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.
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.
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.
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
2. ICV injections
3. CSF and tissue sampling
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 into the intended location. CSF collected will be blue (Figure 1), as will the ventricular system in the brain tissue (Figure 2).
The method was beneficial for a complete study of pharmacokinetics, with samples taken at different time points post-surgery.
Figure 1: Collection of CSF after injection of Evans Blue dye (1.1%) in the anesthetized rat to confirm cannula location. CSF is extracted using a pulled glass pipette (A) and then collected in a tube for snap freezing and storage (B). Please click here to view a larger version of this figure.
Figure 2: Tracing of injection materials in the brain ventricles with Evan's blue dye. Whole brains are sliced with a blade at the injection site (A,B) or at more posterior locations (C) to confirm the successful injection in the ventricular system. Scale bar (B) = 1 mm. Please click here to view a larger version of this figure.
Figure 3: Bodyweight of representative animals. Average body weight (+SD) of n = 174 representative animals (19 cohorts) before surgery (Day 0) and on the day of ICV injection (Day 1). Please click here to view a larger version of this figure.
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 established in the laboratory, the injection location can be confirmed by administering Evans Blue dye through the implanted cannula. This is especially useful and critical if a different strain or age of rats is employed, as different coordinates may be used. An alternative option that can be used in experimental animals is injecting Angiotensin II and observing the animals' drinking behavior after that; however, this method is less reliable38.
Drug-induced neurotoxicity can be a limiting factor for ICV delivery39; however, decreasing the drug delivery rate can significantly reduce adverse events40. This method is not suitable for drugs where a therapeutic dose cannot be administered in small volumes over short periods, such as <10 µL in <1 h in rats. The dose regiment can be adjusted to administer therapeutics in a single dose or over multiple days.
Imaging studies of whole brains or analyzing dissected brain sections can reveal distribution characteristics of the drug. Studying the distribution of the intraventricularly administered drug is crucial to pharmacokinetic analysis such as LC-MS. The exact flow dynamics of CSF are still under investigation and debated41. However, anatomical characteristics within the CNS and the physiochemical properties of the injected drug can impede drugs from distributing equally within the CNS and even between different CSF compartments42. Also, the condition of interest itself can influence the distribution, e.g., the blood-CSF/blood-brain barrier can become leaky in patients and rats with meningitis42,43. Thus, it is recommended that each drug and its distribution be investigated for each relevant condition. If the therapeutic under investigation is shown to distribute unilaterally, i.e., does not efficiently cross to the other hemisphere, researchers may consider implanting bilateral cannulas by modifying the described surgery method.
With this protocol, experienced surgeons can achieve very high survival rates and stable body weights in animals. This is an enhancement compared to previous protocols, one of which suggested inhalation anesthesia as a potential improvement for the wellbeing of animals over the reported median non-survival rate of 2%34. This progress can be further combined with minimally invasive analgesia self-administration through food35,36,37. This results in an optimized prerequisite for good animal welfare and understanding the behavior of a therapeutic agent. With stress being one of the main confounding variables in animal research44,45,46,47, the optimization of invasive protocols is crucial for the 'refinement' factor of the three R's48 and the ability to obtain cleaner data that feeds into the 'reduce' component.
Studying pharmacokinetics is crucial for the safe and effective delivery of any new or repurposed therapeutic. Injecting one-time or repeatedly into the ventricular brain system is an irreplaceable method in translational neuropharmacology. This protocol can aid in studying a wide variety of drugs. It can be complemented by injecting dyes for imaging or can be used to manipulate the CNS environment, e.g., by inducing specific diseases such as brain infections for investigating the therapeutic effect of antibiotics.
The authors have nothing to disclose.
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.
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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