Experimental Techniques

The Neurovascular Research Unit (UIN) has its laboratories in Pavilion III of the Faculty of Medicine at the Complutense University of Madrid. They occupy 240 m² of which 30% are dedicated to offices and meeting places and 70% to 6 laboratories:

1. General Laboratory with refrigerators/freezers (-80ºC and -40ºC), cold room, ultrafiltration water services, radioactive facilities, etc.

2. Laboratory for enzymatic/analytical techniques with HPLC system, spectrophotometers, microplate readers, flow cytometer, etc.

 

3. Molecular biology laboratory with equipment for DNA, RNA and protein analysis such as thermal cyclers, gel documentation systems, PCR and qRT-PCR, Nanodrop, etc.

4. Cell Culture Unit with laminal flow hood, two incubators and nucleofector system.

5. Laboratory for experimental models of stroke with 6 rat/mouse animal surgery stations with all the necessary equipment (inhalation anaesthesia, binocular magnifying glasses, electro-scalpel, micromanipulation equipment, non-invasive pressure transducer, digital camera, video, portable blood autoanalyser, Doppler equipment for measuring brain flow for animals, etc.)

 

6. Microscopy laboratory with digital image analysis equipment, 2 transmitted light and epifluorescence microscopes -one direct and one inverted-, stereology and neuronal reconstruction software (StereoInvestigator and Neurolucida; Microbrightfield; Volocity, Perkin Elmer) and Zeiss LSM-710 spectral confocal microscope.

 

In addition, the Neurovascular Research Unit has the CAIS (Research Assistance Centres) of the UCM, such as the Flow Cytometry and Confocal Microscopy CAI, the Electronic Microscopy CAI, the DNA Sequencing CAI, the Spectroscopy CAI, the Nuclear Magnetic Resonance and Electronic Spin CAI (MRI equipment for animals of 4.7 Teslas and portable ICON equipment), the CAI of Brain Mapping (micro-PET) and the animal CAI of the UCM.

Neurological tests for functional outcome assessment in stroke

(see Zarruk et al., Rev Neurol 2011)

Sensorimotor tests

- Adhesive removal test
- Balance beam test
- Corner test
- Cylinder test
- Elevated body swing test
- Footprint test
- Limb placement test
- Open field test
- Rota-rod test
- Staircase test
- Walking beam test

Memory test

- Barnes maze test
- Morris water maze
- Object recognition and novel object location tests
- Passive avoidance test
- Y Maze test

Neurological Scales
- Modified Neurological Severity Score (mNSS)

Experimental models of stroke

The Neurovascular Research Unit has expertise in different experimental stroke models (the publications of our group where the referenced model is used are underlined):

DISTAL ISCHEMIC STROKE MODELS:

1. Fibrin rich-clot thrombosis (mice and rat):

1. Injection of thrombin into middle cerebral artery (MCA) lumen (Orset et al., Stroke 2007; García-Yébenes et al., Stroke 2011).
2. Recanalization (20 min) using rtPA.

2. Platelet-rich clot thrombosis (mice and rat):

1. By arterial wall injury after local application of FeCl3 (Karatas et al., JCBFM 2011; Peña-Martinez et al., Stroke 2019) or by the photochemical reaction between rose bengal and a laser beam (photothrombotic model; Watson et al., Ann Neurol 1985; Peña-Martinez et al., Stroke 2019).
2. Model resistant to rtPA fibrinolysis

3. Permanent ischemia (mice and rat):

1. By electrocoagulation (Lambertsen et al., JCBFM 2005; Caso et al., Circulation 2007) or ligature (Brint et al., JCBFM 1988; Chen et al., Stroke 1986; Ballesteros et al., J Vis Exp 2014 -see video-) of MCA.
2. Model with no reperfusion

Electrocoagulation of MCA

Ligature of MCA

4. Transient ischemia (mice and rat):

1. By intraluminal thread technique (Longa et al., Stroke 1989; Pérez-Asensio et al., Neurobiol Dis 2005) or by ligature (Ballesteros et al., J Vis Exp 2014 -see video-) of MCA.
2. Model with reperfusion

5. Transient ischemic attack (TIA; mice and rat):

1. Mechanical pressure on MCA (Barone et al., Stroke 1998; Pradillo et al., JCBFM 2005)
2. Occlusion duration accurately controlled
3. Short ischemia duration (< 15 min) mimics TIA

 

HEMORRHAGIC STROKE MODELS:

6. Intracerebral hemorrhages induced by collagenase injection (mice, rat):

1. A stereotaxic injection of collagenase is performed leading to a rupture of the blood vessel’s wall.

 

HEMORRHAGIC TRANSFORMATION STROKE MODELS:

7. Fibrin rich-clot thrombosis (mice and rat):

1. Injection of thrombin into MCA lumen (García-Yébenes et al., Stroke 2011).
2. Delayed recanalization (3h) using rtPA.

 

VASCULAR COGNITIVE IMPAIRMENT MODELS:

8. Focal hypoperfusion (mice):

1. By occlusion of MCA (Chen et al. Stroke 1986; Cuartero et al., J Clin Invest 2019)

9. Chronic hypoperfusion (mice):

1. By bilateral stenosis of common carotid arteries (BCCAs; microcoils; Shibbata et al., Nature Neurosci 2004)

10. High-sodium diet (mice):

1. Mice receive sodium-rich chow (Faraco et al., Nature 2019)