Animal preparation
This is a prospective, randomized experimental study. Male Sprague-Dawley rats weighing between 250 and 280 g were provided with food and water ad libitum and held at a temperature of 22℃ ± 1℃, under a 12-h light/dark cycle for 5 days prior to experiment. Transient global cerebral ischemia was induced via the four-vessel occlusion method described in our prior experiment [16, 17]. To inhibit secretion, atropine (0.01mg/kg) was administered intraperitoneally before surgery, followed by a combination of tiletamine hydrochloride along with zolazepam hydrochloride (Zoletil; Virbac, Carros, France) (30 mg/kg) and xylazine (Rompun; Bayer, Monheim, Germany) (10 mg/kg) 10 min later, intra-abdominally to induce anesthesia. Thereafter, tracheal intubation was performed to maintain airway, and the rats were fixed in a prone position in the stereotactic frame. A thermometer probe was inserted into the rectum of the fixed rats, and an automatic temperature control blanket (Homeothermic blanket system, NP50-7053-r; Harvard Apparatus, Holliston, MA) was used to maintain normothermia (37℃ ± 0.5℃) during surgery.
We shaved and sterilized the areas around the cervical regions, while the rat was fixed in the prone position and made a 3 cm median incision along the centerline from the border of the lower part of occipital bone to the back and cervical region. The muscles of the first cervical vertebra were dissected to expose the bilateral alar foramen. A thin needle-shaped electrocautery (SurgiStatTM II; Covidien, Boulder, CO) was inserted approximately 1 to 1.5 mm through the alar foramen, and the bilateral vertebral arteries were permanently occluded by cauterization. Next, the position was changed to supine, and the ventral cervical region was shaved and sterilized, a 3 cm median incision was made, and the bilateral common carotid arteries were dissected. Polyethylene tubes (PE-10; BD, Franklin Lakes, NJ) were loosely wrapped around the dissected common carotid arteries to allow both ends of the tubes to emerge approximately 3 cm from the skin, which was then sutured.
After the surgery, the rats were kept isolated in individual cages, and left to recover for 24 h under the same environment as before the surgery. On the following day, the rats were restrained without anesthesia, and the polyethylene tubes were pulled to expose the common artery outside the skin. The common carotid arteries were occluded with microvascular clamps (RS-5422; Roboz, Chicago, IL) for 10 min, and global cerebral ischemia was confirmed by the loss of the righting reflex. A thermometer probe was inserted into the rectum to monitor the core temperature and maintain normothermia using a temperature control blanket throughout the ischemic period. The clamps and polyethylene tubes of the rats that survived global cerebral ischemia were removed 10 min later, and normothermia was maintained until the recovery of the righting reflex. We excluded rats maintaining righting reflex during the ischemic period, which meant incomplete global cerebral ischemia. Also, we rejected rats without any available outcome variables because of death during the ischemic period or early death within 2 h post-ischemia (Fig.1).
Study Protocol
Surviving rats were randomly assigned to one of the two treatment groups: paricalcitol group (n = 8), injected intraperitoneally with paricalcitol (Zemplar; Abbott Laboratories, Abbott Park, IL) (1 μg/kg); and normal saline group (n = 8), injected intraperitoneally with an equivalent volume of normal saline. We administered drugs 5 min after the end of the ischemic period. After recovery of righting reflex, rats were returned to the cages and observed until four days after cerebral ischemia. During the observational period, we intraperitoneally injected paricalcitol (1 μg/kg) or an equivalent volume of normal saline on days 1, 2 and 3 post-ischemia.
Neurological outcome
A researcher who was blinded to the treatment measured and recorded the neurological function score (NFS) at 2 h post-ischemia, and then on days 1, 2, 3, and 4 post-ischemia as previously described [18]. The test consists of five categories representing the level of consciousness, respiration, cranial nerves, motor and sensory function, and coordination. The score ranges from 0 (worst) to 500 (normal) (Table1).
Table1. Neurological function scoring in rats .
Parameter
|
Characteristic
|
Score Range
|
General
|
|
|
Consciousness
|
Unresponsive, depressed, normal
|
0, 50, 100
|
Respiration
|
Abnormal, normal (60-120)
|
0, 100
|
Cranial Nerves
|
|
|
Olfactory
|
Orient to smell
|
No = 0, Yes = 20
|
Vision
|
Visual stimulus startle response
|
No = 0, Yes = 20
|
Corneal reflex
|
Blink response to corneal stimulus
|
No = 0, Yes = 20
|
Whisker movement
|
Spontaneous
|
No = 0, Yes = 20
|
Hearing
|
Startle response to loud noise
|
No = 0, Yes = 20
|
Motor
|
|
|
Left forepaw
|
Spontaneous or withdrawal from pain
|
No = 0, Yes = 10
|
Right forepaw
|
Spontaneous or withdrawal from pain
|
No = 0, Yes = 10
|
Left hindpaw
|
Spontaneous or withdrawal from pain
|
No = 0, Yes = 10
|
Right hindpaw
|
Spontaneous or withdrawal from pain
|
No = 0, Yes = 10
|
Tail
|
Spontaneous or withdrawal from pain
|
No = 0, Yes = 10
|
Sensory
|
|
|
Left forepaw
|
Reaction to pain
|
No = 0, Yes = 10
|
Right forepaw
|
Reaction to pain
|
No = 0, Yes = 10
|
Left hindpaw
|
Reaction to pain
|
No = 0, Yes = 10
|
Right hindpaw
|
Reaction to pain
|
No = 0, Yes = 10
|
Tail
|
Reaction to pain
|
No = 0, Yes = 10
|
Coordination
|
|
|
Ledge traverse
|
|
No = 0, Yes = 25
|
Righting reflex
|
|
No = 0, Yes = 25
|
Placing test
|
|
No = 0, Yes = 25
|
Stop to table edge
|
|
No = 0, Yes = 25
|
Total score
|
|
500
|
Rotarod test
We assessed motor coordination using the accelerating rotarod test (Model 7750; Ugo Basile, Comerio, Varese, Italy). Four training sessions were performed 5, 4, 3 days and 1 h prior to the ischemic insult. We placed the rats on the stationary rod. After a while, the rod started to rotate at 2 rpm and accelerated to 40 rpm within 300 s. We recorded the latency to fall from the rotating rod. Rats not falling off within 300 s were scored a maximum of 300. We obtained the baseline score by averaging the two best scores out of the four training sessions. We performed the rotarod test three days after the ischemic insult and determined the post-ischemic latency to fall. We calculated the value relative to baseline for use in data analysis.
Passive-avoidance test
We assessed memory function using passive-avoidance test. The passive-avoidance apparatus (Model 7552; Ugo Basile, Comerio, Varese, Italy) consisted of two sections, the start and escape compartments. The start compartment was illuminated and surrounded by white walls, while the escape compartment was dark, with black walls. The two compartments were connected by an automatic sliding door. Three days after the ischemic insult, the rats were exposed to the passive-avoidance apparatus 60 min before the acquisition trial. During the pre-exposure, the rats were allowed to explore the start compartment for 1 min, without access to the escape compartment. Thereafter, the sliding door was opened and as the rat entered the escape compartment, the door was automatically closed. The rats were then allowed to explore the escape compartment for an additional 1 min. One hour after pre-exposure, the rat was again placed in the start compartment for the acquisition trial, and 10 s later, the door was opened. The latency to step through the door was recorded as baseline retention latency. After entering the escape compartment, the door was closed and an electrical current (0.8 mA, 2 s) was delivered through the grid floor. The next day, we performed the retention trial. The rat was placed in the start compartment, and 10 s later, the door was opened. The retention latency to enter the escape compartment was recorded. No shock was delivered during the retention trial. If the rat failed to enter the escape compartment within 300 s, it was removed from the apparatus and a maximum latency of 300 s was recorded. The data was expressed relative to baseline retention latency and used for data analysis.
Histopathological analysis
After the acquisition trial, tiletamine hydrochloride + zolazepam hydrochloride (1:1 solution, 30 mg/kg) was injected into the abdominal cavity for anesthesia, and the rats were euthanized with 4% paraformaldehyde via transcardiac perfusion fixation. Brains were then post-fixed in 4% paraformaldehyde for more than a day, followed by washing under running water for another day. Finally, they were fixed in paraffin and two 4-μm-thick coronal sections of hippocampal cornu ammonis (CA) 1 region from each rat were obtained for hematoxylin-eosin staining. As each specimen section contained right and left CA1 regions, four histological images of a 1.13-mm-long stratum pyramidale were acquired using an optical microscope (IX71; Olympus, Tokyo, Japan). A blinded researcher calculated the percentage of degenerated pyramidal cells in each image using image analysis software (Image-Pro Premier; Media Cybernetics, Rockville, MD). The median value of 4 images was calculated for each rat.
Statistics
According to our pilot study, we hypothesized that the percentage of injured neurons in the paricalcitol group would be 9 ± 4.5%, while the percentage of injured neurons in the normal saline group would be 30 ± 15%. Assuming a two-sided α of 0.05 at a power of 0.8, the number of rats per group was determined as 8 to reject the null hypothesis. Continuous data were expressed as medians with interquartile ranges (IQR). We conducted Mann-Whitney tests to compare the data between the groups. Log-rank test was used to compare the survival distribution between groups. Survival was presented by Kaplan-Meier curves. Values of p less than 0.05 were considered significant.