A previously reported model of ICH employing autologous blood was used in this study.23 , 24 After mice were anesthetized by intraperitoneal (i.p.) injection of 80 mg/kg phenobarbital, they were fixed on a mouse stereotaxic frame (Stoelting, Wood Dale, IL). Then 20 μL of autologous blood collected from the tail vein and not treated with an anticoagulant was injected into the caudate nucleus using a syringe pump (KD Scientific, Holliston, MA) at 0.8 mm anterior, 2 mm right lateral, and 3.5 mm deep from the bregma. During the procedure, body temperature was kept at 37°C, and the mice had free access to food and water once awake. Mice that died were excluded from the study.
Mouse stereotaxic frame
Manufactured by Stoelting
Sourced in United States
The Mouse Stereotaxic Frame is a precision instrument designed for the accurate positioning and immobilization of mice during neuroscience research and surgical procedures. It provides a stable platform to hold the animal's head in a fixed position, enabling targeted access to specific regions of the brain.
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4 protocols using mouse stereotaxic frame
1
Autologous Blood Injection Mouse ICH Model
2
Autologous Blood Injection into Mouse Brain
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Briefly, mice were anesthetized with an intraperitoneal injection of 400 mg/kg chloral hydrate and fixed on a mouse stereotaxic frame (Stoelting). A 20 μl volume of autologous nonanticoagulated blood was collected from the tail vein of the mouse and then injected into the caudate nucleus at 2 μl/min under stereotactic guidance at the following coordinates relative to bregma: 0.8 mm anterior, 2 mm left lateral, and 3.5 mm deep during a period of 10 min The needle was held in place for 10 min after injection, and the microsyringe was pulled out after the blood had coagulated. The craniotomy was then sealed with bone wax, and the scalp was closed with sutures. Body temperature was maintained at 37 °C throughout the procedure, and the mice were given free access to food and water after they woke up. The mice that died because of anesthesia were excluded.
3
Chemogenetic Manipulation of Neural Circuits
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Mice were deeply anesthetized with ketamine/xylazine (100 mg/kg; 10 mg/kg) and placed in a mouse stereotaxic frame (Stoelting, USA). A 10 µl syringe (7000 series, Hamilton, USA) attached to a micropump was lowered through to skull burr hole into the PFC (AP + 1.8 mm, L ± 0.3 mm, DV −2.7 mm), STR (AP + 1 mm, L ± 1.5 mm, DV −2.7 mm) or BF (AP = + 0.26,ML: ± 1,DV = 5.4). Mice were injected with the following viruses: AAV5-hSyn-DIO-hM3D(Gq)-mCherry, AAV5-hSyn-DIO-hM4D(Gi)-mCherry, or rg-AAV-hSyn-DIO-hM3D(Gq)-mCherry viruses (≥ 7 × 1012 vg/ml, Addgene) within the PFC (0.3 µl per hemisphere), STR (0.5 µl per hemisphere) or BF (0.5 µl per hemisphere) at a flow rate of 50 nl/min. These constructs have been thoroughly validated by us and others in terms of their efficiency at recombining and activating/inactivating neurons [43 (link)–46 (link)]. Clozapine N-oxide (CNO) was injected intraperitoneally at a dose of 3 mg/kg in sterile saline solution 1 hour prior to the behavioral procedures. The response to CNO has been previously shown to be specific, as compared to Cre negative or fluorophore-only injected animals [44 (link), 46 (link)].
4
Controlled Cortical Impact Model in Mice
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Mice were anesthetized with isoflurane (4% for induction, 2–3% for maintenance) and securely positioned in a mouse stereotaxic frame (Stoelting Co). Surgery was performed as described previously (Villapol et al., 2012 ; Yi et al., 2012 (link)). Briefly, an incision was made over the forehead, and the scalp was reflected to expose the skull. A craniotomy was made over the left hemisphere and the bone flap was carefully removed. Mice were injured over the left somatosensory cortex (0 bregma, 2 mm lateral to the suture line) at an impact depth of 1 mm with a 2-mm diameter round impact tip (speed 3.6 m/s, dwell time 100 ms) using an electromagnetically driven CCI injury device (Impact One™ stereotaxic impactor CCI, Leica Microsystems GMBH) (Brody et al., 2007 (link); Pleasant et al., 2011 (link)). These CCI parameters lead to an injury that is considered mild to moderate according to our experience and previous publications (Washington et al., 2012 (link); Yi et al., 2012 (link)). The dura remained intact following craniotomy. Impact caused occasional extradural hemorrhages with mild edema. Following injury, the bone flap was replaced but not secured, and the scalp was sutured closed. Mice were under isoflurane for no longer than 15 min. After recovery from anesthesia, mice were maintained in a warm recovery cage for 1 h and returned to home cages.
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