Animals
This study was approved by the Institutional Animal Care and Use Committee at Loma Linda University. All procedures were carried out in compliance with the guidelines for Animal Experimentation of Loma Linda University. A total of 174 male CD1 mice (8-week-old, weight: 30-35 g; Charles River, Wilmington, MA, USA) were used. The animals were housed in a temperature-controlled environment with a 12 h light/dark cycle.
Experimental protocol
In the present study, all mice were randomly assigned to the following four separate experiments which are shown in the timeline of experimental design. The experimental groups and number of animals used in experiments are listed in Figure 1.
Experiment 1
To evaluate the time course expression of endogenous DKK3 Kremen-1, DVL-1 and phosphorylated-JNK (p-JNK) in ICH mice, 36 animals were randomly divided into 6 groups for western-blot: sham and 3, 6, 12, 24, and 72 h after ICH.
Experiment 2
To assess the short and long-term neurobehavioral function, 48 mice were divided into 8 groups: sham, ICH 24 h, ICH 24 h + rDKK3 0.5 μg, ICH 24 h + rDKK3 1.5 μg, ICH 24 h + rDKK3 5 μg, sham 72 h, ICH 72 h, ICH 72 h + rDKK3 best dosage. The brain edema and neurobehavior tests were evaluated at 24 and 72 h post-ICH. The Morris water maze test was conducted on day 28 post-ICH.
Experiment 3
To determine the DKK3 intracellular localization in ICH mice, 36 animals were randomly divided into 3 groups: sham, ICH, ICH + rDKK3. The cellular localization of DKK3 was evaluated using double-labeling immunofluorescence labeling to co-localize DKK3 with ionized calcium-binding adaptor molecule 1 (Iba-1), neuronal specific nuclear protein (Neun), and Kremen-1.
Experiment 4
To explore the underlying mechanisms of DKK3-mediated anti-inflammatory effects in ICH mice, Kremen-1 siRNA and DVL-1 siRNA were administered by intracerebroventricular (ICV) injection at 24 h before ICH induction. 36 mice were randomly divided into 6 groups: sham, ICH, ICH + rDKK3, ICH + rDKK3 + scramble siRNA, ICH + rDKK3 + Krenam-1 siRNA, ICH + rDKK3 + DVL siRNA. Neurobehavioral tests and western blot analysis were performed at 24 h after ICH.
Intracerebral hemorrhage induction
ICH was induced via injection of collagenase into right basal ganglia as previously described (27). After induction of anesthesia with ketamine (100 mg/kg) and xylazine (10 mg/kg), animals were positioned prone in a stereotaxic frame (David Kopf Instruments, Tujunga, CA, USA). An electronic thermostat-controlled warming blanket was used to maintain the core temperature at 37 °C. ± 0.5°C. The collagenase (0.075 units in 0.5 µl saline, VII-S; Sigma, MO, USA) was injected into the right basal Ganglia. A 26-G needle was inserted with stereotaxic guidance coordinates 0.2 mm anterior, 3.5 mm ventral, and 2.2 mm lateral to the bregma at a rate of 0.1667 μl/min by a micro infusion pump (Harvard Apparatus Inc., South Natick, MA, USA). Following the infusion, the needle was left in position for an additional 10 minutes after injection to prevent the leakage of collagenase, slowly retracted, and the incision was sutured. The sham group operation received only needle insertion.
Assessment of neurological deficits
Neurological functions including modified Garcia test, wire hanging, beam balance and limb placement were evaluated in a blinded fashion (28). The independent researcher blinded to the experimental design at 24 h, 72 h and 28 d after ICH. The Garcia test includes the evaluation of spontaneous activity, axial sensation, vibrissae proprioception, symmetry of limb movement, lateral turning, forelimb walking, and climbing. In the long-term neurological study, we utilized the Morris water maze to test spatial learning capacity and memory function recovery according to previous studies (3, 27). Starting from a semi-random location, each mouse is permitted to search for a partially submerged platform for 60 seconds. Next, the mice were guided to the platform and allowed to remain for 5 seconds. The probe trial was carried out on the last day of the exam. Following learning trial, the platform was removed. The swimming path, frequency of platform crossings, latency of first platform crossing, and frequency of correct quadrant crossings were recorded with a camera and linked to a computer tracking system (Noldus Ethovision, WA, USA).
Evaluation of brain water content
Mice were euthanized at 24 h or 72 h post-ICH. The brains were immediately removed and dissected into 5 parts: ipsilateral and contralateral basal ganglia and cortex. The cerebellum was employed as the internal control. Each part was weighed on an electronic analytical balance (APX-60, Denver Instrument, NY, USA) giving the wet weight (WW) and then dried at 100 °C for 24 h to determine the dry weight (DW). The brain water content was calculated as [(WW–DW) / WW] × 100%.
Drug and siRNA Administration
Three different formats of Kremen-1 siRNA or DVL-1 siRNA (OriGene Technologies, MD, USA) were diluted with transfection reagent (EntranserTM, Engreen Biosystem). ICV administration was performed at 24 h before ICH as previously described (29-31). The Kremen-1 siRNA, DVL-1 siRNA, and scramble siRNA mixture (100 pmol in 2 μL) was delivered into the ipsilateral ventricle, administration at a rate of 0.667 μl/min. rDKK3 (SRP6268, Sigma-Aldrich, MO, USA) was dissolved in 10 μl of saline and three different doses (0.5 μg, 1.5 μg, and 5.0 μg per mouse) were designed. rDKK3 was administered via intranasal route at 1 hr post-ICH.
Western blot analysis
Mice hemispheres were isolated and stored at -80 °C until protein extraction. The ipsilateral brain hemispheres were homogenized in RIPA lysis buffer (sc-24948, Santa Cruz, TX, USA) and then centrifuged (14,000 g at 4 °C for 30 min). Equal amounts of protein (50 μg) were loaded and subjected to electrophoresis on an SDS-PAGE gel. After being transferred to a nitrocellulose membrane, they are blocked with 5% nonfat milk (Bio-Rad Laboratories, Irvine, CA, USA). The membrane was incubated with the primary antibody overnight at 4 °C. The primary antibodies were used as follows: anti-DKK3 (1:1000, ab186409, Abcam, MA, USA), anti-Kremen-1 (1:500, ab86636, Abcam, MA, USA), anti-DVL-1 (1:1000, ab174679, Abcam, MA, USA), anti-AP-1 (1:200, NBP1-89544, Novusbio, CO, USA), anti-caspase-1 (1:1000, NBP1-45433Novusbio, CO, USA), anti-IL-1β (1:500, sc-7884, Santa Cruz, TX, USA)and anti-p-c-Jun N-terminal kinase (p-JNK) (1:500, ab131499, Abcam, MA, USA). The blot bands were quantified using ImageJ (NIH). The results were expressed as ratio of the target band intensity to the band intensity of β-actin (1:1000, sc-58673, Santa Cruz, TX, USA) and then normalized to the mean sham group ratio.
Immunofluorescence staining
Mice were perfused under deep anesthesia with isoflurane, followed by infusion of 4% paraformaldehyde. The brains were then removed and fixed in formalin at 4 °C overnight followed by dehydration with 30% sucrose in PBS. The frozen coronal slices (10 mm thick) were sectioned in cryostat (CM3050S; Leica Microsystems, Bannockburn). Brain slice were hydrated and blocked with 5% normal goat serum. Sections were incubated overnight at 4°C with the following primary antibodies: anti-DKK3 (1:100, ab186409, Abcam, MA, USA), anti-Kremen-1 (1:100, ab86636, Abcam, MA, USA). Then they were incubated by appropriate fluorescence-conjugated secondary antibodies (1:100, AB2337972, AB2338059, AB2340432 or AB233887, Jackson ImmunoResearch Laboratories, PA, USA) at room temperature for 2 h. Sections were observed using an OLYMPUS BX51 microscope.
Statistical Analysis
All values are presented in the text as mean ± Standard Deviation (SD). Western blot data were analyzed using one-way ANOVA with Tukey post hoc tests. Behavior data were analyzed using one-way ANOVA on ranks with Tukey post hoc tests or repeated measures ANOVA when appropriate. All histological data were analyzed using one-way ANOVA with Student-Newman's post hoc tests. Statistical significance implies p < 0.05.