This research was supported by the Australian Research Council through the ARC Centre of Excellence in Vision Science (CE0561903), the National Health and Medical Research Council (1049990), and Progetto regionale speciale multiasse 'Reti per l'Alta Formazione' - PO FSE Abruzzo 2007-2013 - Azione 4.II.iii.

All experiments conducted were in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and with the approval of the University of L&#8217;Aquila Animal Ethics Committee and the Australian National University Animal Experimentation Ethics Committee. Adult rats (P100-200) were used throughout this study.
1. Prepare the Copolymer Resin Pellets
<ol>
	<li>Place 20 Elvax 40W pellets into a small glass beaker in the fume cupboard.</li>
	<li>Fill the beaker with 100% ethanol ensuring to cover all pellets.</li>
	<li>Cover the beaker with Parafilm, and soak the pellets in 100% ethanol at room temperature for 7-10 days. NOTE: From this step onwards, do not use metal or plastic to handle or store the resin.</li>
</ol>
2. Prepare the Drug Solution for Loading
<ol>
	<li>Dissolve the drug of choice in 0.1% dimethyl sulfoxide (DMSO). Make the drug solution up to 40 &#181;l in volume. Load the resin with three times the concentration of the drug that would be given in a single dose.</li>
</ol>
3. Prepare and Load the Drug-resin Complex
<ol>
	<li>Transfer the 20 washed pellets to another small glass beaker. Dissolve the pellets in 4 ml dichloromethane for approximately 45 min. Cover the beaker with Parafilm while the pellets are dissolving.</li>
	<li>Prepare a solution of Fast Green FCF by dissolving it in 0.1% DMSO to a concentration of 0.001 mg/ml.</li>
	<li>Take one pipette, and draw up 40 &#181;l of the drug solution prepared earlier. Load another pipette with 40 &#181;l of Fast Green solution.</li>
	<li>Add the two solutions simultaneously into the beaker. Mix rapidly using a glass stirring rod until the green dye is uniformly distributed throughout the mixture.</li>
	<li>Promptly transfer the beaker onto dry ice for 10 min to fast freeze the resin.</li>
	<li>Set up an &#8220;evaporative chamber&#8221; to evaporate the solvent. Fill a larger container up to one-third with calcium sulfate pebbles. Create a well in the pebbles, and carefully place the beaker in the well so that the pebbles reach up to half of the beaker, to stabilize the beaker in the chamber.</li>
	<li>Cover the larger outside container with Parafilm.</li>
	<li>Transfer the evaporative chamber to the freezer. Keep at -20 &#176;C for 2-3 weeks.</li>
</ol>
4. Prepare the Drug-resin Complex for Surgical Implantation
<ol>
	<li>Remove the evaporative chamber from the freezer. Transfer the solid drug-resin block to a glass dish kept on ice.</li>
	<li>With the aid of a microscope or loupe, cut a piece approximately 0.05 mm in diameter and 0.1 mm in length from the block using a trephine or punch tool.</li>
	<li>Wrap the remainder of the block in freeze-proof material (e.g., aluminum foil) and keep at -20 &#176;C until required.</li>
</ol>
5. Surgically Implant the Drug-resin Complex into the Rat Eye
<ol>
	<li>Prepare the animal for surgery. Anesthetize using an intraperitoneal injection of a mixture of ketamine (100 mg/kg body weight) and xylazine (12 mg/kg body weight). Observe the animal until full anesthesia is achieved. NOTE: The animal should have a loss of movements (except breathing), and a loss of corneal and toe pinch reflexes.</li>
	<li>Transfer the fully anesthetized animal to the surgery table. Use mydriaticum (e.g.,&#160;atropine sulphate), and local anesthetic (e.g.,&#160;tetracaine hydrochloride) drops on the eye(s) undergoing surgery. Apply artificial tears to keep the cornea moist during surgery. Use disposable needles and sterile instruments throughout the surgery.</li>
	<li>Stabilize the eye using a pair of blunt curved forceps after the pupil is fully dilated. Use an operating microscope to make a full depth puncture wound to reach the vitreous, approximately 2 mm from the limbus using a 25 G needle.</li>
	<li>Insert the prepared piece of the drug-resin complex into the puncture wound using a pair of fine, pointy forceps (e.g., tweezers) that are able to penetrate the puncture site.</li>
	<li>As prophylaxis for bacterial infection, apply antibiotic ointment to the eye, to prevent inflammation as well as preventing corneal dryness while the animal is recovering.</li>
	<li>Observe the animal until full recovery. Do not leave the animal unattended until it has regained consciousness. Do not return the animal to the company of other animals until fully recovered. If the animal shows signs of stress or discomfort, analgesics should be used according to the approved ethics protocol.</li>
	<li>Follow the usual experimental procedures for collection and analysis of the tissue.</li>
	<li>At the completion of experiments, euthanize the animal with an intraperitoneal injection of a pentobarbital (60 mg/kg body weight) 2% lidocaine hydrochloride solution.</li>
</ol>
NOTE: Lignocaine Hydrochloride minimizes local discomfort.

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The authors have nothing to disclosure.

This paper demonstrated the use of a drug-resin complex for the slow-release delivery of drugs to the retina.&#160;We aimed to present a method which is relatively inexpensive and easy to apply in a small animal model.
Given that the function of APB is to act as a glutamate analogue, it will block the retinal response in the eye. The results show that APB caused the blockage of the retinal response by one week postimplantation. This indicates that APB was successfully released into the vitreou...

The treatment of chronic diseases such as diabetes and high blood pressure presents many challenges, as these diseases generally require treatment for extended periods of time, often for life. This has called for the development of slow release drug delivery systems, which reduce the need for frequent dosing. The effectiveness of these slow release methods has been demonstrated through the development of insulin pumps, to reduce the number of insulin injections needed to treat diabetes. Chronic diseases of the eye, particularly those affecting its inner layers, require the frequent administering of drugs through invasive procedures. One such chronic disease affecting the human eye is age related macular degeneration (AMD). It affects the central retina, which is a layer of neural tissue situated at the back of the eye responsible for initiating vision. AMD is the leading cause of blindness in the Western World1. A particular challenge with treating retinal diseases is that the drug is required to reach this deep layer in the eye, which often requires invasive methods of delivery. Drugs are usually administered to the vitreous chamber and retina using intravitreal injections. However, with each injection there is a risk of postinjection complications, including endophthalmitis, retinal detachment, cataract and vitreous hemorrhaging2. This risk is multiplied with every reinjection of the drug.
Reducing the need for multiple injections would be a major benefit in the treatment of AMD. To treat the wet form of AMD, where new vessel growth is a hallmark, the established therapeutic strategy is to target the endothelial growth factors (VEGF) using VEGF inhibitors3. At present, these are delivered through repeated intravitreal injections. Similarly, in the treatment of macular oedema, a common complication of diabetic retinopathy, corticosteroids are delivered through repeated injections4. Delivery of these drugs through slow release methods could indeed reduce the risk of postinjection complications.
The notion of a slow release drug delivery system was first described using a silicone rubber vehicle to deliver small molecules into animal tissues5. Since then, other slow release methods have been developed to deliver larger molecules, several of which have been tested in the eye. Particle carriers, such as biodegradable microspheres, poly-lactide-co-glycolide (PLGA) nanoparticles and phospholipid vesicles (liposomes) can be useful as delivery vehicles6,7. PLGA nanoparticles and liposomes have been compared in an in vitro environment for their ability to deliver anti-cancer agents across the sclera over time7. Both vehicles were effective in slowly releasing the drugs. However, the study was only conducted in an in vitro environment. Bochot et al. (2002)8 tested the efficacy of liposomes to deliver molecules to the retina in vivo. They have demonstrated that liposomes successfully deliver small oligonucleotides to the rabbit retina. The authors suggested that liposomes could be beneficial in the treatment of retinal diseases8. However, the nature of these vesicles to float in the vitreous means that they will likely blur or impair vision9.
Okabe et al. (2003)10 used non biodegradable polymer discs made up of 33% ethylene-vinyl acetate to apply beta-methasone in rabbits. They implanted discs into a scleral pocket and demonstrated the effective release of the drug into the vitreous and retina for up to one month10. However, in this particular protocol, the implant was relatively large and rigid, and required a more complex surgical procedure including a large scleral incision and suturing.
An earlier study investigated the tissue response to various polymer vehicles by implanting them into the cornea of rabbit eyes, and found that ethylene-vinyl acetate copolymers washed in alcohol did not cause inflammation or irritation. These complexes were shown to sustain the delivery of larger compounds into animal tissues for extended periods of time, some exceeding up to 100 days dependent on the drug11. One such type of copolymer resin has been developed industrially in the form of Elvax 40W (40% by weight ethylene-vinyl acetate comonomer content with a &#8216;W&#8217; amide additive to improve pellet handling). This copolymer resin is an inert substance which is stable at both room and body temperature. It has not been shown to cause allergies or toxicity in biological tissue. This resin has effectively delivered a large variety of drugs in different experimental models that investigate the functions of various systems, such as the mammary ductal system12, the primary auditory cortex13,14, and the frog visual system15. This resin has also been used in the eye to deliver drugs to developing turtle16,17, chicken embryo18,19, and adult ferret retinas20. In the rat central nervous system, the resin has only been used in the brain21-23, but its use in the rat eye has not been documented.
The advantages of using this copolymer resin to deliver drugs slowly to the retina over other methods, is that it is a stable compound that does not cause inflammation or irritation in the eye. Unlike particle carriers, the drug-resin complex would not impair vision after implantation, as it usually remains at the site of delivery instead of floating in the vitreous. It would only require a simple implantation process into the vitreous cavity close to the limbus of the eye, and would not require suturing after implantation. Recently, there has been an emergence in several novel delivery systems, such as encapsulated cell technology (ECT)24,25, hydrogels26, and microfilms27. However, the method used in the current study for preparing and delivering the drug-resin complex is both easy to follow and inexpensive, thereby being more advantageous for use in a basic research environment. The challenge of using this complex to deliver long-term drug treatments is to determine the optimal concentration of the drug that will maximize the therapeutic benefits of having fewer intravitreal injections.
This paper aims to demonstrate the use of the drug-resin complex for long-term treatment of the adult rat retina. The efficacy of this mode of delivery is tested using the glutamate analogue 2-amino-4-phosphonobutyrate (APB) as the drug. APB blocks the light response of the ON bipolar cells by mimicking glutamate, an endogenous neurotransmitter in the retina28. When APB competes with glutamate for its receptor, it blocks the light response. APB has been used in physiological studies to control retinal function and measure its effect using electrophysiological methods such as electroretinography (ERG). In previous studies, APB has been used for both short-term29 and long-term treatment of the developing retina; the latter involved giving a single dose via intraocular injection daily for 30 days30. The same amount of APB (0.092 mg in sterile saline, at a concentration of 50 &#181;M) was used for all injections as suggested in previous work28,29,31. We chose APB to test the drug-resin complex as a slow-release vehicle to deliver drugs into the eye. The methods outlined in this study are similar to previously described methods involving the preparation of the drug-resin complex16,32; however we also detail its use specifically in the adult rat eye. After surgical implantation of the APB-loaded resin into the eye, ERG was performed to establish whether APB abolishes the retinal light response, and therefore whether APB has been successfully released into the vitreous and retina.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Elvax 40W Pellets</td>
			<td>Du Pont, DE, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich Co. LLC., MO, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2-amino-4-phosphonobutyric acid (APB)</td>
			<td>Sigma-Aldrich Co. LLC., MO, USA</td>
			<td>A1910</td>
			<td></td>
		</tr>
		<tr>
			<td>Dichloromethane</td>
			<td>Sigma-Aldrich Co. LLC., MO, USA</td>
			<td>34856</td>
			<td></td>
		</tr>
		<tr>
			<td>Fast Green FCF</td>
			<td>Sigma-Aldrich Co. LLC., MO, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Drierite, calcium sulfate</td>
			<td>Sigma-Aldrich Co. LLC., MO, USA</td>
			<td>238910</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine, Ilium Ketamil</td>
			<td>Troy Laboratories Pty. Ltd., NSW, Australia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine, Ilium Xylazil-20</td>
			<td>Troy Laboratories Pty. Ltd., NSW, Australia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Atropine Sulphate, Minims eye drops</td>
			<td>Bausch &#38; Lomb Pty. Ltd., NSW, Australia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tetracaine Hydrochloride, Minims eye drops</td>
			<td>Bausch &#38; Lomb Pty. Ltd., NSW, Australia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chloramphenicol, Chlorsig ointment</td>
			<td>Aspen Pharma Pty. Ltd., NSW, Australia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pentobarbital, Lethabarb</td>
			<td>Virbac Australia Pty. Ltd., NSW, Australia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lignocaine Hydrochloride, Ilium Lignocaine-20</td>
			<td>Troy Laboratories Pty. Ltd., NSW, Australia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Uni-Core Punch Tool</td>
			<td>World Precision Instruments Inc., FL, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Curved Forceps</td>
			<td>World Precision Instruments Inc., FL, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Operating Microscope, Zeiss OPMI 99</td>
			<td>Zeiss, West Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>25G Insulin Needle</td>
			<td>Terumo Corp., Tokyo, Japan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont Tweezers</td>
			<td>World Precision Instruments Inc., FL, USA</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Full-field electroretinography (ERG) was used to detect the effect of APB on the retina. For details on how the ERG was performed, refer to the following studies33,34. In brief, after anesthetizing the animal, a gold electrode was placed on the cornea of the eye, and the reference electrode was placed in the anterior scalp between the eyes, to record the electrical activity of the retina. Stimuli were generated using an electronic flash unit and the intensity ranged from 10 to 106 photoisomerization...

Watch this Scientific Journal Video about Slow-release Drug Delivery through Elvax 40W to the Rat Retina: Implications for the Treatment of Chronic Conditions at JoVE.com

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

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 ...

slow-release-drug-delivery-through-elvax-40w-to-rat-retina

Research

JoVE Journal

Medicine

11.5K 조회수.  University of L'Aquila.  이 논문은 Elvax 40W가 성인 쥐의 망막에 약물 전달에 대한 서방의 방법으로 사용 할 수있는 방법을 자세히 설명합니다. 준비, 로딩, 및 눈에 약물 - 수지 복합체를 제공하기위한 프로토콜을 설명한다. 

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

The Measurement and Treatment of Suppression in Amblyopia

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current estimates put the prevalence of amblyopia at approximately 1-3%1-3, the majority of cases being monocular2. Amblyopia is most frequently caused by ocular misalignment (strabismus), blur induced by unequal refractive error (anisometropia), and in some cases by form deprivation.
Although amblyopia is initially caused by abnormal visual input in infancy, once established, the visual deficit often remains when normal visual input has been restored using surgery and/or refractive correction. This is because amblyopia is the result of abnormal visual cortex development rather than a problem with the amblyopic eye itself4,5 . Amblyopia is characterized by both monocular and binocular deficits6,7 which include impaired visual acuity and poor or absent stereopsis respectively. The visual dysfunction in amblyopia is often associated with a strong suppression of the inputs from the amblyopic eye under binocular viewing conditions8. Recent work has indicated that suppression may play a central role in both the monocular and binocular deficits associated with amblyopia9,10 .
Current clinical tests for suppression tend to verify the presence or absence of suppression rather than giving a quantitative measurement of the degree of suppression. Here we describe a technique for measuring amblyopic suppression with a compact, portable device11,12 . The device consists of a laptop computer connected to a pair of virtual reality goggles. The novelty of the technique lies in the way we present visual stimuli to measure suppression. Stimuli are shown to the amblyopic eye at high contrast while the contrast of the stimuli shown to the non-amblyopic eye are varied. Patients perform a simple signal/noise task that allows for a precise measurement of the strength of excitatory binocular interactions. The contrast offset at which neither eye has a performance advantage is a measure of the "balance point" and is a direct measure of suppression. This technique has been validated psychophysically both in control13,14 and patient6,9,11 populations.
In addition to measuring suppression this technique also forms the basis of a novel form of treatment to decrease suppression over time and improve binocular and often monocular function in adult patients with amblyopia12,15,16 . This new treatment approach can be deployed either on the goggle system described above or on a specially modified iPod touch device15.

Amblyopia is a developmental disorder of the visual cortex that is often accompanied by strong suppression of one eye. We present a new technique for measuring and treating interocular suppression in patients with amblyopia that can be deployed using virtual reality goggles or a portable iPod Touch device.

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current ...

A Model of Chronic Nutrient Infusion in the Rat

Chronic exposure to excessive levels of nutrients is postulated to affect the function of several organs and tissues and to contribute to the development of the many complications associated with obesity and the metabolic syndrome, including type 2 diabetes. To study the mechanisms by which excessive levels of glucose and fatty acids affect the pancreatic beta-cell and the secretion of insulin, we have established a chronic nutrient infusion model in the rat. The procedure consists of catheterizing the right jugular vein and left carotid artery under general anesthesia; allowing a 7-day recuperation period; connecting the catheters to the pumps using a swivel and counterweight system that enables the animal to move freely in the cage; and infusing glucose and/or Intralipid (a soybean oil emulsion which generates a mixture of approximately 80% unsaturated/20% saturated fatty acids when infused with heparin) for 72 hr. This model offers several advantages, including the possibility to finely modulate the target levels of circulating glucose and fatty acids; the option to co-infuse pharmacological compounds; and the relatively short time frame as opposed to dietary models. It can be used to examine the mechanisms of nutrient-induced dysfunction in a variety of organs and to test the effectiveness of drugs in this context.

A protocol for chronic infusions of glucose and Intralipid in rats is described. This model can be used to study the impact of nutrient excess on organ function and physiological parameters.

Chronic exposure to excessive levels of nutrients is postulated to  affect the function of several organs and tissues and to contribute to the  ...

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 ...

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 ...

Intravascular Delivery of Biologics to the Rat Kidney

The renal microvascular compartment plays an important role in the progression of kidney disease and hypertension, leading to the development of End Stage Renal Disease with high risk of death for cardiovascular events. Moreover, recent clinical studies have shown that renovascular structure and function may have a great impact on functional renal recovery after surgery. Here, we describe a protocol for the delivery of drugs into the renal artery of rats. This procedure offers significant advantages over the frequently used systemic administration as it may allow a more localized therapeutic effect. In addition, the use of rodents in pharmacodynamic analysis of preclinical studies may be cost effective, paving the way for the design of translational experiments in larger animal models. Using this technique, infusion of rat recombinant Vascular Endothelial Growth Factor (VEGF) protein in rats has induced activation of VEGF signaling as shown by increased expression of FLK1, pAKT/AKT, pERK/ERK. In summary, we established a protocol for the intrarenal delivery of drugs in rats, which is simple and highly reproducible.

The administration of drugs for recovery of kidney function requires control of the localization and distribution of the therapeutic compound. Here, we describe in detail a simple technique for intrarenal delivery of drugs in rats. This procedure may be easily performed with no mortality and high reproducibility.

The renal microvascular compartment plays an important role in the progression of kidney disease and hypertension, leading to the development of End Stage ...

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 ...

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) ...

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.

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 ...