Moorvms ldf1

To validate the model, the blood flow in the RVA was monitored before, during, and after ischemia by using a laser Doppler flow meter (moorVMS-LDF1, Moor Instruments, United Kingdom; Figure 1C).

Transient focal ischemia was induced by occlusion of the middle cerebral artery as previously described [79 (link),80 (link)], with slight modifications [47 (link),48 (link)]. This model of brain ischemia is considered one of the best models to mimic human ischemic stroke [81 (link)], representing an appropriate in vivo procedure to define the protective role of Ngb-NP against stroke.
Briefly, the middle cerebral artery is occluded using a nylon filament suture with a 3–4 mm coating (Doccol Corporation, Redlands, CA, USA), inserted through the right common carotid artery, and advanced until the origin of the middle cerebral artery. A laser-Doppler flow probe (tip diameter 1 mm) attached to a flowmeter (moorVMS-LDF1, Moor Instruments, Axminster, UK) is located over the thinned skull in the MCAO territory (4 mm lateral to bregma) to obtain a continuous measure of relative cerebral brain flow during the surgery. Only animals with a cerebral blood flow reduction over 60% were included in the study. After surgery, anesthesia was discontinued, and rats were returned to their home cages. Animals in the three groups studied were sacrificed 24 h after MCAO procedure.

As previously described⁷⁶ , laser doppler flowmetry was used to assess the effects of CNO on regional cerebral blood flow. Urethane-anesthetized mice had a 3 mm diameter optical probe lowered to ~1 mm above the thinned skull over the right somatosensory cortex (Moor Instruments, MoorVMS-LDF1, PC version 2.2). Perfusion measurements were sampled at 2 Hz over a 60-90 min period. Following 10-15 min of stable baseline measurements, mice were injected with vehicle and/or CNO (0.3–0.5 mg/kg, i.p.). Perfusion measurements collected after injection were normalized to the average baseline value. To determine the effect of saline/CNO injection or C0₂ inhalation on cerebral blood flow, perfusion units were averaged over 5 min period starting 15–20 min after injection, or 3 min after initiating CO2 inhalation and expressed relative to baseline.

The effect of intranasal rhMANF (10 µg) on cerebral blood flow (CBF) was measured with Laser Doppler flowmetry (LDF; moorVMS-LDF, Moor Instruments, Axminster, UK) during and after dMCAo (n = 19). The skull was thinned by drilling a small hole on the cortex [A/P  − 2.0; L/M 4.0 relative to bregma [53 ]] and the moorVMS-LDF1 optical fibre probe was attached on the skull with a PH-DO single fibre holder (Moor Instruments) and dental cement. The baseline CBF was monitored for 10 min before the occlusion of CCAs and dMCA, and monitoring was continued for 10 min after reperfusion. Body temperature was maintained at 37°C with an automated heating pad. One animal was excluded due to insufficient (< 65%) reduction of CBF after stroke induction.

CBF was monitored using a laser doppler perfusion and temperature monitor (Moor VMS-LDF1, Moor Instruments, UK). The rats of each group were anesthetized with chloral hydrate (3.5 mL/kg, i.p.). A paramedian skin incision was performed, and the subcutaneous tissue and cranial fascia were dissected to reach the skull bone. The probe of the monitor was placed at the settled skull bone area to detect the CBF (Cuccione et al., 2016 (link)). CBF changes in the rats were monitored before 2VO operation at the following successive periods: 1, 6, 24 h, 3, 7, 14, and 28 days after operation, as well as 1, 7, 14, and 28 days after drug treatment.