Name: delivery of antibodies into the murine brain via convection-enhanced delivery
Uploaded: 2019-07-18T13:00:00
Description: Watch this Scientific Journal Video about Delivery of Antibodies into the Murine Brain via Convection-enhanced Delivery at JoVE.com

This work was supported by grants of the University of Zurich (FK-15-057), the Novartis Foundation for medical-biological Research (16C231) and Swiss Cancer Research (KFS-3852-02-2016, KFS-4146-02-2017) to Johannes vom Berg and BRIDGE Proof of Concept (20B1-1_177300) to Linda Schellhammer.

All methods described here have been approved by the Swiss Cantonal Veterinary Office under license number ZH246/15.
1. Preparation of the Step Catheters
<ol>
	<li>Preparation of a fused silica tubing for the step of the catheter
	<ol>
		<li>Cut the fused silica capillary with inner diameter of 0.1 mm and wall thickness of 0.0325 mm tubing to a length of 30 mm.</li>
		<li>Examine the tubing for cracks and heat polish the ends using a microforge to ensure the tubing openings ...

<ol>
	<li>Scherrmann, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+delivery+via+the+blood-brain+barrier.">Drug delivery via the blood-brain barrier.</a> Vascular Pharmacology. 38 (6), 349-354 (2002).</li><li>Barua, N. U., Gill, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Convection-enhanced+drug+delivery:+prospects+for+glioblastoma+treatment.">Convection-enhanced drug delivery: prospects for glioblastoma treatment.</a> CNS Oncology. 3 (5), 313-316 (2014).</li><li>Bobo, R. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Convection-enhanced+delivery+of+macromolecules+in+the+brain.">Convection-enhanced delivery of macromolecules in the brain.</a> Proceedings of the National Academy of Sciences of the United States of America. 91 (6), 2076-2080 (1994).</li><li>Morrison, P. F., Laske, D. W., Bobo, H., Oldfield, E. H., Dedrick, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-flow+microinfusion:+tissue+penetration+and+pharmacodynamics.">High-flow microinfusion: tissue penetration and pharmacodynamics.</a> American Journal of Physiology. 266 (1 Pt 2), R292-R305 (1994).</li><li>Zhou, Z., Singh, R., Souweidane, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Convection-Enhanced+Delivery+for+diffuse+intrinsic+pontine+glioma+treatment.">Convection-Enhanced Delivery for diffuse intrinsic pontine glioma treatment.</a> Current Neuropharmacology. 15 (1), 116-128 (2017).</li><li>Barua, N. U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrastriatal+convection-enhanced+delivery+results+in+widespread+perivascular+distribution+in+a+pre-clinical+model.">Intrastriatal convection-enhanced delivery results in widespread perivascular distribution in a pre-clinical model.</a> Fluids and Barriers of the CNS. 9 (1), 2 (2012).</li><li>Shoji, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+convection-enhanced+delivery+of+an+anti-CD40+agonistic+monoclonal+antibody+induces+antitumor+effects+in+mouse+glioma+models.">Local convection-enhanced delivery of an anti-CD40 agonistic monoclonal antibody induces antitumor effects in mouse glioma models.</a> Neuro-Oncology. 18 (8), 1120-1128 (2016).</li><li>Souweidane, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Convection-enhanced+delivery+for+diffuse+intrinsic+pontine+glioma:+a+single-centre,+dose-escalation,+phase+1+trial.">Convection-enhanced delivery for diffuse intrinsic pontine glioma: a single-centre, dose-escalation, phase 1 trial.</a> The Lancet Oncology. , (2018).</li><li>Zhang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+immune+checkpoints+in+malignant+glioma.">Targeting immune checkpoints in malignant glioma.</a> Oncotarget. 8 (4), 7157-7174 (2017).</li><li>Barua, N. U., Gill, S. S., Love, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Convection-enhanced+drug+delivery+to+the+brain:+therapeutic+potential+and+neuropathological+considerations.">Convection-enhanced drug delivery to the brain: therapeutic potential and neuropathological considerations.</a> Brain Pathology. 24 (2), 117-127 (2014).</li><li>Mehta, A. M., Sonabend, A. M., Bruce, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Convection-Enhanced+Delivery.">Convection-Enhanced Delivery.</a> Neurotherapeutics. 14 (2), 358-371 (2017).</li><li>Krauze, M. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reflux-free+cannula+for+convection-enhanced+high-speed+delivery+of+therapeutic+agents.">Reflux-free cannula for convection-enhanced high-speed delivery of therapeutic agents.</a> Journal of Neurosurgery. 103 (5), 923-929 (2005).</li><li>Nash, K. R., Gordon, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Convection+Enhanced+Delivery+of+Recombinant+Adeno-associated+Virus+into+the+Mouse+Brain.">Convection Enhanced Delivery of Recombinant Adeno-associated Virus into the Mouse Brain.</a> Methods in Molecular Biology. 1382, 285-295 (2016).</li><li>Ohlfest, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combinatorial+antiangiogenic+gene+therapy+by+nonviral+gene+transfer+using+the+sleeping+beauty+transposon+causes+tumor+regression+and+improves+survival+in+mice+bearing+intracranial+human+glioblastoma.">Combinatorial antiangiogenic gene therapy by nonviral gene transfer using the sleeping beauty transposon causes tumor regression and improves survival in mice bearing intracranial human glioblastoma.</a> Molecular Therapy. 12 (5), 778-788 (2005).</li><li>Yin, D., Forsayeth, J., Bankiewicz, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+cannula+design+and+placement+for+convection-enhanced+delivery+in+rat+striatum.">Optimized cannula design and placement for convection-enhanced delivery in rat striatum.</a> Journal of Neuroscience Methods. 187 (1), 46-51 (2010).</li><li>Mamot, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extensive+distribution+of+liposomes+in+rodent+brains+and+brain+tumors+following+convection-enhanced+delivery.">Extensive distribution of liposomes in rodent brains and brain tumors following convection-enhanced delivery.</a> Journal of Neuro-Oncology. 68 (1), 1-9 (2004).</li><li>Saito, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+affinity+of+the+infusate+affects+the+distribution+volume+during+convection-enhanced+delivery+into+rodent+brains:+implications+for+local+drug+delivery.">Tissue affinity of the infusate affects the distribution volume during convection-enhanced delivery into rodent brains: implications for local drug delivery.</a> Journal of Neuroscience Methods. 154 (1-2), 225-232 (2006).</li><li>Oh, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+distribution+of+small+molecules+and+viral+vectors+in+the+murine+brain+using+a+hollow+fiber+catheter.">Improved distribution of small molecules and viral vectors in the murine brain using a hollow fiber catheter.</a> Journal of Neurosurgery. 107 (3), 568-577 (2007).</li><li>Barua, N. U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+implantable+catheter+system+with+transcutaneous+port+for+intermittent+convection-enhanced+delivery+of+carboplatin+for+recurrent+glioblastoma.">A novel implantable catheter system with transcutaneous port for intermittent convection-enhanced delivery of carboplatin for recurrent glioblastoma.</a> Drug Delivery. 23 (1), 167-173 (2016).</li><li>Rosenbluth, K. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+of+an+in-dwelling+cannula+for+convection-enhanced+delivery.">Design of an in-dwelling cannula for convection-enhanced delivery.</a> Journal of Neuroscience Methods. 196 (1), 118-123 (2011).</li><li>Debinski, W., Tatter, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Convection-enhanced+delivery+for+the+treatment+of+brain+tumors.">Convection-enhanced delivery for the treatment of brain tumors.</a> Expert Review of Neurotherapeutics. 9 (10), 1519-1527 (2009).</li><li>MacKay, J. A., Deen, D. F., Szoka, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+in+brain+of+liposomes+after+convection+enhanced+delivery;+modulation+by+particle+charge,+particle+diameter,+and+presence+of+steric+coating.">Distribution in brain of liposomes after convection enhanced delivery; modulation by particle charge, particle diameter, and presence of steric coating.</a> Brain Research. 1035 (2), 139-153 (2005).</li><li>Chen, Z. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+realistic+brain+tissue+phantom+for+intraparenchymal+infusion+studies.">A realistic brain tissue phantom for intraparenchymal infusion studies.</a> Journal of Neurosurgery. 101 (2), 314-322 (2004).</li><li>Sampson, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poor+drug+distribution+as+a+possible+explanation+for+the+results+of+the+PRECISE+trial.">Poor drug distribution as a possible explanation for the results of the PRECISE trial.</a> Journal of Neurosurgery. 113 (2), 301-309 (2010).</li><li>Wick, W., Weller, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trabedersen+to+target+transforming+growth+factor-beta:+when+the+journey+is+not+the+reward,+in+reference+to+Bogdahn+et+al.+(Neuro-Oncology+2011;13:132-142).">Trabedersen to target transforming growth factor-beta: when the journey is not the reward, in reference to Bogdahn et al. (Neuro-Oncology 2011;13:132-142).</a> Neuro-Oncology. 13 (5), 559-560 (2011).</li><li>Saito, R., Tominaga, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Convection-enhanced+delivery+of+therapeutics+for+malignant+gliomas.">Convection-enhanced delivery of therapeutics for malignant gliomas.</a> Neurologia Medico-Chirurgica. 57 (1), 8-16 (2017).</li><li>Bedussi, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clearance+from+the+mouse+brain+by+convection+of+interstitial+fluid+towards+the+ventricular+system.">Clearance from the mouse brain by convection of interstitial fluid towards the ventricular system.</a> Fluids Barriers CNS. 12, 23 (2015).</li><li>Noroxe, D. S., Poulsen, H. S., Lassen, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hallmarks+of+glioblastoma:+a+systematic+review.">Hallmarks of glioblastoma: a systematic review.</a> ESMO Open. 1 (6), e000144 (2016).</li><li>Boucher, Y., Salehi, H., Witwer, B., Harsh, G. R. t., Jain, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interstitial+fluid+pressure+in+intracranial+tumours+in+patients+and+in+rodents.">Interstitial fluid pressure in intracranial tumours in patients and in rodents.</a> British Journal of Cancer. 75 (6), 829-836 (1997).</li><li>Glushakova, O. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospective+clinical+biomarkers+of+caspase-mediated+apoptosis+associated+with+neuronal+and+neurovascular+damage+following+stroke+and+other+severe+brain+injuries:+Implications+for+chronic+neurodegeneration.">Prospective clinical biomarkers of caspase-mediated apoptosis associated with neuronal and neurovascular damage following stroke and other severe brain injuries: Implications for chronic neurodegeneration.</a> Brain Circulation. 3 (2), 87-108 (2017).</li><li>Vom Berg, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+IL-12/IL-23+signaling+reduces+Alzheimer's+disease-like+pathology+and+cognitive+decline.">Inhibition of IL-12/IL-23 signaling reduces Alzheimer's disease-like pathology and cognitive decline.</a> Nature Medicine. 18 (12), 1812-1819 (2012).</li><li>Vom Berg, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intratumoral+IL-12+combined+with+CTLA-4+blockade+elicits+T+cell-mediated+glioma+rejection.">Intratumoral IL-12 combined with CTLA-4 blockade elicits T cell-mediated glioma rejection.</a> Journal of Experimental Medicine. 210 (13), 2803-2811 (2013).</li><li>Kurdi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+administration+of+the+mTORC1+inhibitor+everolimus+induces+tolerance+and+decreases+autophagy+in+mice.">Continuous administration of the mTORC1 inhibitor everolimus induces tolerance and decreases autophagy in mice.</a> British Journal of Pharmacology. 173 (23), 3359-3371 (2016).</li></ol>

Convection-enhanced delivery, or pressure-mediated drug infusion into the brain, was first proposed in the early 19903. This approach promises perfusion of large brain volumes behind the blood brain barrier in a controlled manner2. However, so far, only a few clinical trials have been performed using this approach, partially because CED in a clinical setup has shown to be technically demanding24,25. Recent developme...

The blood brain barrier (BBB) forms a semipermeable border separating the central nervous system (CNS) from the blood circulation. Reaching the CNS with therapeutics is however necessary in context of various diseases, like brain tumors, Alzheimer&#8217;s disease (AD) or Parkinson&#8217;s disease (PD) among others1. This becomes important in the development of new therapies, especially if the tested drug exhibits poor BBB permeability or its systemic exposure can lead to dangerous toxicity1,2. Some of the clinically used antibodies display both of these features. A solution to this problem would be to deliver the therapeutics directly behind the BBB.
Convection-enhanced delivery (CED) is a neurosurgical technique enabling effective perfusion of large brain volumes. This is achieved by surgically installing one or more catheters in the target area. During the drug application, a pressure gradient is formed at the opening of the catheter, which becomes the driving force of the infusate dispersion in the tissue3,4. It is thus the duration of infusion and not the diffusion coefficients that determine the perfusion range2,4,5. This provides uniform delivery of the infusate over a much larger brain volume compared to conventional, diffusion based intracerebral injection methods2,6. At the same time, this delivery modality has a lower risk of tissue damage2. Accordingly, CED can enable safe and efficacious administration of conventional chemotherapeutics for treatment of CNS tumors, as well as delivery of immunomodulatory agents or agonistic and antagonistic antibodies in a multitude of other CNS disorders2,7,8,9. CED is currently tested in therapies of Parkinson&#8217;s disease, Alzheimer&#8217;s disease, as well as high-grade glioma2,7,8,10,11.
Catheter design and the injection regimen are among the most important factors influencing the outcome of CED 10,12,13,14,15,16. Furthermore, it requires specific physicochemical properties of the infusate, including moderate size of the particles, an anionic charge, and low tissue affinity 10,17. Each of these parameters has to be potentially adjusted according to the histological features of the brain region to be targeted2,10,17.
Here we describe methodology for performing CED of an antibody solution into the caudate putamen (striatum) of mice. Furthermore, the protocol includes preparation of step catheters in a laboratory setup, testing them in vitro and performing the CED.
There are multiple catheter designs available in the literature, differing by the shape of the cannula, the materials used and the number of catheter openings12,15,18,19,20,21,22. We are using a step catheter made of a fused silica capillary protruding 1 mm from a blunt end metal needle. This catheter design can be easily manufactured in a research laboratory and reproducibly gives good CED results when tested in vitro with agarose blocks with physical parameters resembling brain parenchyma in vivo23.
Moreover, we implement a ramping regimen for delivering 5 &#181;L of infusate in vivo. In such a protocol the injection rate is increased from 0.2 &#181;L/min to a maximum of 0.8 &#181;L/min, thus minimizing chances of infusate reflux along the catheter as well as risk of tissue damage16. Using this protocol, we have successfully administered mice with up to 20 &#181;g of antibody in 5 &#181;L of PBS over the course of 11 min 30 s.
The protocol can be readily adjusted for other infusion volumes or for injecting various other substances, e.g. chemotherapeutics, cytokines, viral particles or liposomes2,10,14,18,22. In case of using infusate with drastically different physicochemical properties compared to a phosphate buffered saline (PBS) or artificial cerebrospinal fluid (aCSF) solution of antibodies, additional validation steps are recommended. For catheter assembly, validation and CED, we describe all steps using a stereotactic robot with a drill and injection unit mounted onto a regular stereotactic frame. This procedure can also be performed with a manual stereotactic frame connected to programmable microinfusion pump that can drive the described glass microsyringes.

<table>
	<tbody>
		<tr>
			<td>10 &#956;L syringe</td>
			<td>Hamilton</td>
			<td>7635-01</td>
			<td></td>
		</tr>
		<tr>
			<td>27 G blunt end needle</td>
			<td>Hamilton</td>
			<td>7762-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Promega</td>
			<td>V3121</td>
			<td></td>
		</tr>
		<tr>
			<td>Atipamezol</td>
			<td>Janssen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bone wax</td>
			<td>Braun</td>
			<td>1029754</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>Indivior Schweiz AG</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Carprofen</td>
			<td>Pfizer AG</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dental drill bits, steel, size ISO 009</td>
			<td>Hager &#38; Meisinger</td>
			<td>1RF009</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 100%</td>
			<td>Reuss-Chemie AG</td>
			<td>179-VL03K-/1</td>
			<td></td>
		</tr>
		<tr>
			<td>Fentanyl</td>
			<td>Helvepharm AG</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FITC-Dextran, 2000 kDa</td>
			<td>Sigma Aldrich</td>
			<td>FD2000S</td>
			<td></td>
		</tr>
		<tr>
			<td>Flumazenil</td>
			<td>Labatec Pharma AG</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Sigma Aldrich</td>
			<td>F8775-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>High viscosity cyanoacrylate glue</td>
			<td>Migros</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Iodine solution</td>
			<td>Mundipharma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Medetomidin</td>
			<td>Orion Pharma AG</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microforge</td>
			<td>Narishige</td>
			<td>MF-900</td>
			<td></td>
		</tr>
		<tr>
			<td>Midazolam</td>
			<td>Roche Pharma AG</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ophthalmic ointment</td>
			<td>Bausch + Lomb</td>
			<td>Vitamin A Blache</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>ThermoFischer Scientific</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyclonal goat anti-rat IgG (H+L) antibody coupled with Alexa Fluor 647</td>
			<td>Jackson Immuno</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpels</td>
			<td>Braun</td>
			<td>BB518</td>
			<td></td>
		</tr>
		<tr>
			<td>Silica tubing internal diameter 0.1 mm, wall thickness of 0.0325 mm</td>
			<td>Postnova</td>
			<td>Z-FSS-100165</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotactic frame for mice</td>
			<td>Stoelting</td>
			<td>51615</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotactic robot</td>
			<td>Neurostar</td>
			<td>Drill and Injection Robot</td>
			<td></td>
		</tr>
		<tr>
			<td>Succrose</td>
			<td>Sigma Aldrich</td>
			<td>S0389-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Topical tissue adhesive</td>
			<td>Zoetis</td>
			<td>GLUture</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>ThermoFischer Scientific</td>
			<td>15250061</td>
			<td></td>
		</tr>
		<tr>
			<td>Water</td>
			<td>Bichsel</td>
			<td>1000004</td>
			<td></td>
		</tr>
	</tbody>
</table>

delivery of antibodies into the murine brain via convection-enhanced delivery

Convection-enhanced delivery (CED) is a neurosurgical technique enabling effective perfusion of large brain volumes using a catheter system. Such an approach provides a safe delivery method by-passing the blood brain barrier (BBB), thus allowing treatment with therapeutics with poor BBB-permeability or those for which systemic exposure is not desired, e.g., due to toxicity. CED requires optimization of the catheter design, injection protocol, and properties of the infusate. With this protocol we describe how to perform CED of a solution containing up to 20 &#181;g of an antibody into the caudate putamen of mice. It describes preparation of step catheters, testing them in vitro and performing the CED in mice using a ramping injection program. The protocol can be readily adjusted for other infusion volumes and can be used for injecting various tracers or pharmacologically active or inactive substances, including chemotherapeutics, cytokines, viral particles, and liposomes.

This protocol enables preparation of step catheters (Figure 1) for use in the CED procedure in a laboratory environment. In order to control the catheters for leakage, reflux along the needle tract and clogging, we recommend performing injections of a dye, e.g., trypan blue solution, into an agarose block. Figure 3 depicts a cloud of trypan blue forming after injection of 1 &#181;L at 0.5 &#181;L/minute using a CED catheter (Figure 3A</stron...

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Delivery of Antibodies into the Murine Brain via Convection-enhanced Delivery

Convection-enhanced delivery (CED) is a method enabling effective delivery of therapeutics into the brain by direct perfusion of large tissue volumes. The procedure requires the use of catheters and an optimized injection procedure. This protocol describes a methodology for CED of an antibody into a mouse brain.

Convection-enhanced delivery (CED) is a neurosurgical technique enabling effective perfusion of large brain volumes using a catheter system. Such an ...

This protocol can be used to perform convection-enhanced delivery in small rodents using a step catheter system for the adjustable uniform perfusion of target brain regions. The advantage of convection-enhanced delivery is that it allows delivery of substances into the region of interest bypassing the blood-brain barrier with little tissue damage or reflux. This technique is primarily useful for delivery of therapeutic substances such as antibodies into the brain.And can therefore be used to target many neurological conditions. Demonstrating the procedure will be Michal Beffinger, a post-doc from my laboratory. Begin by cutting a piece of fused silica capillary tubing with an inner diameter of 0.1 millimeter and a wall thickness of 0.325 millimeter to a length of 30 millimeters.After examining the tubing for cracks use a microforge to heat polish the ends to ensure the tubing openings have a smooth surface. Next, mount a 27-gauge needle onto a 10 micro liter syringe and place the syringe in a stereotactic robot. Use the robot to move the syringe over a hard surface and touch the surface with the needle tip.After noting this position elevate the needle to enable placement of the fused silica capillary inside of the needle such that 20 millimeters of the capillary protrudes from the needle. Use a pipette to evenly spread two microliters of high viscosity cyanoacrylate adhesive over the capillary starting from the metal needle and finishing ten millimeters above the lower end of the capillary. Use the stereotactic robot to lower the metal needle until the tip of the needle is one millimeter over the reference surface to fix the fused silica capillary in the metal needle forming a one millimeter step from the tip of the metal needle.Remove any excess glue forming at the end of the metal needle to avoid planting the catheter step. Check the tip under a microscope to confirm that all of the excess glue has been removed. Then wait 15 minutes for the glue to harden and remove the syringe with the catheter from the robot.For step catheter testing cut solidified 0.6%agarose gel into 20 by 20 millimeter blocks and manually fill the step catheter syringe with 10 microliters of filtered 0.4%trypan blue solution. Using the stereotactic robot, dispense one microliter of dye at 0.2 micro liters per minute to assess the sealing of the step of the catheter during the fixation procedure. The trypan blue solution should be visible solely on the tip of the catheter.Wipe away the dye with a paper tissue and place an agarose block in the stereotactic robot. Calibrate the robot said that the tip of the catheter is referenced against the surface of the agarose block. Set a proportion sequence of injection volumes according to the specific experimental plan.To inject the solution into the murine caudate putamen, set the injection to a one millimeter frontal and one and a half to two millimeter lateral from bregma position at a depth of 3.5 millimeters. When the needle is in position start the convection enhanced delivery procedure and inject five microliters of trypan blue solution into the agarose block. Assess the shape of the trypan blue cloud in the agarose and for potential leakage along the catheter tract.No major backflow over the tip of the metal needle should be visible. After the injection leave the catheter in place for two minutes before retracting the needle at one millimetre per minute to ensure a proper dispersion of the fluid into the brain and sealing of the injection tract during the removal. Place a new agarose block into the robot and start a second injection of one microliter at 0.2 micro liters per minute to assess clogging of the catheter within the agarose.The trypan blue should again form a cloud from the tip of the catheter immediately after the start of the injection. Then assess whether the leftover volume in the syringe corresponds to 3 microliters as any variations might indicate a leakage of fluid through the catheter mounting or syringe plunger. For antibody injection into the murine striatum confirm a lack of response to skin pinch in an anesthetized Mouse and shave the head with a hair trimmer.Disinfect the skin with cotton swabs soaked in iodine solution. Use a scalpel to make a 10 millimeter skin incision along the cranial midline finishing at the eye level. Fix the mouse in the stereotactic frame using the nose clamp and ear bars, taking care that the skull surface is horizontal and tightly secured.Place the syringe in the stereotactic robot and synchronize the drill bit with the tip of the catheter on a reference point. Use forceps to retract the skin and localize bregma on the skull surface. Reference bregma in the software using the tip of the drill bit and move the drill to a one millimeter frontal and two millimeter lateral from bregma position.Drill a burr hole taking care not to damage the dura mater and move the syringe over the burr hole. Dispense 0.5 to one microliter from the syringe to ensure that no air bubbles are left in the catheter. Start the convection enhanced delivery as demonstrated, observing the skull surface for any traces of fluid backflow from the injection spot.At the end of the delivery and catheter removal start the injection pump at 0.2 micro liters per minute to check for evidence of catheter clogging during the injection. If no clogging occurred a droplet of injection mix should immediately be observed coming from the catheter tip. In this image of a cloud of trypan blue forming after the injection of one microliter of dye at 0.5 micro liters per minute using a convection enhanced catheter as demonstrated, no reflux along the needle tract was visible over the beginning of the catheter step.And the dispersed cloud formed a desired spherical shape. In this image using a blunt end needle however, significant reflux could be observed. Notably, convection enhanced delivery enables the perfusion of large volumes into the murine brain in a uniform and less tissue damaging manner compared to conventional injection.In both types of delivery there is a typical distribution profile of antibody and dextran particles over the corpus callosum. However, the dispersion profile of the injected antibody is more diffused than is observed for the high molecular weight dextran after convection enhanced delivery, exemplifying differences in distribution between different infusates. As the catheter can potentially clog during the brain infusion, it is important to check it immediately after by slowly dispensing a small volume of infusate.This procedure can serve as a way to deliver pharmacologically active compounds to the brain and should be followed by close monitoring of disease symptoms and expected side effects. This technique enables the infusion of a precise brain region with therapeutics including antibodies, opening new possibilities for the development of central nervous system targeted therapies.

Research

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Neuroscience

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