Under general anesthesia with pentobarbital sodium (0.5 g/kg, i.p.), rats (250–300 g) were placed in a stereotaxic frame (Kopf Instruments, Tujunga, CA, USA) with the incisor bar adjusted to achieve a flat skull position relative to lambda and bregma. Using bregma as the reference point, guiding cannulas (RWD Life Science) were implanted bilaterally (anterior-posterior (AP) -2.3 mm; lateral (L) ± 4.5 mm; dorsal-ventral (DV) -7.0 mm) according to coordinates obtained from Paxinos and Watson (1997) [79 ]. The bilateral cannulas were permanently secured to the skull surface using dental acrylic anchored with 4 skull screws. Following surgery, animals were allowed a total recovery period of 5 days. Simultaneous bilateral microinjection of drugs or vehicle into the amygdala in conscious rats were made using 2-μl Hamilton syringes connected to infusion a cannula (RWD Life Science) via PE-50 tubing. The infusion cannula projected 1.0 mm below the tip of the guiding cannula. A total of 0.5 μl solution was in vivo injected into each side of the CeA over a 60-s time period, with the infusion cannula left in place for an additional 60 s to prevent backflow of drug up to the guiding cannula.
Guiding cannula
Manufactured by RWD Life Science
Sourced in China
Guiding cannulas are hollow, cylindrical instruments used in various medical and research applications. They are designed to provide a stable and guided pathway for the insertion and manipulation of other tools or devices within the body or a research setup. The core function of guiding cannulas is to facilitate controlled access and direction during procedures or experiments.
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5 protocols using guiding cannula
1
Bilateral Amygdalar Drug Microinjection in Rats
2
Stereotaxic Viral Vector Injection and In Vivo Peptide Administration in Mice
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For stereotaxic surgery, the 2-month-old C57BL/6 mice were placed in a stereotaxic apparatus and anesthetized with 2% isoflurane (RWD Life Science, R510-22) through a nose cone (300–500 mL/min, RWD Life Science, China, R500). Then, AAV-CaMKII-eGFP-2A-vector-3xFlag or AAV-CaMKII-eGFP-2A-GSK-3β WT-3xFlag or AAV-CaMKII-eGFP-2A-GSK-3β K15Q-3xFlag (1 μl, 4.0 × 1012 viral particles per ml) was bilaterally injected into the hippocampal CA1 region (posterior 1.82 mm, lateral 1.0 mm, and ventral 1.25 mm relative to bregma) at a rate of 0.1 μL/min. The needle was kept in place for 10 min before withdrawal. After the skin was sutured, the mice were placed on a heater for analepsia.
For in vivo peptide administration, guiding cannulas (RWD, Shenzhen, China) were implanted into the lateral ventricle (posterior 0.22 mm, lateral ± 1.0mm from the bregma, ventral -2.5 from the skull) of 12-month-old S129 mice and 3xTg mice, the peptides (1mM, 5 μL) were delivered using an automatic microinjection system (World Precision Instruments, USA), once every 2 days. Mice were restricted in a custom-designed device and stayed awake during drug administration. Upon deep anaesthesia (loss of the pedal pain, slowing of breathing and heart rate), these mice were euthanized by excising the heart for further analysis after behavioral experiments.
For in vivo peptide administration, guiding cannulas (RWD, Shenzhen, China) were implanted into the lateral ventricle (posterior 0.22 mm, lateral ± 1.0mm from the bregma, ventral -2.5 from the skull) of 12-month-old S129 mice and 3xTg mice, the peptides (1mM, 5 μL) were delivered using an automatic microinjection system (World Precision Instruments, USA), once every 2 days. Mice were restricted in a custom-designed device and stayed awake during drug administration. Upon deep anaesthesia (loss of the pedal pain, slowing of breathing and heart rate), these mice were euthanized by excising the heart for further analysis after behavioral experiments.
3
Evaluating BBB-Penetrability and Tau Dephosphorylation
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For 2-month male C57BL/6 mice, 10 mM FITC-labeled or unlabeled-DEPTAC were directly injected through the tail vein or lateral ventricle for the evaluation of BBB-penetrability. For 9-month 3×Tg AD mice or Tau368 (20–30 g body weight), unlabeled-DEPTAC (5 mM, 1 μL for each time) was delivered for once into the lateral ventricle through direct stereotaxic injection, or repeatedly administrated through guiding cannulas (RWD, Shenzhen, China) implanted into the lateral ventricle once every 3 days for a consecutive month to test its dephosphorylation efficiency. Mice were restricted in a custom designed device and stayed awake during drug administration, and sacrificed at 24 h post the single or last DEPTAC administration.
4
Intracerebral Injection of MeCP2-HDO in Mice
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Mice were anesthetized with isoflurane and fixed to the stereotaxic apparatus. Drugs were injected into the right lateral ventricle following the stereotaxic coordinates: 0.8 mm lateral, −2.1 mm ventral, and 0.74 mm from Bregma. For injection two times, a guiding cannula (RWD Life Science, China) was implanted using the coordinates as described above. The concentration of MeCP2-HDO was 200 nM. The volume was 2 μl per injection. Drugs were injected by an injection cannula through a guiding cannula.
5
Intranasal α-Syn-HDO Injection in Mouse Model
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Mice were anesthetized with isoflurane and fixed to a stereotaxic apparatus. AAV9-hSyn-human SNCA (6.58 × 1013 vg/mL, Vigenebio Biosciences, Jinan, China) or PFFs were injected into the substantia nigra (1.2 mm lateral, −4.3 mm ventral, and −3.1 mm from bregma).1 (link) Virus (2 μL) or PFFs (2.5 μL) were injected into each site using a 10 μL Hamilton syringe with a fixed needle at a rate of 0.25 μL/min using a microinjector pump (KDS, Stoelting). The needle remained in place for 5 min after the viral suspension or PFFs were completely injected followed by slow removal (over 2 min). The mice were placed on a heating pad until recovery from anesthesia.
α-Syn-HDO was injected into the right lateral ventricle using the following stereotaxic coordinates: 0.8 mm lateral, −2.1 mm ventral, and 0.74 mm from bregma following anesthetization. For multiple injections of α-Syn-HDO over 4 weeks (α-Syn-HDO: 200 nM/2 μL/week, total four times), a guiding cannula (RWD Life Science, China) was implanted using the coordinates described above. The drugs were injected by an injection cannula through a guiding cannula.
α-Syn-HDO was injected into the right lateral ventricle using the following stereotaxic coordinates: 0.8 mm lateral, −2.1 mm ventral, and 0.74 mm from bregma following anesthetization. For multiple injections of α-Syn-HDO over 4 weeks (α-Syn-HDO: 200 nM/2 μL/week, total four times), a guiding cannula (RWD Life Science, China) was implanted using the coordinates described above. The drugs were injected by an injection cannula through a guiding cannula.
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