Microinfusion pump

To induce a cholinergic deficit, 192IgG-saporin was injected into the medial septum area (MSA) with a microinfusion pump (Stoelting Co., Wood Dale, IL, USA) at a rate of 0.2 μL/min using a Hamilton syringe (Hamilton Company, Reno, NV, USA) as previously described in [11 (link)]. The rats were anesthetized using chloral hydrate (400 mg/kg, i.p.). For additional anesthetic and analgesic effects, the ears and wound surface of each animal were treated with lidocainum (Pharmstandart, Moscow, Russia). The stereotaxic coordinates were as follows: +0.4 mm anteroposterior, −1.5 mm lateral, and 14° [42 (link)]. The drug was infused at a concentration of 1.5 μg/per rat. The rats were allowed to recover for 21 days. The injection sites were confirmed for each experiment. The animals, in which the number of cholinergic neurons in the medial septal area after 192IgG-saporin injection was >50% that of the controls, were excluded from further experiments.

Stroke-like focal brain injury model was performed as we previously described 16 (link). Briefly, under general anesthesia, a burr hole was placed in the skull and a needle (length 15 mm, gauge 33) connected to a 10 μl syringe (Hamilton, Switzerland) was inserted into brain at coordinates: A 0.5; L 3.8; D 4.7 mm. Then, 1 μl of 5 mmol ouabain solution (Sigma, Poland) was injected over 1 minute using a microinfusion pump (Stoelting, USA) and five minutes later the needle was withdrawn and the skin was closed with a suture. After the procedure, each animal was injected with an antibiotic (Baytril; Bayer; 0.4 mg/ml) and an analgesic (Rycarfa; Krka; 5 mg/ml).

Rats were anesthetized with ketamine (90 mg/kg) and xylazine (10 mg/kg), given by i.p. injection, and immobilized in a stereotaxic apparatus (Stoelting). A small burr hole was drilled in the cranium over the right hemisphere. The needle (length 15 mm, gage 33), connected to a 10-μl syringe (Hamilton, Switzerland), was lowered into the right striatum (coordinates A 0.5, L 3.8, D 4.7 mm). To minimize brain shift, there was a delay of 5 min between the needle insertion and the injection of the active substance. Then, 1 μl of 5 nmol ouabain (Sigma, Poland) was injected into the brain at a rate of 0.5 μl/min using a microinfusion pump (Stoelting, USA) mounted on a stereotaxic apparatus (Stoelting), as previously described [6 (link)]. The needle was then withdrawn and the skin was closed with a suture.

Three days after ouabain injection, rats were anesthetized with ketamine (90 mg/kg) and xylazine (10 mg/kg), given by i.p. injection, and immobilized in a stereotactic apparatus (Stoelting). Then, an incision was made through the skin overlying the sagittal suture, and a small burr hole was drilled in the cranium over the right hemisphere. The needle (length 15 mm, gage 33), connected to a 5-μl Hamilton syringe, was lowered into the corpus callosum (coordinates A 0.0, L 4.0, V 3.0 mm where the bregma was adjusted to the same horizontal plane, and the ventral coordinates were calculated from the dura), and 2 μl of HUCB-NSCs labeled with CMFDA (2 × 10⁵) was injected at a rate of 0.5 μl/min via a microinfusion pump (Stoelting) mounted on stereotactic apparatus. After injection, the needle was left in situ for 5 min to avoid the leakage of injected cells through the needle tract. Then, the needle was withdrawn and the skin closed with a suture.

Rats were anesthetized with ketamine (75 mg/kg) and xylazine (10 mg/kg) and secured in a stereotaxic frame (Stoelting, USA). Vertical microdialysis guide cannulas (Intracerebral Guide Cannula with stylet; BAS Bioanalytical, USA) were implanted in the hippocampus (HIP) according to the following stereotaxic coordinates: A / P − 5.5, L/M + 5.0 and V / D − 4.8 mm from bregma and the dura (G. Paxinos and C.H. Watson). Seven days after surgery, microdialysis probes (length 2 mm) were inserted into the cannulas, and the HIP was perfused with artificial cerebrospinal fluid (aCSF), which consisted of 140 mM NaCl, 2.7 mM KCl, 1.2 mM CaCl₂, 1 mM MgCl₂, 0.3 mM NaH₂PO₄, and 1.7 mM Na₂HPO₄ (Ph 7.4), at a flow rate of 1.5 μL/min maintained with a microinfusion pump (Stoelting, IL USA). Three basal samples were collected from freely moving rats at 30 min intervals after a 2 h wash-out period. Then, drugs (saline, MK-801 0.1 mg/kg, 1 MeTIQ 50 mg/kg) were injected. In the combined groups, 1MeTIQ was given 30 min before MK-801 injection. After drug administration, 6 experimental samples at 30 min intervals were collected. All dialysates were immediately frozen on dry ice (− 70 °C) until they were used in a biochemical assay.

ZIP (Abcam, Cambridge, MA, USA) was dissolved in sterile saline to a concentration of 10 nmol/μl. ZIP or saline were infused into the dorsal hippocampus (1 μl per hemisphere) via a microinjector (28 gauge) connected to a microinfusion pump (Stoelting Co., Wood Dale, IL, USA) at a rate of 0.25 μl per min. The injector remained connected for an additional 1 min to allow for drug diffusion away from the tip of the cannula.

Drug administration was performed using standard stereotaxic methods. Animals were anesthetized with isoflurane (Baxter, United States) and mounted in a Kopf stereotaxic frame. 192IgG-saporin (1.5 mg) or PBS was injected into the medial septum area (0.4 mm anterior, 1.5 lateral to bregma, angle 14°). This dose of the toxin was chosen based on our previous data that the immunotoxin dose of 1.5 mg induced loss of choline acetyltransferase (ChAT)–positive neurons in the medial septum area (Dobryakova et al., 2020b (link), a (link)). AAV suspension was bilaterally injected into the CA1 area of the hippocampus (2.9 mm posterior, 1.7 lateral to bregma, 3.3 mm depth; 1 μL/side). All injections were performed through the Hamilton syringe (Hamilton company, United States) using a microinfusion pump (Stoelting Co., United States) at a rate of 0.5 μL/min. After each injection, the needle was left in situ for 10 min. Rats were allowed to recover for 21 days after the surgery.