Role of flunarizine hydrochloride in secondary brain injury following intracerebral hemorrhage in rats.

This study aimed to explore the role and mechanism(s) of flunarizine hydrochloride in the intracerebral hemorrhage (ICH) rats. The 32 adult male Sprague Dawley (SD) rats were randomly assigned into four groups: control group, sham group, ICH group, and FLU + ICH group. The effects of flunarizine hydrochloride were assessed on the basis of hematoma volume, blood-brain barrier (BBB) integrity, and brain water content in the ICH rat models. The role of flunarizine hydrochloride in cell recovery was assessed by behavioral scores, quantitative real-time polymerase chain reaction (qRT-PCR), and western blot assay. Involvement of PI3K/AKT pathway in exerting the effect of flunarizine hydrochloride was also determined. Results showed that the hematoma volume, BBB integrity, and brain water content were significantly decreased in the FLU + ICH group. Cell apoptosis significantly increased in the ICH model group, while flunarizine hydrochloride decreased this increase. The expressions of glial cell line-derived neurotrophic factor (GDNF), neuroglobin (NGB), and p-AKT were increased after flunarizine hydrochloride treatment in ICH rats. In conclusion, flunarizine hydrochloride has protective effects against ICH by reducing brain injury, cell apoptosis, and the activation of P13K/AKT pathway. These findings provide a theoretical basis for the treatment of flunarizine hydrochloride in ICH.

Introduction

Intracerebral hemorrhage (ICH) is a life-threatening disease, mainly caused by head trauma. It is reported that its incidence is twice as much as subarachnoid hemorrhage (SAH) and is believed to be cause severe disabilities.[1] Currently, there is no effective therapy to prolong the survival rate of ICH patients and improve the quality of life of survivors.[2] Therefore, it is necessary to improve or update the newer therapies of ICH.

Flunarizine hydrochloride is a calcium-ion blocker, which can be resistant to oxidation, anti-infarction, and regulate neurotransmitter and calcium channels.[3] Gulati and his colleagues reported that flunarizine significantly reduced ischemic reperfusion-associated cognitive dysfunction in aged mice.[3] Furthermore, flunarizine also showed a marked vasodilatory effect and prevented vasospasm in the rabbit models of SAH.[4] Although flunarizine has a potential role in experimental animals and in vitro cell culture models, more studies are required to evaluate the clinical effects of flunarizine in ICH management.

This study aimed to explore the effect and mechanism(s) of flunarizine hydrochloride on secondary brain injury in ICH rat models. The animal models were designed to mimic the clinical efficacy of ICH.[5] The initial setback was induced by the hematoma expansion (HE) and intracerebral bleeding.[6,7] The later phases included the infiltration of systemic immunological cells into the brain leading to loss of blood–brain barrier (BBB) integrity and enhancement of brain edema (BE).[8] This study assessed the neuro-protective effects of flunarizine hydrochloride on HE, BBB integrity, BE, and established rat models based on ICH. Furthermore, we also investigated the underlying mechanism(s) of flunarizine hydrochloride, and the PI3K/AKT pathway might be involved in reducing neuronal apoptosis.

Materials and methods

Animals and grouping

A total of 32 adult male Sprague Dawley (SD) rats weighing from 320 to 350 g were used in this study. After 3 days of adaptive feeding, the SD rats were randomly divided into four groups as follows: control group (n = 8), sham group (n = 8), ICH group (n = 8), and flunarizine hydrochloride (FLU) + ICH group (n = 8). Animals were housed under a 12 h light/dark cycle with free access to food and water. All experimental protocols were approved by the Animal Care and Use Committee of the Shanxi Academy of Medical Science, Shanxi Dayi Hospital (approval no. 101-34), in accordance with guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory.

Behavioral tests

The neurological severity score (NSS) was evaluated at 3, 6, and 12 h after reperfusion using Zea Longa 5-grade scale. A score of 0 indicates no neurological deficit; a score of 1 indicates a mild focal neurologic deficit demonstrated by failure to extend forepaw completely; a score of 2 suggests a moderate focal neurologic deficit demonstrated by circling movement to the left; and scores of 3 and 4 indicate severe focal deficits demonstrated by falling to the left and no spontaneous walk, respectively. The behavioral assessments were performed by investigators unaware of the treatment subjected to the animals.

ICH model preparation

Three days before the surgery, the rats were injected intraperitoneally with 10 mL/kg of flunarizine hydrochloride once daily in the FLU + ICH group. Then, the rats in the other groups were fed normally. All rats were anesthetized by intraperitoneal injection of 10 mL/kg 3.6% chloral hydrate and fixed on a stereotaxic apparatus for further processes. The anterior fontanelle of the rat was exposed by about 10 mm incision along the scalp midline. A small hole (0.5 mm) was drilled at a 3 mm distance on the left side of the midline by a micro-hand drill. In the ICH group and FLU + ICH group, about 50 μL of autologous blood from the tail was injected into the hole at a constant rate of 20 μL per min. At the meanwhile, the control group and the sham group did not inject blood. Furthermore, normal saline (15 μL) containing 10 U hirudin was injected into the hematoma in the sham group, ICH group, and FLU + ICH group, whereas 15 μL of normal saline was used in the control group. Then, the rats were decapitated and their brains were harvested at 24 h in each of the groups, but in the sham group, the brains were harvested at 2 h after injection.

In addition, the samples from the FLU + ICH group were divided into two groups. In order to demonstrate the role of the PI3K/AKT pathway in the neuro-protective effect of flunarizine hydrochloride, LY294002, a specific inhibitor of PI3K, was randomly added to selected group and cultured at 37°C for 36 h. Then, the samples were used for the subsequent experiment.

Measurements of hematoma and hemorrhagic volumes

The hematoma volume was assessed by morphometric measurement on day 1 post-ICH.[9,10] The harvested rat brains were sliced serially at coronal plane into 2-mm-thick slices. The images of the slices were captured by a digital camera and quantified by Image J. The hematoma volume of each slice (with blood clot) was calculated by multiplying the section thickness (2 mm) and the blood clot area (mm2).

BBB disruption

A modified Evans blue extravasation method was used to evaluate BBB integrity in the ICH rat model.[11] Briefly, a pre-prepared warm 2% Evans blue dye was infused into femoral vein after 22 h post-ICH. After 2 h, the rats were perfused with normal saline to wash out residual dye in the blood vessels. The infiltrated Evans blue dye in the right and left hemispheres was quantified with a spectrophotometer at an absorbance of 610 nm, respectively. And the dye in the cerebellum was used as an internal control. The content of the dye was calibrated with a standard curve of known dye.

BE assay

A common wet/dry method was used to evaluate BE in this study. The contralateral and ipsilateral brain hemispheres were immediately weighed to obtain the wet weight and dried in a 100°C oven for 24 h to obtain dry weight on day 1 post-ICH. The water content was calculated by the following formula: water content (wet weight %) = [(wet weight) − (dry weight)]/ (wet weight) × 100%. Furthermore, the increase in ipsilateral water content (Δ) was expressed as follows: (% of ipsilateral water content) − (% of contralateral water content).

Cell apoptosis

TUNEL staining was performed following the manufacturer’s protocol using a TUNEL staining kit. The apoptotic index was expressed as the average percentage of TUNEL positive cells in each of 10 fields and was used to evaluate the extent of brain impairment.

Quantitative real-time polymerase chain reaction

TRIzol reagent was used for the total RNA isolation. The RNA PCR Kit (AMV) Version 3.0 was used to test the expression level of messenger RNA (mRNA). The ABI 7500 Real-Time PCR system was used to measure the mRNA expression of GADPH, which is as the internal control. The PCR conditions used were as follows: 95°C, 10 min (1 cycle), then 95°C, 15 s, 60°C, 1 min (40 cycles).

Western blot assay

The frozen samples were homogenized using the Extract buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 1 mM NaF, 2 mM Na3VO4, and 1 mM phenylmethanesulfonyl fluoride (PMSF)). Each protein sample was quantified by DC protein assay and loaded on each lane equally on an SDS polyacrylamide (SDS-PAGE) gel. Then, the gel was transferred to a polyvinylidene difluoride (PVDF) membrane, which was blocked with 5% non-fat milk for 2 h at room temperature and incubated overnight at 4°C with primary antibodies. The membrane was then washed and incubated with goat anti-rabbit antibody (1:5000) for 2 h at room temperature, and washed again. The protein bands were visualized using enhanced chemiluminescence (ECL) western blot detection reagents and exposed to X-ray film.

Statistical analysis

All experiments were repeated three times. The data analysis results from multiple experiments were presented as mean ± standard deviation (SD) in the study. One-way analysis of variance (ANOVA) was used to compare the statistical differences between the control and the treatment groups. The significance of differences between the groups was determined by Bonferroni analyses. All the statistical analysis was performed using SPSS 17.0. A P-value of less than 0.05 was considered to indicate a statistically significance result.

Results

Effects of flunarizine hydrochloride on ICH

In order to explore the effects of flunarizine hydrochloride on ICH, the rats were assigned into four groups as described. The four groups were subjected to receive different treatments. The results in Figure 1(a)–(d) showed that the hematoma volume, BBB integrity, and brain water content were significantly increased in the ICH group than the sham group (P < 0.01 or P < 0.001). Then, flunarizine hydrochloride decreased the increase of these three indicators in the FLU + ICH group (P < 0.05).

Figure 1.

Effects of flunarizine hydrochloride on ICH. (a, b) Effect of flunarizine hydrochloride on hematoma volume, (c) blood–brain barrier (BBB) integrity, and (d) brain water content were detected. Data were represented as the mean ± standard deviation (SD) of three independent experiments.

*P < 0.05; **P < 0.01; ***P < 0.001.

Effects of flunarizine hydrochloride on cell apoptosis

As shown in Figure 2(a), the apoptotic cells were not detected in control group, but their number was significantly enhanced (P < 0.01) in the ICH group than the sham group. Then, flunarizine hydrochloride significantly decreased the cell apoptosis of ICH rats (P < 0.05). We further found that the mRNA levels of B-cell leukemia-2 associated X protein (Bax) and B-cell leukemia-2 (Bcl-2) significantly increased and reduced in the ICH cells, respectively (P < 0.001, Figure 2(b)–(d)). Moreover, flunarizine hydrochloride decreased the expression of Bax and increased Bcl-2 expression (P < 0.05 or P < 0.01, Figure 2(b)–(d)). The results indicated that flunarizine hydrochloride could protect against cerebral hemorrhage by reducing neuronal apoptosis.

Figure 2.

Effects of flunarizine hydrochloride on cell apoptosis. (a) Percentage of TUNEL positive neurons, (b) relative Bax mRNA expression level, and (c) relative Bcl-2 mRNA expression level were detected in ICH rat models, respectively. (d) Expression of Bax and Bcl-2 in the ICH rat models was measured using western blot assay. Data were represented as mean ± standard deviation (SD) of three independent experiments.

Bax: B-cell leukemia-2 associated X protein; Bcl-2: B-cell leukemia-2.

*P < 0.05; **P < 0.01; ***P < 0.001.

Effects of flunarizine hydrochloride on cell recovery

As shown in Figure 3(a), the behavioral scores were significantly enhanced in ICH group (P < 0.001) than the sham group, while this increase was decreased by the flunarizine hydrochloride intervention. In addition, we found that the mRNA and protein levels of glial cell line-derived neurotrophic factor (GDNF) and neuroglobin (NGB) clearly increased in the FLU + ICH group compared with the ICH group (Figure 3(b)–(d)). The results indicated that flunarizine hydrochloride could protect against cerebral hemorrhage by promoting cell recovery.

Figure 3.

Effects of flunarizine hydrochloride on cell recovery. (a) Behavioral score, (b) relative GDNF mRNA expression level, and (c) relative NGB expression level in the ICH rat models were detected, respectively. (d) Expression of GDNF and NGB in the ICH rat models was measured using western blot assay. Data were represented as the mean ± standard deviation (SD) of three independent experiments.

GDNF: glial cell line-derived neurotrophic factor; NGB: neuroglobin.

*P < 0.05; **P < 0.01; ***P < 0.001.

Effects of flunarizine hydrochloride on P13K/AKT pathway

As shown in Figure 4(a), the expression of p-AKT significantly increased in the FLU + ICH group than the ICH group. Furthermore, p-AKT expression decreased in FLU + ICH + LY294002 group (Figure 4(b)). The results demonstrated that LY294002 could inhibit the neuro-protective affects of flunarizine hydrochloride partially, indicating a possible involvement of PI3K/AKT pathway in the mode of action of flunarizine hydrochloride against ICH.

Figure 4.

Effects of flunarizine hydrochloride on AKT pathway. (a) Expression of p-AKT, (b) including inhibitor LY294002, in the ICH rat models was detected using western blot assay.

Discussion

In this study, we explored the effect of flunarizine hydrochloride on the secondary brain injury in ICH rat models. We found that flunarizine hydrochloride significantly decreased the increase in hematoma volume, BBB integrity, brain water content, cell apoptosis, and behavioral scores caused by ICH.

Due to HE, loss of nerve tissue, and other rapid development of the etiology, ICH is difficult to effectively treat, and even leads to nerve inflammation or death. The related study has showed that flunarizine hydrochloride could block calcium channels to prevent Ca2+ from into the cells to shutdown ICH level in rats.[12] Moreover, our study confirmed that flunarizine hydrochloride could reduce the neuronal damage in ICH rats.

ICH therapy could lead to changes in the expression of apoptosis-related genes (Bcl-2 and Bax) in the brain tissues. Then, some studies reported that flunarizine hydrochloride may promote neural regeneration of the disease model and help the cells recover, but also cause up-regulated GDNF expression.[13] In addition, it also could be used to regulate PI3K/AKT pathway to relieve cerebral hemorrhage.[14] According to the results of this study, we found that the findings were consistent with previous studies. The expression of Bcl-2, GDNF, NGB, and p-AKT was up-regulated after flunarizine hydrochloride treatment in ICH rats.

Furthermore, we used LY294002 to verify the protective effects of flunarizine hydrochloride. The results suggested that flunarizine hydrochloride could attenuate apoptosis by modulating P13K/AKT pathway in ICH rats.

In conclusion, flunarizine hydrochloride has protective effects against ICH by reducing hematoma volume, BBB integrity, brain water content, and activation of P13K/AKT pathway. These findings provide a theoretical basis for the treatment of flunarizine hydrochloride. More researches are required to determine the long-term effects of flunarizine hydrochloride on ICH-induced neuronal disorders in the future.