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1

Intracerebral Delivery of Neurotrophic Factors

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The rats were balanced into groups based on the body swing and Bederson’s score on day 4
post-stroke. We chose to give the protein injection on day 7 post-stroke since there are
not yet many phagocytic cells in the thalamus at that time point⁶⁰. The stereotaxic surgery was performed under isoflurane anesthesia (4.5% during
induction, 2.5% during maintenance). After placing the animal in a stereotaxic frame
(Stoelting, Wood Dale, IL, USA), the skull was exposed and a small hole was made with a
dental drill. Using coordinates according to The Rat Brain in Stereotaxic Coordinates⁶¹, 4 µl of vehicle (phosphate buffered saline; PBS), rhCDNF (2.5 µg/µl) or rhMANF
(2.5 µg/µl) was injected into the right thalamus (A/P –3.0; M/L –3.0; D/V –6.0) at speed
0.5 µl/min with 33G blunt needle (Nanofil; World Precision Instruments, Sarasota, FL,
USA). The needle was kept in place for 4 min after the injection to prevent backflow.
RhCDNF and rhMANF (Icosagen, Tartu, Estonia) were produced in a Chinese hamster ovarian
(CHO)-based cell line.
The distribution of rhCDNF in the brain after intra-thalamic injection was tested in
naïve rats by injecting rhCDNF as described above, and sacrificing the animals 2 h after
the stereotaxic injection for immunohistochemistry with anti-hCDNF antibody.

Anttila J.E., Pöyhönen S, & Airavaara M. (2019). Secondary Pathology of the Thalamus after Focal Cortical Stroke in Rats is not Associated with Thermal or Mechanical Hypersensitivity and is Not Alleviated by Intra-Thalamic Post-Stroke Delivery of Recombinant CDNF or MANF. Cell Transplantation, 28(4), 425-438.

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2

Intraoperative Hindlimb Stimulation and Tracer Injection

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Rats were anaesthetized with isoflurane and ketamine, head-fixed and positioned identical to electrode implantation. After craniotomy, intraoperative stimulation (50 Hz, 0.5 ms) was performed using a 33-gauge needle (NanoFil, World Precision Instruments) attached to a 10 μl syringe (Hamilton) driven by an electric microinjection pump (World Precision Instruments, UMC4). Needle positioning was adjusted in −0.1 mm steps starting at AP −7.8/ML +2.0/DV −4.7/0° in relation to Bregma until proper hindlimb stepping was initiated upon stimulation. Fast Blue (FB), 2 × 50 nl (EMS Chemie, 2% in DMSO; 100 nl/s), was injected 200 µm above the site showing the best hindlimb response. The needle was left in place for 5 min between injections and 15 min after the last injection to prevent tracer backflow, followed by skin suturing. Animals received Bactrim and Rimadyl daily and were perfused after 7 days.

Hofer A.S., Scheuber M.I., Sartori A.M., Good N., Stalder S.A., Hammer N., Fricke K., Schalbetter S.M., Engmann A.K., Weber R.Z., Rust R., Schneider M.P., Russi N., Favre G, & Schwab M.E. (2022). Stimulation of the cuneiform nucleus enables training and boosts recovery after spinal cord injury. Brain, 145(10), 3681-3697.

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3

AAV9 Injection in Mouse Pups

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P0 HSA_LR mouse pups were placed on wet ice for 30–60 s for anaesthesia. Using a dissection microscope, the superficial temporal vein was identified, and the tip of a 36 gauge needle (NANOFIL; World Precision Instruments) was inserted into the temporal vein. AAV9 (40 μl, 10¹¹ vg) was then injected using a 50 μl Hamilton syringe and a digital infusion pump (Microinjector MINJ-PD; Tritech Research) at a rate of 1 μl s⁻¹. After vector delivery, the needle remained in the vein for 15 s to prevent backflow of the vector. After removing the needle, gentle pressure was applied using a cotton swab to stop the bleeding. The pups were rewarmed in the investigator’s gloved hands to provide appropriate warmth for 2–3 min. When the pups were fully recovered, they were returned to the cage.

Batra R., Nelles D.A., Roth D.M., Krach F., Nutter C.A., Tadokoro T., Thomas J.D., Sznajder Ł.J., Blue S.M., Gutierrez H.L., Liu P., Aigner S., Platoshyn O., Miyanohara A., Marsala M., Swanson M.S, & Yeo G.W. (2020). The sustained expression of Cas9 targeting toxic RNAs reverses disease phenotypes in mouse models of myotonic dystrophy type 1. Nature biomedical engineering, 5(2), 157-168.

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4

Targeted Optogenetic Manipulation in Mice

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All AAV was produced by the University of North Carolina Chapel Hill Vector Core. Adult female C57BL/6 mice (Charles River Laboratories, Inc.) or Chat-Cre mice (Chat-cre;129S6-Chattm2(cre)Lowl/J, the Jackson Laboratory), 8–12 weeks at the time of surgery, were used for all experiments. AAV-Syn-SomArchon (5.9e12 genome copies (GC)/ml) or AAV-syn-SomArchon-P2A-CoChR-Kv2.1 (2.19e13 GC/ml) was injected into the motor cortex (AP: +1.5, ML: +/−1.5, DV: −0.3, 0.5uL virus), visual cortex (AP: −3.6, ML: +/−2.5, DV: −0.3, 0.5uL virus), hippocampus (AP:−2.0, ML:+1.4, DV:−1.6, 1uL virus) or striatum (AP:+0.8, ML:−1.8, DV:−2.1, 1uL virus). Viral injection occurred at 50–100nL/min (ten minutes total) using a 10uL syringe (NANOFIL, World Precision Instruments LLC) fitted with a 33 gauge needle (World Precision Instruments LLC, NF33BL) and controlled by a microinfusion pump (World Precision Instruments LLC, UltraMicroPump3–4). The syringe was left in place for an additional 10 minutes following injection to facilitate viral spread. About one week following the viral injection, mice underwent a second surgery to implant the cranial window for in vivo imaging.

Piatkevich K.D., Bensussen S., Tseng H.A., Shroff S.N., Lopez-Huerta V.G., Park D., Jung E.E., Shemesh O.A., Straub C., Gritton H.J., Romano M.F., Costa E., Sabatini B.L., Fu Z., Boyden E.S, & Han X. (2019). Population imaging of neural activity in awake behaving mice. Nature, 574(7778), 413-417.

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5

Intravitreal Tracer Administration in Mice

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Mice were anesthetized using intraperitoneal injection of 3 mg/20 g of body weight ketamine and 0.3 mg/20 g of body weight xylazine. Before injection of substrates into the eye, 1% atropine sulfate drops were applied topically to provide better visualization of the needle and internal eye structures.
Aqueous humor injections were performed by puncturing the cornea with a 27-gauge needle, drawing out endogenous aqueous with an ophthalmic sponge, and injecting approximately 5 μL of uptake buffer containing GSH-(glycine-¹³C₂,¹⁵N) using a 10-μL syringe equipped with a 33-gauge needle (Nanofil; World Precision Instruments, Sarasota, FL, USA). A small air bubble was injected with solution in order to prevent solution from leaking out of the puncture site.
Vitreous humor injections were performed by puncturing mouse sclera with a 10-μL syringe (Nanofil) equipped with a 33-gauge needle at a 45° angle in order to avoid puncturing the lens, and injecting 1 μL of uptake buffer containing GSH-(glycine-¹³C₂,¹⁵N) directly into the vitreous body.

Whitson J.A., Sell D.R., Goodman M.C., Monnier V.M, & Fan X. (2016). Evidence of Dual Mechanisms of Glutathione Uptake in the Rodent Lens: A Novel Role for Vitreous Humor in Lens Glutathione Homeostasis. Investigative Ophthalmology & Visual Science, 57(8), 3914-3925.

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6

Striatal AAV9-Luciferase Gene Delivery

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FVB/NJ mice (5 females, 6 weeks old, Jackson Laboratory, Bar Harbor, ME) were anesthetized with 250 mg/kg tribromoethanol prior to surgery, placed on a stereotactic frame and injected with 0.25 μL 1e13 GC AAV9-CMV-Luc2 by micropump syringe (NanoFil, World Precision Instruments) at the lateral edge of the striatum/cortex border (anterior 1 mm, lateral 3 mm and ventral 2 mm from bregma). Imaging was performed at least two weeks after AAV injection.

Evans M.S., Chaurette J.P., Adams ST J.r., Reddy G.R., Paley M.A., Aronin N., Prescher J.A, & Miller S.C. (2014). A synthetic luciferin improves bioluminescence imaging in live mice. Nature methods, 11(4), 393-395.

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7

Serratia marcescens Injection in Flies

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Serratia marcescens (ATCC 13880, Thermo Fisher Scientific) was inoculated into 5 ml of sterile Nutrient Broth (Oxoid, CM0001) and incubated overnight (approximately 16 h) at 26°C with shaking at 200 rpm. The bacterial culture was centrifuged at 10,000 g at 4°C for 2 min. The supernatant was discarded, and the bacterial pellet washed twice using 1× phosphate buffered saline (PBS; Sigma-Aldrich, cat. no P4417) to remove any trace of the medium. The bacterial pellet was resuspended to a target concentration of OD₆₀₀=0.025 in sterile PBS.
One day after adult eclosion, flies were cold anaesthetized at −20°C for 2 min and placed on a Petri dish on an MK20 Dry Bath at −10°C. Injections were performed using a 10 µl syringe (NanoFil) connected to a microinjector (World Precision Instruments) with a delivery speed of 50 nl s⁻¹. A volume of 0.2 µl of the bacterial solution, yielding a dose of approximately 1680 cells, was injected into the fly's coxa of the third right leg. PBS-injected (i.e. sham-treated) flies were used as controls.

Dinh H., Lundbäck I., Kumar S., Than A.T., Morimoto J, & Ponton F. (2022). Sugar-rich larval diet promotes lower adult pathogen load and higher survival after infection in a polyphagous fly. The Journal of Experimental Biology, 225(16), jeb243910.

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8

Optogenetic Manipulation of Cholinergic Neurons

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ChAT-IRES-Cre mice or double transgenic, CalDAG-GEFI-EGFP-BAC;ChAT-IRES-Cre mice and CalDAG-GEFI-EGFP-BAC;ChAT-ChR2-EYFP BAC mice were maintained in deep anesthesia with a continuous flow of 2% isoflurane (Southmedic Inc.) in an oxygen mixture, delivered by a nose-cone attached to a stereotaxic frame. Mice were given bilateral intrastriatal injections, via NanoFil microsyringe (World Precision Instruments), of AAV5 encoding EF1αDIO hChR2(H134R)-mCherry (University of N. Carolina vector core), 0.75 μl per site, at the following stereotactic coordinates (AP = 0.9 mm, ML = −1.9 mm and +1.9 mm, DV = 2.0 mm and 2.7 mm, relative to bregma). Mice were allowed to fully recover from surgery for 2 weeks prior to the initiation of saline or D-amphetamine injections.

Crittenden J.R., Lacey C.J., Weng F.J., Garrison C.E., Gibson D.J., Lin Y, & Graybiel A.M. (2017). Striatal Cholinergic Interneurons Modulate Spike-Timing in Striosomes and Matrix by an Amphetamine-Sensitive Mechanism. Frontiers in Neuroanatomy, 11, 20.

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9

Stereotactic Injection of Anti-CD15-SPIONs

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Imaging probe, anti-CD15-SPIONs were stereotactically injected into the left cerebral lateral ventricle at a dosage of 7 μL per animal. Anti-CD15-SPIONs were injected into the anterior portion of the lateral ventricles on right side with a stereotaxic coordinate: 0.95 mm lateral to bregma, 0.02 mm rostral to bregma and 2.6 mm deep from the pial surface by the same operator (F.Z., with five years of experience with microsurgical procedures). The anti-CD15-SPIONs was slowly injected with a constant rate of 0.5 μL/min using a 28 gauge needle (NanoFil; World Precision Instruments, Sarasota, FL, USA) mounted on a microinjector. After injection, the needle was left in the place for 5 min and then slowly withdrawn.

Zhang F., Duan X., Lu L., Zhang X., Zhong X., Mao J., Chen M, & Shen J. (2016). In Vivo Targeted MR Imaging of Endogenous Neural Stem Cells in Ischemic Stroke. Molecules, 21(9), 1143.

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10

Targeted Optogenetic Manipulation in Mice

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All AAV was produced by the University of North Carolina Chapel Hill Vector Core. Adult female C57BL/6 mice (Charles River Laboratories, Inc.) or Chat-Cre mice (Chat-cre;129S6-Chattm2(cre)Lowl/J, the Jackson Laboratory), 8–12 weeks at the time of surgery, were used for all experiments. AAV-Syn-SomArchon (5.9e12 genome copies (GC)/ml) or AAV-syn-SomArchon-P2A-CoChR-Kv2.1 (2.19e13 GC/ml) was injected into the motor cortex (AP: +1.5, ML: +/−1.5, DV: −0.3, 0.5uL virus), visual cortex (AP: −3.6, ML: +/−2.5, DV: −0.3, 0.5uL virus), hippocampus (AP:−2.0, ML:+1.4, DV:−1.6, 1uL virus) or striatum (AP:+0.8, ML:−1.8, DV:−2.1, 1uL virus). Viral injection occurred at 50–100nL/min (ten minutes total) using a 10uL syringe (NANOFIL, World Precision Instruments LLC) fitted with a 33 gauge needle (World Precision Instruments LLC, NF33BL) and controlled by a microinfusion pump (World Precision Instruments LLC, UltraMicroPump3–4). The syringe was left in place for an additional 10 minutes following injection to facilitate viral spread. About one week following the viral injection, mice underwent a second surgery to implant the cranial window for in vivo imaging.

Piatkevich K.D., Bensussen S., Tseng H.A., Shroff S.N., Lopez-Huerta V.G., Park D., Jung E.E., Shemesh O.A., Straub C., Gritton H.J., Romano M.F., Costa E., Sabatini B.L., Fu Z., Boyden E.S, & Han X. (2019). Population imaging of neural activity in awake behaving mice. Nature, 574(7778), 413-417.

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Nanofil

Lab products found in correlation

27 protocols using nanofil

Intracerebral Delivery of Neurotrophic Factors

Intraoperative Hindlimb Stimulation and Tracer Injection

AAV9 Injection in Mouse Pups

Targeted Optogenetic Manipulation in Mice

Intravitreal Tracer Administration in Mice

Striatal AAV9-Luciferase Gene Delivery

Serratia marcescens Injection in Flies

Optogenetic Manipulation of Cholinergic Neurons

Stereotactic Injection of Anti-CD15-SPIONs

Targeted Optogenetic Manipulation in Mice

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