE.com

The work described in this article was supported by a NCI RO1 CA166907, NIDCD RO1-DC 002396 and RO3 DC011621.

Male Wistar rats were handled in accordance with the National Institutes of Health animal use guidelines and a protocol approved by the Southern Illinois University School of Medicine Laboratory Animal Care and Use Committee. Auditory brainstem response (ABR) was performed on rats while under anesthesia prior to drug administration and 72 h after to verify the effect of the trans-tympanic drug delivery.
1. Auditory Brainstem Response (ABR)
Note:&#160;...

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The trans-tympanic administration route allows for localized delivery of drugs and other agents to the cochlea which could otherwise produce significant systemic side effects if administered systemically. This method of drug administration allows rapid access of drugs to the site of action at significantly higher doses than would be achieved through the systemic route. Results presented here and published previously showed that trans-tympanic administration of [R]-N-phenyl isopropyl adenosine (R-PIA) pr...

The peripheral auditory system is exquisitely sensitive to drugs such as cisplatin and aminoglycoside antibiotics. Cisplatin is a widely used chemotherapeutic agent for the treatment of a variety of solid tumors, such as ovarian, testicular, and head and neck cancers. Ototoxicity experienced with the use of this drug is dose-limiting and quite common, affecting 75-100% of patients treated1. Other drugs, such as carboplatin and oxaliplatin, have emerged as alternatives to cisplatin2,3,4,5, but their usefulness is limited to a few cancers.
Early studies have shown the critical role of reactive oxygen species (ROS) in mediating the ototoxicity produced by cisplatin and aminoglycosides. Subsequent studies showed that the NOX3 isoform of NADPH oxidase is the primary source of ROS in the cochlea, and is activated by cisplatin6,7. The generation of ROS compromises the antioxidant buffering capacity of cells, leading to increased lipid peroxidation of cellular membranes8. Furthermore, cisplatin increases the production of hydroxyl radicals which generate highly toxic aldehyde 4-hydroxynonenal (4-HNE), an initiator of cell death9,10. Based on these findings, several antioxidants have been examined for the treatment of cisplatin ototoxicity. These include N-acetyl cysteine (NAC), sodium thiosulfate (STS), amifostine, and D-methionine. However, a major concern of antioxidant therapy is that these antioxidants could reduce cisplatin chemotherapeutic efficacy when administered systemically11 through the interaction of cisplatin with thiol groups in the antioxidant molecules.
In view of these problems with antioxidant therapy, the goal of this study was to examine the trans-tympanic route of delivering antioxidants and other drugs to the cochlea to reduce hearing loss. The trans-tympanic route of drug and short interfering (si) RNA, described below, appears particularly promising.

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			<td height="19" style="height:19px;width:423px;">Ketathesia (100 mg/ml) 10 ml</td>
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			<td height="19" style="height:19px;">(&#8722;)-N6-(2-Phenylisopropyl)adenosine</td>
			<td>Sigma Aldrich</td>
			<td>P4532</td>
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			<td height="19" style="height:19px;">8-Cyclopentyl-1,3-dipropylxanthine</td>
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			<td height="22" style="height:22px;">siRNA pSTAT1</td>
			<td>Qiagen</td>
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			<td>Kaur et al. 201120</td>
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			<td height="22" style="height:22px;">siRNA NOX3</td>
			<td>Qiagen</td>
			<td>Custome Made</td>
			<td>Kaur et al. 201120</td>
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			<td>Qiagen</td>
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			<td>Kaur et al. 201120</td>
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trans-tympanic drug delivery for the treatment of ototoxicity

The systemic administration of protective agents to treat drug-induced ototoxicity is limited by the possibility that these protective agents could interfere with the chemotherapeutic efficacy of the primary drugs. This is especially true for the drug cisplatin, whose anticancer actions are attenuated by antioxidants which provide adequate protection against hearing loss. Other current or potential otoprotective agents could pose a similar problem, if administered systemically. The application of various biologicals or protective agents directly to the cochlea would allow for high levels of these agents locally with limited systemic side effects. In this report, we demonstrate a trans-tympanic method of delivery of various drugs or biological reagents to the cochlea, which should enhance basic science research on the cochlea and provide a simple way of directing the use of otoprotective agents in the clinics. This report details a method of trans-tympanic drug delivery and provides examples of how this technique has been used successfully in experimental animals to treat cisplatin ototoxicity.

ABR responses measured in rats at three days following cisplatin administration showed a significant elevation in thresholds. Elevation of these thresholds was significantly reduced in rats administered with trans-tympanic [R]-N-phenylisopropyladenosine (R-PIA), adenosine A1 receptor agonist15, prior to cisplatin. The specificity of the action of R-PIA at the adenosine A1 receptor was demonstrated by the observation ...

Watch this Scientific Journal Video about Trans-Tympanic Drug Delivery for the Treatment of Ototoxicity at JoVE.com

Trans-Tympanic Drug Delivery for the Treatment of Ototoxicity

We present a technique for localized administration of drugs through the trans-tympanic route into the cochlea. Drug delivery through this route would not interfere with the anti-cancer efficacy of the chemotherapeutic drugs such as cisplatin.

The systemic administration of protective agents to treat drug-induced ototoxicity is limited by the possibility that these protective agents could ...

The overall goal of this trans-tympanic procedure, is to locally deliver high concentrations of antioxidants, otoprotective agents, or biological reagents directly into the cochlea for treatment of hearing loss. The trans-tympanic route of drug delivery allows for the localized delivery of drugs and other agents into the cochlea that may cause significant side effects if administered systemically. The main advantage of this matter is that it permits rapid administration of drugs in significantly higher doses into the cochlea than would be achieved by systemic delivery.This technique has the potential for the prophylactic delivery of reagents to prevent against cisplatin-induced hearing loss without interfering with the anti-tumor effect. After confirming a lack of response to toe pinch, apply ointment to the eyes of the rat. To asses the auditory brainstem response, place the rat in the prone position on a 37 degree Celsius heating pad inside a controlled acoustical booth and insert a stainless steel ground electrode into the rear flank, a stainless steel positive electrode into the top of the skull between the ears, and one stainless steal negative electrode below the pinna of each ear.Carefully insert high-frequency transducer tubes into the rat's ears. Then, using high-frequency transducers, apply acoustics stimuli as a five millisecond tone burst at 8, 16, and 32 kilohertz. Place the animal in the left lateral decubitus position.Insert a 2.5 millimeter disposable ear specula into the ear canal and use a surgical scope to position the specula so that the tympanic membrane is visible. It's important to orient the needles so that the ear drum is still visible, because the ear specula is so small and a successful injection requires visualization of all materials and tissue under the microscope. Using a 29 and a half gauge 0.5 milliliter insulin syringe, aspirate 50 microliters of the drug or reagent of interest into the syringe barrel.And use the specula to direct the needle into the anterior inferior of the tympanic membrane. Use the needle to poke a hole through the membrane and administer the entire 50 microliter volume of the drug. Allow the rat to rest in this position for 15 minutes.Then, place the rat in the right lateral decubitus position and administer the drug into the right ear as just demonstrated. For rats receiving cisplatin, place the rat in the supine position on a 37 degree Celsius heating pad and draw cisplatin into a 21 and three-quarter gauge butterfly needle connected to a 12-inch piece of tubing. Then, use a syringe pump to administer one milligram per milliliter of cisplatin via intraperitoneal injection over 30 minutes.When all of the cisplatin has been delivered, place the rat back into its cage in the prone position, taking care that its breathing is not obstructed, and monitor the animal until it is fully recovered. Following the measurement of the post-treatment ABR, sacrifice the rat and dissect out temporal bone. Place the harvested cochlea in a seven milliliter glass scintillation vial containing four percent paraformaldehyde in PBS and store it overnight at four degrees Celsius.The next morning, wash the cochlea in room temperature PBS. Followed by complete immersion in 120 millimolar EDTA. Then, place the vial on a rotator to decalcify the cochlea for two to three weeks at room temperature.Upon completion of the decalcification, place the cochlea in sequential immersions of seven milliliters of sucrose for 24 hours per sucrose solution at four degrees Celsius. After the last submersion, add fresh optimal cutting temperature compound to a 15 by 15 by 5 millimeter disposable embedding mold, and place the cochlea on its side in the mold so that the sample is parallel to the bottom of the mold. Immediately place the mold on dry ice to solidify the OCT and store the mold at negative 80 degrees Celsius overnight.The next morning, transfer the mold onto dry ice and set a cryostat to negative 30 degrees Celsius. Then, use sharp microtone blade to acquire two ten-micrometer sections of cochlea sample per polylysine coated slide. Insert the slides at four degrees Celsius.To analyze the sections by immunohistochemistry, place the slides in a glass microscope slide staining dish on a slide rack and wash the section three times with 350 milliliters of PBS for five minutes per wash. After the last watch, use a lab wipe to remove the excess PBS around each sample, and use hydrophobic barrier pen to surround each section. Block the tissues for one hour at room temperature with 150 microliters of blocking solution per sample.At the end of the incubation, top off the excess blocking solution and incubate the samples with 150 microliters of the primary antibody of interest overnight at four degree Celsius in a humidified chamber. The next morning, return the slides to the staining dish on the side rack and wash the slides three times in PBS as just demonstrated. Incubate the sections with the appropriate secondary antibodies for two to three hours at room temperature in the humidified chamber in the dark.At the end of the incubation, return the slides to the staining dish and wash the slides three times in fresh PBS. After the last wash, tap the slides to remove the excess PBS and dry the slides with a lab wipe. Place a drop of mounting agent supplemented with DAPI directly onto each tissue section and carefully place a cover slip onto each slide taking care not to create bubbles.Then, allow the slides to cure overnight at room temperature in the dark before imaging the samples by confocal microscopy. Auditory brainstem responses measured in rats at three days post-cisplatin administration shows significant elevation in thresholds that is reduced in rats treated with transtympanic delivery of the adenosine A1 receptor agonist, R-PIA, prior to cisplatin administration. Conversely, treatment with DPCPX, an A1 receptor-specific antagonist, potentiates the cisplatin-induced auditory brainstem response threshold shifts, demonstrating the specificity of the R-PIA agonist for the A1 receptor.Cisplatin also increases outer hair cell loss and damage as assessed by scanning electron microscopy, an effect that is significantly reduced by transtympanic R-PIA administration. Further, transtympanic R-PIA reduces basal and cisplatin-induced phospho stat1 immunoreactivity in the outer hair cells of the cochlea, in accordance with the anti inflammatory property of this drug. The transtympanic route can also be used to administer other biologics, such as siRNAs, to reduce stat1 levels and to block the activation of this transcription factor to phospho stat1 by cisplatin, for example.Addition studies show that transtympanic administration of NOX3 siRNA reduces the ability of drugs, such as capsaicin, to increase the expression of some downstream mediators that are implicated in capsaicin-induced hearing loss. When administering this technique in intratympanic injection, it is imperative that the animal be adequately anesthetized so it does not move during the procedure. Once mastered, this drug delivery system could be used to screen a large number of compounds for efficacy against drug-induced hair loss in experimental animals.

trans-tympanic-drug-delivery-for-the-treatment-of-ototoxicity

Research

JoVE Journal

Medicine

Osmotic Drug Delivery to Ischemic Hindlimbs and Perfusion of Vasculature with Microfil for Micro-Computed Tomography Imaging

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to stimulate microcirculation. The choice of delivery method for these agents is important because the route of administration profoundly affects the bioactivity and efficacy of these agents1,2. In this article, we demonstrate how to locally administer a substance in ischemic hindlimbs by using a catheterized osmotic pump. This pump can deliver a fixed volume of aqueous solution continuously for an allotted period of time. We also present our mouse model of unilateral hindlimb ischemia induced by ligation of the common femoral artery proximal to the origin of profunda femoris and epigastrica arteries in the left hindlimb. Lastly, we describe the in vivo cannulation and ligation of the infrarenal abdominal aorta and perfusion of the hindlimb vasculature with Microfil, a silicone radiopaque casting agent. Microfil can perfuse and fill the entire vascular bed (arterial and venous), and because we have ligated the major vascular conduit for exit, the agent can be retained in the vasculature for future ex vivo imaging with the use of small specimen micro-CT3.

We show here the in vivo insertion of an osmotic pump for constant local drug delivery and the creation of hindlimb ischemia in a mouse model. Moreover, the hindlimb vasculature is perfused with Microfil, a silicone radiopaque agent, to prepare for micro-computed tomography (micro-CT) imaging.

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to ...

Heterotopic Mucosal Engrafting Procedure for Direct Drug Delivery to the Brain in Mice

Delivery of therapeutics into the brain is impeded by the presence of the blood-brain barrier (BBB) which restricts the passage of polar and high molecular weight compounds from the bloodstream and into brain tissue. Some direct delivery success in humans has been achieved via implantation of transcranial catheters; however this method is highly invasive and associated with numerous complications. A less invasive alternative would be to dose the brain through a surgically implanted, semipermeable membrane such as the nasal mucosa that is used to repair skull base defects following endoscopic transnasal tumor removal surgery in humans. Drug transfer though this membrane would effectively bypass the BBB and diffuse directly into the brain and cerebrospinal fluid. Inspired by this approach, a surgical approach in mice was developed that uses a donor septal mucosal membrane engrafted over an extracranial surgical BBB defect. This model has been shown to effectively allow the passage of high molecular weight compounds into the brain. Since numerous drug candidates are incapable of crossing the BBB, this model is valuable for performing preclinical testing of novel therapies for neurological and psychiatric diseases.

A mouse model of human endoscopic skull base reconstruction has been developed that creates a semipermeable interface between the brain and nose using nasal mucosal grafts. This method allows researchers to study delivery to the central nervous system of high molecular weight therapeutics which are otherwise excluded by the blood-brain barrier when administered systemically.

Delivery of therapeutics into the brain is impeded by the presence of the blood-brain barrier (BBB) which restricts the passage of polar and high ...

Slow-release Drug Delivery through Elvax 40W to the Rat Retina: Implications for the Treatment of Chronic Conditions

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these diseases. Chronic retinal diseases such as retinal oedema or neovascularization usually require multiple intraocular injections to effectively treat the condition. However, the risks associated with these injections increase with repeated delivery of the drug. Therefore, alternative delivery methods need to be established in order to minimize the risks of reinjection. Several other investigations have developed methods to deliver drugs over extended time, through materials capable of releasing chemicals slowly into the eye. In this investigation, we outline the use of Elvax 40W, a copolymer resin, to act as a vehicle for drug delivery to the adult rat retina. The resin is made and loaded with the drug. The drug-resin complex is then implanted into the vitreous cavity, where it will slowly release the drug over time. This method was tested using 2-amino-4-phosphonobutyrate (APB), a glutamate analogue that blocks the light response of the retina. It was demonstrated that the APB was slowly released from the resin, and was able to block the retinal response by 7 days after implantation. This indicates that slow-release drug delivery using this copolymer resin is effective for treating the retina, and could be used therapeutically with further testing.

This paper details how Elvax 40W can be used as a slow-release method for drug delivery to the adult rat retina. The protocol for preparing, loading, and delivering the drug-resin complex to the eye is described.

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these ...

Intraluminal Drug Delivery to the Mouse Arteriovenous Fistula Endothelium

Delivery of therapeutic agents to enhance arteriovenous fistula (AVF) maturation can be administered either via intraluminal or external routes. The simple murine AVF model was combined with intraluminal administration of drug solution to the venous endothelium at the same time as fistula creation. Technical aspects of this model are discussed. Under general anesthesia, an abdominal incision is made and the aorta and inferior vena cava (IVC) are exposed. The infra-renal aorta and IVC are dissected for clamping. After proximal and distal clamping, the puncture site is exposed and a 25 G needle is used to puncture both walls of the aorta and into the IVC. Immediately after the puncture, a reporter gene-expressing viral vector was infused in the IVC via the same needle, followed by 15 min of incubation. The intraluminal administration method enabled more robust viral gene delivery to the venous endothelium compared to administration by the external route. This novel method of delivery will facilitate studies that explore the role of the endothelium in AVF maturation and enable intraluminal drug delivery at the time of surgical operation.

After puncturing the aorta through the inferior vena cava (IVC) to create an aorto-caval fistula in the mouse, solution containing a drug is infused into the IVC via the same needle, followed by incubation. This method enables more robust drug delivery to the venous endothelium compared to the external route.

Delivery of therapeutic agents to enhance arteriovenous fistula (AVF) maturation can be administered either via intraluminal or external routes. The ...

Network Analysis of Foramen Ovale Electrode Recordings in Drug-resistant Temporal Lobe Epilepsy Patients

Approximately 30% of epilepsy patients are refractory to antiepileptic drugs. In these cases, surgery is the only alternative to eliminate/control seizures. However, a significant minority of patients continues to exhibit post-operative seizures, even in those cases in which the suspected source of seizures has been correctly localized and resected. The protocol presented here combines a clinical procedure routinely employed during the pre-operative evaluation of temporal lobe epilepsy (TLE) patients with a novel technique for network analysis. The method allows for the evaluation of the temporal evolution of mesial network parameters. The bilateral insertion of foramen ovale electrodes (FOE) into the ambient cistern simultaneously records electrocortical activity at several mesial areas in the temporal lobe. Furthermore, network methodology applied to the recorded time series tracks the temporal evolution of the mesial networks both interictally and during the seizures. In this way, the presented protocol offers a unique way to visualize and quantify measures that considers the relationships between several mesial areas instead of a single area.

This protocol describes a procedure to track the evolution of mesial network measures in temporal lobe epilepsy (TLE) patients. It is based on the combination of intracranial recordings with a novel numerical technique for data analysis. Specifically, we present a protocol for network analyses of foramen ovale recordings.

Approximately 30% of epilepsy patients are refractory to antiepileptic drugs. In these cases, surgery is the only alternative to eliminate/control ...

Pancreatic Duct Infusion: An Effective and Selective Method of Drug and Viral Delivery

The pancreas is a bifunctional organ with both endocrine and exocrine components. A number of pathologies can afflict the pancreas, including diabetes, pancreatitis, and pancreatic cancer. All three of these diseases mark active areas of study, not only to develop immediate therapy, but also to better understand their pathophysiology. There are few tools to further these areas of study. Pancreatic duct infusion is an important technique that can allow for lineage tracing, gene introduction, and cell line-specific targeting. The technique requires the intricate dissection of the second portion of the duodenum and ampulla, followed by the occlusion of the bile duct and the cannulation of the pancreatic duct. Although the technique is technically challenging at first, the applications are myriad. Ambiguity in the specifics of the procedure between groups highlighted the need for a standard protocol. This work describes the expression of a green fluorescent protein (GFP) within the pancreas after the pancreatic duct infusion of a viral vector expressing GFP versus a sham surgery. The infusion and therefore expression is specific to the pancreas, without expression present in any other tissue type.

Pancreatic duct infusion is an important technique that can allow for lineage tracing, gene introduction, and cell line-specific targeting. A pancreatic duct infusion technique for drug and viral delivery to pancreatic cells is presented here.

The pancreas is a bifunctional organ with both endocrine and exocrine components. A number of pathologies can afflict the pancreas, including diabetes, ...

Optimized LC-MS/MS Method for the High-throughput Analysis of Clinical Samples of Ivacaftor, Its Major Metabolites, and Lumacaftor in Biological Fluids of Cystic Fibrosis Patients

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening pulmonary manifestations. Ivacaftor (IVA) and ivacaftor-lumacaftor (LUMA) combination are two new breakthrough CF drugs that directly modulate the activity and trafficking of the defective CFTR-protein. However, there is still a dearth of understanding on pharmacokinetic/pharmacodynamic parameters and the pharmacology of ivacaftor and lumacaftor. The HPLC-MS technique for the simultaneous analysis of the concentrations of ivacaftor, hydroxymethyl-ivacaftor, ivacaftor-carboxylate, and lumacaftor in biological fluids in patients receiving standard ivacaftor or ivacaftor-lumacaftor combination therapy has previously been developed by our group and partially validated to FDA standards. However, to allow the high-throughput analysis of a larger number of patient samples, our group has optimized the reported method through the use of a smaller pore size reverse-phase chromatography column (2.6 &#181;m, C8 100 &#197;; 50 x 2.1 mm) and a gradient solvent system (0-1 min: 40% B; 1-2 min: 40-70% B; 2-2.7 min: held at 70% B; 2.7-2.8 min: 70-90% B; 2.8-4.0 min: 90% B washing; 4.0-4.1 min: 90-40% B; 4.1-6.0 min: held at 40% B) instead of an isocratic elution. The goal of this study was to reduce the HPLC-MS analysis time per sample dramatically from ~15 min to only 6 min per sample, which is essential for the analysis of a large amount of patient samples. This expedient method will be of considerable utility for studies into the exposure-response relationships of these breakthrough CF drugs.

Ivacaftor and ivacaftor-lumacaftor combination are two new CF drugs. However, there is still a dearth of understanding on their PK/PD and pharmacology. We present an optimized HPLC-MS technique for the simultaneous analysis of ivacaftor and its major metabolites, and lumacaftor.

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

Novel Methods for Intranasal Administration Under Inhalation Anesthesia to Evaluate Nose-to-Brain Drug Delivery

Intranasal administration has been reported to be a potential pathway for nose-to-brain delivery of therapeutic agents that circumvents the blood-brain barrier. However, there have been few reports regarding not only the quantitative analysis but also optimal administration conditions and dosing regimens for investigations of nose-to-brain delivery. The limited progress in research on nose-to-brain pathway mechanisms using rodents represents a significant impediment in terms of designing nose-to-brain delivery systems for candidate drugs.
To gain some headway in this regard, we developed and evaluated two novel methods of stable intranasal administration under inhalation anesthesia for experimental animals. We also describe a method for the evaluation of drug distribution levels in the brain via the nose-to-brain pathway using radio-labeled [14C]-inulin (molecular weight: 5,000) as a model substrate of water-soluble macromolecules.
Initially, we developed a pipette-based intranasal administration protocol using temporarily openable masks, which enabled us to perform reliable administration to animals under stable anesthesia. Using this system, [14C]-inulin could be delivered to the brain with little experimental error.
We subsequently developed an intranasal administration protocol entailing reverse cannulation from the airway side through the esophagus, which was developed to minimize the effects of mucociliary clearance (MC). This technique led to significantly higher levels of [14C]-inulin, which was quantitatively detected in the olfactory bulb, cerebrum, and medulla oblongata, than the pipette method. This appears to be because retention of the drug solution in the nasal cavity was substantially increased by active administration using a syringe pump in a direction opposite to the MC into the nasal cavity.
In conclusion, the two methods of intranasal administration developed in this study can be expected to be extremely useful techniques for evaluating pharmacokinetics in rodents. The reverse cannulation method, in particular, could be useful for evaluating the full potential of nose-to-brain delivery of drug candidates.

Here, we describe two novel methods of stable intranasal administration under inhalation anesthesia with minimal physical stress for experimental animals. We also describe a method for quantitative evaluation of drug distribution levels in the brain via the nose-to-brain pathway using radiolabeled [14C]-inulin as a model substrate of water-soluble macromolecules.

Intranasal administration has been reported to be a potential pathway for nose-to-brain delivery of therapeutic agents that circumvents the blood-brain ...

Preclinical Model of Hind Limb Ischemia in Diabetic Rabbits

Peripheral vascular disease is a widespread clinical problem that affects millions of patients worldwide. A major consequence of peripheral vascular disease is the development of ischemia. In severe cases, patients can develop critical limb ischemia in which they experience constant pain and an increased risk of limb amputation. Current therapies for peripheral ischemia include bypass surgery or percutaneous interventions such as angioplasty with stenting or atherectomy to restore blood flow. However, these treatments often fail to the continued progression of vascular disease or restenosis or are contraindicated due to the overall poor health of the patient. A promising potential approach to treat peripheral ischemia involves the induction of therapeutic neovascularization to allow the patient to develop collateral vasculature. This newly formed network alleviates peripheral ischemia by restoring perfusion to the affected area. The most frequently employed preclinical model for peripheral ischemia utilizes the creation of hind limb ischemia in healthy rabbits through femoral artery ligation. In the past, however, there has been a strong disconnect between the success of preclinical studies and the failure of clinical trials regarding treatments for peripheral ischemia. Healthy animals typically have robust vascular regeneration in response to surgically induced ischemia, in contrast to the reduced vascularity and regeneration in patients with chronic peripheral ischemia. Here, we describe an optimized animal model for peripheral ischemia in rabbits that includes hyperlipidemia and diabetes. This model has reduced collateral formation and blood pressure recovery in comparison to a model with a higher cholesterol diet. Thus, the model may provide better correlation with human patients with compromised angiogenesis from the common co-morbidities that accompany peripheral vascular disease.

We describe a surgical procedure used to induce peripheral ischemia in rabbits with hyperlipidemia and diabetes. This surgery acts as a preclinical model for conditions experienced in peripheral artery disease in patients. Angiography is also described as a means to measure the extent of introduced ischemia and recovery of perfusion.

Peripheral vascular disease is a widespread clinical problem that affects millions of patients worldwide. A major consequence of peripheral vascular ...