Intraventricular Drug Delivery and Sampling for Pharmacokinetics and Pharmacodynamics Study

Summary

Delivery of therapeutics directly into the central nervous system is one way of circumventing the blood-brain barrier. The present protocol demonstrates intracerebroventricular injection for subsequent collection of cerebrospinal fluid and bodily organs. This facilitates the investigation of drug pharmacokinetics and pharmacodynamics in animal models for developing new treatments.

Abstract

Although the blood-brain barrier (BBB) protects the brain from foreign entities, it also prevents some therapeutics from crossing into the central nervous system (CNS) to ameliorate diseases or infections. Drugs are administered directly into the CNS in animals and humans to circumvent the BBB. The present protocol describes a unique way of treating brain infections through intraventricular delivery of antibiotics, i.e., polymyxins, the last-line antibiotics to treat multi-drug resistant Gram-negative bacteria. A straightforward stereotaxic surgery protocol was developed to implant a guide cannula reaching into the lateral ventricle in rats. After a recovery period of 24 h, rats can be injected consciously and repeatedly through a cannula that is fitted to the guide. Injections can be delivered manually as a bolus or infusion using a microinjection pump to obtain a slow and controlled flow rate. The intraventricular injection was successfully confirmed with Evans Blue dye. Cerebrospinal fluid (CSF) can be drained, and the brain and other organs can be collected. This approach is highly amenable for studies involving drug delivery to the CNS and subsequent assessment of pharmacokinetic and pharmacodynamic activity.

Introduction

The blood-brain barrier (BBB) is a crucial protective mechanism for the central nervous system (CNS). The selectively-permeable, anatomic barrier separates the circulating blood and its solutes from the brain's extracellular fluid, thus preventing most molecules from entering the brain^1,2,3,4, depending on their size, lipophilicity⁵, and the availability of an active transport mechanism².

This protective barrier is beneficial for the effective regulation of intricate brain homeostasis and CNS health^4,6. However, it also makes it difficult to deliver drugs to treat infections in the brain or other CNS diseases^4,7. Apart from disrupting the BBB using a variety of methods^8,9, the primary approach to circumvent the BBB is to deliver a drug directly into the brain by releasing it into the cerebrospinal fluid (CSF)⁴. Even though it is a relatively invasive practice, it has been used successfully to deliver targeted therapeutics to patients and laboratory animals. In humans, drugs can be delivered into the intraventricular system or CSF and subsequently sampled using the Ommaya reservoir, a reservoir residing under the scalp, attached to a catheter inserted into the lateral ventricle^10,11. Similar techniques have been established in laboratory animals such as rodents to achieve equivalent goals. Micro-osmotic pumps were implanted in mice^12,13,14,15 and rats^16,17 for continuous drug delivery into the ventricular system or brain parenchyma. Additionally, direct intracerebroventricular injections were conducted in anesthetized mice using a disposable needle^18,19 and conscious rats via a surgically implanted cannula^20,21,22,23. Drug delivery to the CNS has been an invaluable method to enhance understanding in various fields^{20,24,25,26,27,28}.

CNS infections are one such field that urgently needs new therapeutics and an enhanced understanding of existing anti-infective therapies. CNS Infections caused by multi-drug resistant Gram-negative bacteria are particularly concerning⁷. Polymyxins are the last-line antibiotics increasingly used to treat infections due to these 'superbugs'²⁹. When polymyxins are administered intravenously as per the current dosing guidelines³⁰, their penetration into the CNS is very low, while higher doses increase the risk of nephrotoxicity. Therefore, intravenous polymyxin therapy is of little use to treat CNS infections⁷. Establishing a safe and effective dosage regimen for polymyxins delivery to the CNS is an urgent unmet medical need^31,32,33. Therefore, the present protocol was established and is described with a focus on injecting antibiotics directly into the CSF of rats. It can, however, be used to administer any drug that is not neurotoxic and where therapeutic concentrations can be administered in small volumes (e.g., up to 10 µL in rats). The techniques described can also be modified to target different brain regions and deliver multiple injections.

The present protocol presents a straightforward surgery and injection technique that allows for efficient pharmacokinetics and distribution post-ICV administration of drugs. The surgery involves implanting a guide cannula. As it is a less invasive procedure than the implantation of a micro-osmotic pump^{12,13,14,15,16,17}, this is an advanced option suitable for the short-term administration of drugs into CSF. This protocol is simplified and can produce very high survival rates and stable body weights 24 h post-surgery, which is an improvement compared to existing methods³⁴. After surgery, conscious rats received either a manual bolus ICV injection or slower delivery using a micropump to lower the peak plasma concentrations. At the same time, they could freely move in their cage. To establish safe and effective drug dosage regimens, samples of CSF, brain, spinal cord, kidney, plasma, etc., were then used to study pharmacokinetics and drug distribution following intracerebroventricular (ICV) administration. Drug distribution can also be investigated visually, e.g., using immunohistochemistry or matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). If necessary, a bilateral cannula can be implanted, e.g., to inject drugs that would otherwise distribute unilaterally into both hemispheres.

Protocol

All experiments were conducted following the Australian code for the care and use of animals for scientific purposes. Experiments were approved by the University of Melbourne ethics committee (application #1914890). 8-14 weeks old male and female Sprague-Dawley rats were used for the experiments. 1. Stereotaxic surgery for lateral ventricle cannulation Use autoclaved cotton tips and surgical drapes during the surgery. Set up the following for the…

Representative Results

The surgical protocol presented is highly successful, with trained surgeons reaching >99.8% survival rate and animals showing stable body weight post-surgery on Day 1, compared to their pre-surgery weight on Day 0 (mean ± SD of 315.8 g ± 42.1 g for Day 0 and 314.1 g ± 43.0 g for Day 1, Figure 3). Before collecting CSF, an injection of 1.1% Evans Blue dye into the implanted cannula can aid as confirmation of the injection having been delivered in…

Discussion

Researchers and clinicians employ ICV injections to circumvent the protective mechanism of the BBB and deliver drugs directly into the CNS^{12,18,19,21,24}. The present work is a complete ICV protocol for delivering drugs efficiently into the CNS and extracting CSF for pharmacokinetic analysis. At the start of the experimentation, when this protocol is being esta…

Materials

Acetone	Terumo, Japan	SS+01T
5 mL syringes	Terumo, Japan	SS+05S
Acetone	Merck, Germany	67641
Bench protector sheets	Halyard, USA	2765-C
Betadine	Mundipharma, Netherlands	1015695
Buprenorphine; Temgesic	Clifford Hallam Healthcare, Australia	1238366
Carprofen	Zoetis, Australia	10001132
Chlorhexidine	Tasman Chemicals, Australia	890401
Chux superwipes (or equivalent)	Chux, Australia	n/a	autoclaved
Clippers	n/a	n/a
Cotton swabs	LP Italiana, Italy	112191	autoclaved
Dental cement powder (Vertex Self cure powder)	Henry Schein, USA	VX-SC500GVD5
Dental cement solvent (Vertex Self cure liquid)	Henry Schein, USA	VX-SC250MLLQ
Disposable needles: 18 G, 26 G, 30 G	Terumo, Japan	NN+2525RL
Disposable surgical blades	Westlab, Australia	663-255
Dissector scissors	F.S.T.	14082-09
Dummy cannulas	Bio Scientific, Australia	C313DC/SPC	cut to 4.05 to fit the guide cannula
Ethanol 80%	Merck, Australia	10107
Evan's blue dye	Sigma	E2129 – 50G
Eye lube	Clifford Hallam Healthcare, Australia	2070491
Felt tip pen	Sharpie, USA	D-4236
Fibre optic light source	n/a	n/a
Flattened needle (18 G) or similar to apply superglue	n/a	n/a
Glass pipettes, pulled	Hirschmann Laborgeraete, Germany	9100175
Glass syringe 10 uL	Hamilton, USA	701 LT and 1701 LT
Guide cannulas	Bio Scientific, Australia	C313G/SPC	22 G, cut 4 mm below the pedestal for lateral ventricle cannulation in adult Sprague Dawley rats
Haemostat
Heat bead steriliser	Inotech, Switzerland	IS-250
Heat pad	n/a	n/a
Hydrogen peroxide 3%	Perrigo, Australia	11383
Induction chamber (Perspex 300 mm x 200 mm)	n/a	n/a
Injector cannula	Bio Scientific, Australia	C313I/SPC	cut to fit the 4 mm cannula + 0.5 mm projection
Isoflurane	Clifford Hallam Healthcare, Australia	2093803
Isoflurane vaporiser and appropriate scavenging system	n/a	n/a
Medium size weighing boats	n/a	n/a
Metal spatula	Met-App, Australia	n/a
Micro syringe pump	New Era, USA	NE-300
Microdrill	RWD Life Science Co, China	87001
Polymyxin B	Beta Pharma, China	86-40302
Protein LoBind tubes, 0.5 mL	Eppendorf, Germany	Z666491
Ropivacaine 1%; Naropin	AstraZeneca, UK	PS09634
Scissors, large	F.S.T.	14511-15
Scissors, small	F.S.T.	14079-10
Screwdriver	n/a	n/a
Screws	Mr. Specs, Australia	n/a
Stereotaxic frame	RWD Life Science Co, China	n/a	Necessary components: rat ear bars, tooth bar, anaesthesia nose cone, arm with digital readout (X, Y, Z) and cannula holder
Sterile saline 0.9%	Baxter, USA	AHB1323
Super etch (37% phosphoric acid) gel	SDI Limited, Australia	8100045
Superglue	UHU, Germany	n/a
Tissue forceps with hooks	F.S.T.	11027-12
Tubing, PE-50	Bio Scientific, Australia	C313CT