eoe sub articles

EoE Sub Articles

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Preparation of Micro-Osmotic Pump Infusion Systems
NOTE: Prepare the pump, artificial cerebrospinal fluid (aCSF) buffer, and drug (Met-CCL5/RANTES protein solution (10 ng/mL in aCSF)) under sterile conditions using buffers filtered with 0.2 &#181;m filters and conduct all the procedures under the culture hood with gloves. The surgery procedures are conducted as follows:
<ol>
	<li>Prepare the micro-osmotic pumps one day before surgery: Fill the brain micro-osmotic pump with artificial cerebrospinal fluid (aCSF) with a 1 mL syringe and blunt-head needle provided along with the kit. Immerse the micro-osmotic pump in aCSF and place it on a shaker and gently shake overnight. 
	CAUTION: The pump should be filled with aCSF, and air bubbles should be avoided inside the pump (Figure 1A).</li>
	<li>Before starting surgery, prepare the recombinant Met-CCL5/RANTES protein solution (10 ng/mL, diluted in aCSF) to be used in the experiment. Remove aCSF from the pump and fill the pump with the drug solution slowly until excess leaks out. 
	NOTE: 15 mL aCSF or Met-CCL5/RANTES solution is sufficient for 5-8 pumps. 
	CAUTION: Repeat the procedure to ensure that the pump is completely filled with the drug without bubbles inside.</li>
	<li>Cut the catheter tubes into the desired length and attach them with the blunt-end brain infusion needle in the brain infusion kit. Fill the infusion kit and tubes with the drug.</li>
	<li>Finally, assemble and attach the brain infusion kit to the micro-osmotic pump. 
	CAUTION: No bubbles should be formed in the tube or the pump (Figure 1A).</li>
	<li>Immerse the entire osmotic pump-brain fusion set in aCSF in a sterilized 50 mL tube to prevent the pump from drying out. The osmotic pump-brain fusion set is now ready to be used for surgery. 
	NOTE: The micro-osmotic pump systems can be used for long-term drug infusion. This ensures a safe and convenient mode of drug delivery into the mouse brain.</li>
</ol>
2. Intracerebral Ventricular Surgery &#8211; implantation of the micro osmotic pump
CAUTION: Sterilize the surgical environment with 75% ethanol and ensure that the people involved in the study are wearing sterile gloves and a clean lab coat. Surgical tools/ instruments must be autoclaved and dried before use, and subsequently sterilized with 75% ethanol in-between mice surgeries.
<ol>
	<li>Weigh the mouse and anesthetize it using intra-peritoneal injection (IP) with Ketamine/Xylazine (Ketamine 50 mg/kg, Xylazine 10 mg/kg). 
	CAUTION: Mice body weights lower than 24 g are not recommended for osmotic pump implantation surgery.</li>
	<li>Mount and fix the mouse head onto the stereotactic apparatus (Figure 1B).</li>
	<li>Use a pair of surgical scissors and pincers to cut open the outer skin covering the skull. Use iodine to clean the peripheral skull.</li>
	<li>Separate the outermost layer of skin from the subcutaneous skin with the help of a pair of blunt-head pincers near the neck region for the osmotic pump-brain fusion set implantation (Figure 1C).</li>
	<li>Mark the infusion point with reference to the brain map (Figure 1D) using the stereotactic apparatus. In this experiment, the needle needs to be implanted in the 3rd&#160;ventricle region (Bregma: 0.0 mm lateral, 1.3 mm posterior, 5.7 mm ventral).</li>
	<li>Drill a hole using a nail drill around the area marked on the skull (Figure 1E). 
	CAUTION: Be careful not to break the mouse meninges and blood vessels, thus avoiding the disruption of micro-blood vessels in the brain.</li>
	<li>Place the micro-osmotic pump-brain fusion set containing aCSF (as control) or drug (Met-CCL5/RANTES protein solution) under the skin behind the neck region and insert the brain infusion needle into the drilled hole to infuse the drug into the mouse brain (Figure 1E). The needle will penetrate the meninges and get into the ventricle. Fix the needle in place on the skull using surface desensitizing gel (Figure 1F) and wait 1-2 min until the glue dries. Next, cut off the projecting part on top of the needle (Figure 1G-H).</li>
	<li>Use a tissue adhesive glue to heal the operation wound on the head. Apply 50 &#181;L of the glue on top of the wound, pull both sides of the skin together, and hold on for 30 s to allow the skin to seal (Figure 1I). 
	CAUTION: Use 100% alcohol pad to clean the wound after surgery and 100 ul penicillin with streptomycin to prevent infection.&#160;NOTE: Mouse skin will form scar tissue and heal in a few days following the administration of the surgical glue. The main advantage of the glue is the avoidance of surgical stitches which may cause skin irritation or inflammation.</li>
	<li>Place the mouse in a clean cage kept on a warm plate (heated up to 37 &#176;C) and wait until the mouse recovers from the anesthetic effect.&#160; 
	CAUTION: It is critical to maintain the mouse&#39;s body temperature to enhance the chance of survival after surgery.</li>
	<li>After a one-week recovery period, the mice will be ready for further experiments, such as the Oral Glucose Tolerance Test (OGTT) and Insulin Tolerance Test (ITT).</li>
</ol>

<table><tbody><tr><td>Vetbond Tissue Adhesive&amp;nbsp;</td><td>3M&amp;nbsp;</td><td>#1049SB&amp;nbsp;</td><td>The glue used to seal the lesion site on the mouse head</td></tr><tr><td>LOCTITE 454 instant adhesive&amp;nbsp;</td><td>Durect Corporation&amp;nbsp;</td><td>#8670&amp;nbsp;</td><td>The glue used to fix the needle on the mouse skull</td></tr><tr><td>Alzet Micro- Osmotic Pump&amp;nbsp;</td><td>Durect Corporation&amp;nbsp;</td><td>#9922&amp;nbsp;</td><td>0.11 &amp;mu;l per hour, 28 days</td></tr><tr><td>Brain infusion system&amp;nbsp;</td><td>Durect Corporation&amp;nbsp;</td><td>#8851&amp;nbsp;</td><td>1-3 mm, used to perfuse the drug in to the mice brain</td></tr><tr><td>MIO NE116 CONTROL UNIT (nail drill)&amp;nbsp;</td><td>Mio System&amp;nbsp;</td><td>#E235-015&amp;nbsp;</td><td>To drill a hole in the skull of the mouse</td></tr><tr><td>aCSF formula&amp;nbsp;</td><td>119 mM NaCl&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 26.2 mM NaHCO3 2.5 mM KCl&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 1 mM NaH2PO4&amp;nbsp;&amp;nbsp; 1.3 mM MgCl2&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 10 mM glucose&amp;nbsp;</td><td></td><td>Filter sterilize with a 0.22 &amp;mu;m filter apparatus, and store at 4&amp;deg;C. aCSF is stable for 3-4 weeks</td></tr></tbody></table>

continuous drug infusion system in mouse model: a surgical procedure to implant micro-osmotic pump infusion system in the mouse brain for continuous drug delivery

Source: Ajoy, R., Chou, S. Y. <a href="https://www.jove.com/v/56410">Studying the Hypothalamic Insulin Signal to Peripheral Glucose Intolerance with a Continuous Drug Infusion System into the Mouse Brain.</a> J. Vis. Exp. (2018)
In this video, we demonstrate the assembly of a micro-osmotic pump brain infusion system followed by its surgical implantation in the mouse skull. This system enables continuous delivery of potential drugs and facilitates the study of their effects on the brain and brain tissue.

<img alt="Figure 1" src="/files/ftp_upload/20792/20792_Figure1.jpg" /> 
Figure 1. Osmotic pump preparation and implantation surgical procedure in mouse. (A) The brain infusion kit and pump preparation perfused with drug solution. Red arrows indicate the catheter tubes filled with liquid. (B) Fix and mount the mouse head onto the stereotactic apparatus. (C) Separate the outermost layer of the skin from the subcutaneous skin for the im...

Continuous Drug Infusion System in Mouse Model: A Surgical Procedure to Implant Micro-osmotic Pump Infusion System in the Mouse Brain for Continuous Drug Delivery

Source: Ajoy, R., Chou, S. Y. Studying the Hypothalamic Insulin Signal to Peripheral Glucose Intolerance with a Continuous Drug Infusion System into the ...

Drug delivery into the brain through cerebrospinal fluid, or CSF, is limited by the blood-brain barrier, or BBB, which significantly reduces the drug concentration. A micro-osmotic pump-based brain infusion system is a small implantable device that overcomes BBB, enabling continuous drug delivery into the brain.
To begin, take a micro-osmotic pump coated with an artificial CSF or aCSF, a vehicle for drug delivery. Now, fill the pump with the desired drug solution. Next, take a brain infusion assembly comprising a catheter attached to an infusion needle. Fill the assembly with the drug. Finally, connect this assembly to the micro-osmotic pump to complete the brain infusion system.
Now, prep an anesthetized mouse on a stereotactic apparatus. Remove the outer skin of the head and disinfect the exposed skull. Using a microdrill, create a hole in the skull to form a passage for the infusion system. Open the skin behind the neck region and place the micro-osmotic pump underneath. Simultaneously, insert the infusion needle into the drilled hole.
The needle penetrates the meninges, and reaches the ventricles to infuse the drug into the brain. Now, apply a suitable adhesive gel to fix the needle. Then, detach the top of the needle. Allow the mouse to recover for further experiments.

continuous-drug-infusion-system-mouse-model-surgical-procedure-to

Research

JoVE Encyclopedia of Experiments

Biology

Rodent Models

Adult

2.2K Views. Source: Ajoy, R., Chou, S. Y. Studying the Hypothalamic Insulin Signal to Peripheral Glucose Intolerance with a Continuous Drug Infusion System into the Mouse Brain. J. Vis. Exp. (2018) In this video, we demonstrate the assembly of a micro-osmotic pump brain infusion system followed by its surgical implantation in the mouse skull. This system enables continuous delivery of potential drugs and facilitates the study of their effects on the brain and brain tissue. 

Video: Continuous Drug Infusion System in Mouse Model: A Surgical Procedure to Implant Micro-osmotic Pump Infusion System in the Mouse Brain for Continuous Drug Delivery - Concept

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

JoVE Journal

Neuroscience

Implantation of Miniosmotic Pumps and Delivery of Tract Tracers to Study Brain Reorganization in Pathophysiological Conditions

Pharmacological treatment in animal models of cerebral disease imposes the problem of repeated injection protocols that may induce stress in animals and result in impermanent tissue levels of the drug. Additionally, drug delivery to the brain is delicate due to the blood brain barrier (BBB), thus significantly reducing intracerebral concentrations of selective drugs after systemic administration. Therefore, a system that allows both constant drug delivery without peak levels and circumvention of the BBB is in order to achieve sufficiently high intracerebral concentrations of drugs that are impermeable to the BBB. In this context, miniosmotic pumps represent an ideal system for constant drug delivery at a fixed known rate that eludes the problem of daily injection stress in animals and that may also be used for direct brain delivery of drugs. Here, we describe a method for miniosmotic pump implantation and post operatory care that should be given to animals in order to successfully apply this technique. We embed the aforementioned experimental paradigm in standard procedures that are used for studying neuroplasticity within the brain of C57BL6 mice. Thus, we exposed animals to 30 min brain infarct and implanted with miniosmotic pumps connected to the skull via a cannula in order to deliver a pro-plasticity drug. Behavioral testing was done during 30 days of treatment. After removal the animals received injections of anterograde tract tracers to analyze neuronal plasticity in the chronic phase of recovery. Results indicated that neuroprotection by the delivered drug was accompanied with increase in motor fibers crossing the midline of the brain at target structures. The results affirm the value of these techniques for drug administration and brain plasticity studies in modern neuroscience.

In order to study brain reorganization under pathological conditions we used miniosmotic pumps for direct protein delivery into the brain circumventing the blood brain barrier. Tract tracers are then injected to study alterations in brain connectivity under the influence of the protein.

Pharmacological treatment in animal models of cerebral disease imposes the problem of repeated injection protocols that may induce stress in animals and ...

Osmotic Pump-based Drug-delivery for In Vivo Remyelination Research on the Central Nervous System

Demyelination has been identified in not only multiple sclerosis (MS), but also other central nervous system diseases such as Alzheimer's disease and autism. As evidence suggests that remyelination can effectively ameliorate the disease symptoms, there is an increasing focus on drug development to promote the myelin regeneration process. Thus, a region-selectable and result-reliable drug delivery technique is required to test the efficiency and specificity of these drugs in vivo. This protocol introduces the osmotic pump implant as a new drug delivery approach in the lysolecithin-induced demyelination mouse model. The osmotic pump is a small implantable device that can bypass the blood-brain barrier (BBB) and deliver drugs steadily and directly to specific areas of the mouse brain. It can also effectively improve the bioavailability of drugs such as peptides and proteins with a short half-life. Therefore, this method is of great value to the field of central nervous system myelin regeneration research.

Osmotic Pump-based Drug-delivery for In Vivo Remyelination Research on the Central Nervous System

Demyelination takes place in multiple central nervous system diseases. A reliable in vivo drug delivery technique is necessary for remyelinating drug testing. This protocol describes an osmotic pump-based method that allows long-term drug delivery directly into the brain parenchyma and improves the drug bioavailability, with broad application in remyelination research.

Demyelination has been identified in not only multiple sclerosis (MS), but also other central nervous system diseases such as Alzheimer's disease and ...

Continuous Drug Infusion System in Mouse Model: A Surgical Procedure to Implant Micro-osmotic Pump Infusion System in the Mouse Brain for Continuous Drug Delivery

Transcript

Continuous Drug Infusion System in Mouse Model: A Surgical Procedure to Implant Micro-osmotic Pump Infusion System in the Mouse Brain for Continuous Drug Delivery

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Implantation of Miniosmotic Pumps and Delivery of Tract Tracers to Study Brain Reorganization in Pathophysiological Conditions

Osmotic Pump-based Drug-delivery for In Vivo Remyelination Research on the Central Nervous System