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 brain1,2,3,4, 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 health4,6. However, it also makes it difficult to deliver drugs to treat infections in the brain or other CNS diseases4,7. Apart from disrupting the BBB using a variety of methods8,9, 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 ventricle10,11. Similar techniques have been established in laboratory animals such as rodents to achieve equivalent goals. Micro-osmotic pumps were implanted in mice12,13,14,15 and rats16,17 for continuous drug delivery into the ventricular system or brain parenchyma. Additionally, direct intracerebroventricular injections were conducted in anesthetized mice using a disposable needle18,19 and conscious rats via a surgically implanted cannula20,21,22,23. Drug delivery to the CNS has been an invaluable method to enhance understanding in various fields20,24,25,26,27,28.
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 need31,32,33. 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 pump12,13,14,15,16,17, 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
Representative Results
Discussion
Researchers and clinicians employ ICV injections to circumvent the protective mechanism of the BBB and deliver drugs directly into the CNS12,18,19,21,24. 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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors thank the Biomedical Science Animal Facility at the University of Melbourne for the provision and care of animals. This research was supported by a research grant from the National Institute of Allergy and Infectious Diseases of the National Institute of Health (R01 AI146241, GR, and TV). JL is an Australian National Health Medical Research Council (NHMRC) Principal Research Fellow. The content is solely the authors' responsibility and does not necessarily represent the official views of the National Institute of Allergy and Infectious Diseases or the National Institute of Health.
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 |
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