Chronic Remote Ischemic Conditioning Is Cerebroprotective and Induces Vascular Remodeling in a VCID Model

Vascular contributions to cognitive impairment and dementia (VCID) make up 50% of the cases of dementia. The purpose of this study was to determine the effect of chronic remote ischemic conditioning (C-RIC) on improving long-term (6 months) outcomes and cerebral blood flow (CBF) and collateral formation in a mouse model of VCID. Adult C57BL/6J male mice (10 weeks) were randomly assigned to four different groups: (1) sham-bilateral carotid artery stenosis (BCAS), (2) BCAS + sham RIC, (3) BCAS+C-RIC for 1 month (1MO), and (4) BCAS+C-RIC-4 months (4MO). CBF, cognitive impairment, and functional outcomes were performed up for 6 months after BCAS surgery. The expression of CD31, α-SMA, and myelin basic protein (MBP) was assessed by immunohistochemistry (IHC). Additional set of mice were randomized to sham, BCAS, and BCAS+C-RIC. The cerebrovascular angioarchitecture was studied with micro-CT. RIC therapy for either 1 or 4 months significantly improved CBF, new collateral formation, functional and cognitive outcomes, and prevented white matter damage. There was no difference between C-RIC for 1 or 4 months; IHC studies at 6 months showed an increase in brain CD31 and α-SMA expression indicating increased angiogenesis and MBP indicating preservation of white matter in animals receiving RIC. One month of daily RIC is as effective as 4 months of daily RIC in improving CBF, angiogenesis, and long-term functional outcomes (6 months) in a VCID model. This suggests that 1 month of RIC is sufficient to reduce cognitive impairment and induce beneficial cerebrovascular remodeling. Electronic supplementary material The online version of this article (doi:10.1007/s12975-017-0555-1) contains supplementary material, which is available to authorized users.


Bilateral carotid artery stenosis (BCAS) Surgery
Bilateral Common Carotid Artery Stenosis (BCAS) procedure in the mouse was performed as described elsewhere [1,2]. Briefly, mice were subjected to BCAS and subsequent cerebral chronic hypoperfusion using microcoils specially designed for the mouse (microcoil specifications: piano wire diameter 0.08mm, internal diameter 0.18mm, coiling pitch 0.5mm, and total length 2.5mm; Sawane Spring Co Ltd, Japan). To induce VCID in the Buprenex sedated mice under isofluorane placed on a thermoregulated bed to maintain body temperature at 37 o C during surgery; a midline cervical skin incision was made ventrally. The two common carotid arteries (CCA) were exposed one-by-one, freed from their sheaths, and a microcoil was twined by rotating it around the each CCA, as published elsewhere. The site of surgery was closed, and mice were observed and taken care of post-surgery until conscious and recovered to freely access food and water ad libitum. In Sham group, the skin was incised and the two common carotid arteries were exposed one by one.

Non-Invasive RIC Therapeutic methods
Non-invasive RIC therapy using a BP-cuff was performed as reported earlier [3,4] with certain modifications by us [2]. An automated blood pressure instrument (SC1000 rodent BP instrument remodeled as a RIC-BP dual mode instrument with software and computer) was modified with a multi-channel RIC device for multiple rodents (RC2000, V1.14; a customized product developed by Hatteras Instrument; Cary NC, for our laboratory) to produce cuff-based, non-invasive, bilateral RIC in the mouse hind limbs. After 1 week post-BCAS surgery, RIC was started daily. RIC was performed in isoflurane anesthetized mice with the modified instrument.. Briefly, mice were placed on a thermoregulated bed to maintain body temperature at 37 o C and slightly stretched limbs were secured using paper tape. Customized mouse limb cuffs were wrapped on each hind limb and RIC was performed as 4 cycles x 5 minutes/cycle at 200 mmHg using a 5-min inflate and deflate in each cycle. Sham RIC mice were secured in the same fashion, but the BP cuff was not inflated, controlling for any effects of stress or handling. Following the RIC therapy, mice were placed into a clean home cage and monitored until conscious.

Estimation of nitrite in plasma
Plasma NOx (NO2 -+ NO3 -) levels were measured by NO-specific chemiluminescence, as described previously [5] with slight modification. Blood were collected from each groups of mice in isoflurane anesthetized condition with cardiac puncture. The plasma sample were prepared by centrifuging the blood sample for 20 min at 2000 rpm / 4 ºC. Plasma (100 ul) was mixed with twice volumes of ethanol (100%) and kept at -20 C for 40-60 min followed by centrifuged at maximum rpm (13000 rpm) for 10 min to remove protein as pellet. The supernatant (100 ul) was taken and injected to measure nitrite. The nitrite levels were measured by NO Analyzer 280i (GE Analytical instruments, Colorado, USA). The level of nitrite was expressed in nM.

Novel object test
Behavioral assessment by novel object recognition (NOR) test was performed as reported earlier by us [2, 6]. The 2-trial novel object recognition task was also performed in which a mouse was placed in an enclosed box (40 inch x 40 inch) with 2 identical objects situated within a 4-inch diameter circle and located a set distance apart. The mouse was then removed from the environment for a set amount of time and 1 of the 2 previously used (familiar) objects was replaced with a novel object that was different from the familiar object in texture and appearance. The mouse's behavior on exposure to the novel object was then recorded. This test is based on the natural tendency of mice to investigate a novel object rather than a familiar one, which reflects the use of learning and recognition memory processes. The capability of the mouse to discriminate between a familiar versus novel object was determined as the discrimination index, DI= (Tn-Tf)/ (Tn+Tf), where Tn is the time spent by the mouse with the novel object and Tf indicates the time spent with the familiar object.

Wire hanging test
This test was used as a measure of grasping ability, forelimb strength and coordination movements described earlier [7]. Mice used their forelimbs to suspend their body on a single 1 mm diameter cord stretched mid-way between two metal stands/post (55 cm long suspended horizontally, 50 cm above a standard mouse cage was filled with soft cloths and under the cord in between the stands). The latency to fall was measured five times (in seconds) for each mouse. The first two trials served to familiarize the mouse with the testing conditions. The subsequently three trials were taken at five minute intervals to allow a recovery period. The latency time measurements began from the point when the mouse was hanging free on the wire and ended with the animal falling to the cage underneath the cord. No attempt was made to force compliance during any of the various trials. If an animal adopted a strategy that permitted an extended hanging time, this was allowed and the true latency to fall time was recorded (in sec). In cases where an animal was unable to hang from the wire for at least one second, a fall time of zero was noted for that trial.

Beam Walk test
Motor balance and coordination test was performed by using the balance beam as reported earlier [8] with a little modification. The beam apparatus consists of 1.25 meter beams (scale marked) with a flat surface of 6 mm width) resting 20 cm above the table top on two poles. A black box is placed at the end of the beam as the finish point. (Plywood and other types of wood for the beams, poles, and box can be used). Nesting material from home cages is placed in the black box to attract the mouse to the finish point. A lamp (with 60 watt light bulb) is used to shine light above the start point and serves as an aversive stimulus. Approximately 10-15 min prior to training/testing, the mice can be transported to the room to acclimatization where the beam apparatus are located. Mice were trained before the real test for three times. During training, mice were encouraged to keep moving across the beam by prodding, poking, or pushing it from behind with gloved fingers. Training trials were repeated until each animal crossed the beam three times without stopping or turning around. On a final testing, three trials were performed per mouse to cross a beam. The time (in seconds) to cross the beam (100 cm) is counted by timer (one at 25 cm that starts a timer and one at 100 cm that stops the timer). A soft bed /cloths is stretched below the beam above the table top, to cushion any falls. Trials in which the animal stopped or turned around were repeated. The average of the trials was calculated in seconds.

Y-maze test
A continuous spontaneous alternation test was performed at 4 and 6 months in all groups after the sham surgery and BCAS surgery using a Y-maze (San Diego Instruments, San Diego, CA) as described previously [9]. The Y-maze consisted of three arms at 120∘ and was made of beige plastic. Each arm was 7.5 cm wide and 38 cm long, and its three sides (except for the side adjoining the other arms) were surrounded by 12.5-cm high walls. The floor of the Y-maze was covered with a sawdust bedding material. Between each trial, the sawdust was mixed and redispersed to remove or randomize odor trails. Distal visual cues were placed around the Ymaze. A mouse was placed in one arm and allowed to explore for 7 min freely. Mouse behavior was monitored, recorded, and analyzed by a webcam (C920, Logitech, Newark, CA) and the Any-Maze software (Stoelting, Wood Dale, IL). A mouse was considered to have entered an arm if the whole body (except for the tail) entered the arm and to have exited if the whole body (except for the tail) exited the arm. If an animal consecutively entered three different arms, it was counted as an alternating triad. Because the maximum number of triads is the total number of arm entries minus 2, the score of alternation was calculated as "the number of alternating triads/ (the total number of arm entries minus 2)."

Histological and Immunohistochemical Assessment
For histological and immunohistochemical staining, a standard paraffin block and frozen block were used to get the coronal sections of thickness 6 um and 10 um respectively. Each slide was mounted with 4 sections. Histochemical-staining for Fiber density of Luxol-Fast-blue (LFB, a demyelination marker)-neutral red staining was performed to detect the severity of WM lesion [2, 10, 11] with slightly modification. Briefly, de-waxed rehydrated sections were immersed in the LFB solution (Solvent Blue 38, Sigma) at 60 °C overnight. Excess stain was removed by 95% ethanol treatment followed by washing with deionized water. Grey and white matter differentiation was initiated with the treatment of 0.05% aqueous lithium carbonate (Sigma) for 30 second followed by 70% ethanol until the nuclei are decolorized. Sections were immersed in neutral red solution (Sigma) for 30 min and washed in deionized water. They were dehydrated in ethanol gradient (70 -100%), and finally cleared in xylene and mounted with cytoseal. For immunostaining, mice brain sections were deparaffinized and pretreated with citrate buffer (pH 6.0) for antigen retrieval at 120 C followed by cooling (30 min at room temperature) and subsequently washed with PBS twice. The sections were incubated with anti-MBP (C-16 clone, SC-13914, Santa Cruz, CA, USA; 1:100 dilutions) overnight at 4 C, followed by incubation with appropriate biotinylated secondary antibody (Vector Laboratories, Burlingame, CA; 1:200 dilution) for 1 hour. To visualize the immunoreactivity, sections were finally incubated with an avidin-biotin peroxidase complex solution (Vectastain ABC kit, Vector Laboratories, diluted 1:100) for 30 min, followed by diaminobenzidine (DAB) as the substrate (Vector Laboratories). After staining, the sections were counterstained with Harris hematoxylin (cat. #HHS35-1L; Sigma) for few seconds. The sections were then dehydrated rapidly through ethanol and xylene, and mounted with VectaMount medium (Vector). Images were captured with Zeiss microscope integrated with Axio Vision software. For immunofluorescence staining, frozen sections of mouse brain were air dried for 20 min prior to acetone fixation at -20 C for 5 min and washed immediately twice in PBS with 0.05% Tween-20. The sections were then blocked with 5% goat serum in PBS with 0.1% Tween-20 for 1 hr at room temperature. The section were first labeled with rat anti-platelet endothelial cell adhesion molecule