Acknowledgements
We thank SAI Infusions Technologies and Dr. V. Karicheti (Associate Director, Surgical Model Solutions Scientist, Charles River Canada) for their input and advice for implantation of the CAB and cannula. Funding for this study was provided by the National Hockey League Player’s Association to MJS. The study is reported in accordance with ARRIVE guidelines (https://arriveguidelines.org)
Data set availability: All datasets generated or analyzed during this study are included in this published article.
Contributions: Inception and study design, data analyses and manuscript preparation: KV; AUP preparation: KV, ES, ET; animal care and surgery: ES, NSD, DG, ET, LM; extracellular vesicle isolations and western blots: FA; proofreading and editing of manuscript: all authors; funding: MJS.
Online Methods
Animal husbandry: Sprague-Dawley rats (RRID:RGD_737891) were group housed with enrichment in open-air conventional cages and fed LabDiet ProLab IsoPro RMH3000 and powdered Teklad 2018 ad libitum with free access to water. All animals were handled in accordance to CCAC regulations and all methods were performed in accordance with relevant guidelines and regulations. The study was conducted under an Animal Care Committee approved AUP (#2022-196) at the University of Western Ontario (London, Ontario, Canada). Animals were group housed on a 12h light/dark cycle.
Pre-surgical preparation: Rats were anesthetized with 4% isoflurane (Fresenius Kabi Canada Ltd) induction and maintained on 1.5% isoflurane. Prophylactic saline (10ml/kg/hr; Pfizer) and 1.0mg/kg buprenorphine S/R (ACVS, University of Western Ontario) were given subcutaneously. Bupivicain (Aspen Pharmacare Canada Inc, Sensorcaine) was injected subcutaneously between the ears at the cranial surgical site and into the ventral neck surgical site. The surgical sites (base of the skull, between the shoulder blades and the ventral neck) was prepared by clipping and scrubbing with chlorhexidine (Partnar 2% chlorhexidine solution) and surgical scrub (EcoLab Bacti-Stat Antibacterial Hand Soap). The animal was placed on a circulating water heating pad to maintain body temperature and monitored with PhysioSuite (Kent Scientific PS-03). The plane of anesthesia was monitored and adjusted as required.
Jugular vein catheterization: A 5cc syringe was placed under the back of the neck for support while the rat was in dorsal recumbency. The external jugular vein was exposed through a 1cm incision made approximately 1cm lateral to the midline. 1 drop of lidocaine (Teligent) was applied to prevent vasoconstriction and the vein isolated with three 4-0 silk ties (Ethicon, Perma-Hand Silk, 683G) at each end of the exposed vessel. The cranial suture was tightened to occlude blood flow and a small incision made in the vessel to allow insertion of a heparinized saline-filled (2 units heparin/ml 0.9% sodium chloride) catheter (SAI Infusions Technologies, Rat Jugular Vein Catheter, Rounded, Beads at 3cm & 3.5cm, RCMC-7.6-02). The catheter was advanced past the caudal suture, aspirated to confirm patency and then secured by the caudal suture and flushed with heparinized saline. The cranial tie was then used to secure the catheter and the catheter tunneled to the dorsum of the animal after the placement of the dual channel Catheter Access Button (CAB; below). The incision was then closed with 4-0 monocryl (Ethicon, 4-0 Monocryl Y494G) suture in a subcuticular pattern, and tissue glue (3M Vetbond).
Placement of the CAB: The Dual-Channel Catheter Access Button (SAI infusion technologies, Catheter Access Button for Rat 22G, SAI CAB22-R2) was placed between the shoulder blades of the animal through an incision. The jugular vein catheter was tunneled subcutaneously and externalized in the mid-scapular region for connection to the Catheter Access Button (CAB) for blood collection.
Surgical catheterization of the cisterna magna and connection to the CAB: A Kopf stereotaxic frame (Stoelting) with blunt ear bars was used with the anaesthetized animal positioned with the body lower than the head. A 2-3 cm midline incision was made through the scalp. The skin and muscle were retracted laterally. Soft tissues were kept moist throughout the procedure. The surface of the skull was etched with a scalpel blade to allow for adherence of dental cement. Using a sterile 1.35 mm stereotaxic drill bit (Stoelting, 514555), a craniotomy/burr hole was made into the skull over the occipital crest, allowing cautious insertion of a 22g cannula (SAI Infusion Technologies, Rodent CM Cannula, 90 degrees with 7.6 mm tip, SAI RCMC-7.6-02) into the cisterna magna. Free flow of CSF from the unplugged catheter indicated correct placement of the catheter. The plug was replaced and dental cement (Bisco BisCem Self-Adhesive Resin Cement, Dual-Cured, D-45011P) was carefully applied around the cannula and any exposed cranium to secure it in place and allowed to dry completely. The acrylic was smoothed, trimmed and filed to remove any sharp edges and extra acrylic was cleaned from the skin. Screws were not needed. The cannula was tunneled subcutaneously to the CAB and the incision closed using 4-0 monocryl and tissue glue.
Catheter patency, flushing and post-operative care: Catheter and cannula patencies were confirmed prior to trimming and connection to the CAB. The Cannulock of the CAB was moistened with sterile saline, the catheter/cannula connected and implanted subcutaneously. The CAB was positioned rostrally and the incision closed in a simple interrupted pattern using 4-0 monocryl and tissue glue. Patency was confirmed via CSF and blood withdrawal from the CAB ports. The jugular catheter was locked using 236 ml of catheter locking solution (heparin-glycerol, SAI Infusions, SAI HGS-10). Access ports were covered with magnetic caps (SAI Infusions, CAB-RCR, CAB-BCR or CAB-GCR). The rat was removed from the stereotaxic apparatus and recovered from anesthesia with supplemental heat and monitoring of vital parameters/reflexes until fully recovered and ambulatory. Rats were monitored post-operatively three times daily for four days and then at least once daily until endpoint. Animals were co-housed upon recovery from anesthesia. Additional subcutaneous saline and/or Buprenorphine S/R were provided if necessary. Blood and CSF collections were started 72 hours after the CAB placement and continued daily for 14 days.
Blood collection: Each rat was weighed and the volume of blood to be collected was calculated as: 0.75% of the animal’s body weight in grams = weekly limit of blood collection, with 1g blood being equivalent to 1g weight 9. Rats were wrapped in a towel for restraint, the magnetic cap removed from the CAB and the ports accessed using a syringe fitted with a blunt tipped adaptor (SAI Infusions, SAI CLPAD-100). Using aseptic techniques, twice the volume of the catheter was withdrawn and discarded and the sample then collected into an EDTA-blood collection tube. The catheter was flushed with 4 times the catheter volume (945 ml) with heparinized saline and then locked with 236 ml locking solution with positive pressure to prevent any blood clotting at the end of the catheter.
CSF collection: Rats have a total CSF volume of 200-275 ml and CSF is produced at 3.7 ml/min 10. Using syringes fitted with the catheter access port adaptor as above, first the volume of the cannula was collected (~15 ml), then the sample of CSF collected (50 ml-150 ml volume) and the catheter flushed with 15 ml of PBS.
Euthanasia: Endpoint for these animals was at 14 days post recovery (17 days from CAB placement). Anaesthetic overdose was used.
Isolation of extracellular vesicles (EVs) from blood and CSF samples: Blood collected into EDTA tubes was spun at 3000 rpm for 10 minutes to remove cells and the plasma saved for EV extraction. Blood samples were then pooled to obtain >10 ml of blood. CSF samples were pooled from individual animals to obtain 500 ml total volume. EVs were isolated using ultracentrifugation at 100,000g for 120 min at 4oC in polycarbonate tubes (Beckmann, 8x51mm #355657). The supernatant was removed from the tube, and the pellet was resuspended in 100 ml cold phosphate buffered saline (PBS pH 7.2; Invitrogen) containing cOmplete proteinase inhibitor (Roche; 1 tablet/50 ml PBS) and RNAseOUT (Invitrogen; 5 ml/50 ml PBS).
Western blotting for exosomal markers: EV fractions isolated by ultracentrifugation were loaded to 12% SDS-PAGE gels, transferred to nitrocellulose membrane, blocked for 1h at room temperature in TBS+0.1% Tween 20 containing 10% skim milk powder and probed for exosomal markers with RαAlix (Proteintech, #12422-1-AP; RRID:AB_2162467), RαCD81 (Abcam, #ab109201; RRID:AB_10866464), RαHsp90 (Proteintech, #13171-1-AP; RRID:AB_212-924), MαSyntenin-1 (Santa Cruz, #sc-515538), or RαTSG101 (GeneTex, #GTX64349) overnight at 4oC in blocking buffer. Membranes were washed in TBS+0.1%Tween 20, and probed with secondary antibodies (HRP-conjugated GαM (BioRad, #1706516; RRID:AB_11125547) or HRP-conjugated GαR (Invitrogen, #65-6120; RRID:AB_2533967)) for 1 hour at room temperature. Blots were washed and then signal developed with the Western Lightning Plus ECL reagent (FroggaBio, #NEL104001EA) and visualized with a BioRad Gel Documentation Centre. When required, membranes were stripped with ReStore Stripping Buffer (ThermoFisher, #PI21059)