Hydrochloride fasudil attenuates brain injury in ICH rats

2.1 Animals and treatment groups

Adult male Wistar rats (250 ± 20 g) were purchased from the Experimental Animal Center of the First People’s Hospital Affiliated to Shanghai Jiao Tong University (Shanghai, China). One hundred seventeen rats were randomly divided into four groups: normal group (n = 9), sham-operated group (n = 36), ICH group (n = 36), and ICH/HF group (n = 36). The rats were fasted for 12 h before the experiments but had free access to water.

1. Ethical approval: The research related to animal use has been complied with all the relevant national regulations and institutional policies for the care and use of animals.

2.2 Administration of saline and HF

Rats were used in this study, and they were divided into groups receiving intraperitoneal injection of either saline or HF (12 mg/Kg). Then, the intravenous injection was changed to intraperitoneal injection, which was 11.15–11.81 mg/kg/d (the ratio of intraperitoneal injection to intravenous dose was 1.18–1.25). HF (MedChemExpress, Newark, NJ, USA) was dissolved in 2 mL of 0.9% saline solution and administered intraperitoneally once a day (12 mg/kg/d), starting 12 h after the onset of ICH. Rats in the sham-operated and ICH groups received an equal volume of normal saline.

2.3 ICH model

In rats, ICH was induced by injection of non-anticoagulant autologous arterial blood into the right caudate nucleus, as previously described [14]. Briefly, the rats were anesthetized by intraperitoneal injection of 10% chloral hydrate (0.4 mL/100 g) and placed in a brain stereotactic apparatus (Huaibei Zhenghua Biological Instrument Equipment Co., Ltd, Huaibei, China). The right caudate nucleus (0.2 mm anterior to coronal, 3.0 mm lateral to the bregma, and 6.0 mm ventral) was positioned in accordance with the “Stereotaxic Atlas of Rat Brain” [15]. Non-anticoagulant autologous blood (100 μL) was collected from the right femoral artery of the rats using a microsyringe and immediately injected into the right caudate nucleus at a rate of 10 μL/min. The needle was left in place for 15–20 min and then slowly removed. The skull was sealed using medical bone wax to prevent blood reflux, and the incision was sutured. The sham-operated rats underwent the same surgical procedure, except that no blood was injected.

When the rats had recovered from the anesthetic, the behavioral abnormalities were assessed by a blinded observer using Zea Longa scores [16] (0: rat has no neurological deficit; 1: rat is unable to completely extend contralateral forelimb; 2: rat circles to left when walking; 3: rat tumbles to left when walking; and 4: rat is unconscious and unable to walk). Scores of 1–3 were used for modeling.

2.4 Serum interleukin-6 and tumor necrosis factor-α levels

On days 0, 1, 3, 7, and 14, the rats were anesthetized by intraperitoneal injection of 10% chloral hydrate (0.4 mL/100 g). After appropriate skin preparation and sterilization, the chest was opened and blood was collected from the right atrium. Serum was isolated using standard procedures, and the levels of interleukin (IL)-6 and tumor necrosis factor (TNF)-α were determined using a commercial enzyme-linked immunosorbent assay (ELISA) kit (Affymetrix Inc., Santa Clara, CA, USA), according to the manufacturer’s protocol.

2.5 Quantitative real-time polymerase chain reaction

The levels of ROCK2 mRNA were measured using quantitative real-time polymerase chain reaction (qRT-PCR) on days 0, 1, 3, 7, and 14 after induction of ICH. Total RNA was isolated from the homogenates of 100 mg tissue surrounding the hematoma (not including hematomas) using a total RNA extraction kit (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. First-strand cDNA was synthesized from RNA using a first-strand cDNA synthesis kit (Thermo Scientific, Foster City, CA, USA). PCR was carried out using Maxima SYBR Green qPCR Master Mix (Thermo Scientific, Waltham, MA, USA). The primers (ROCK2, 5ʹ-AGATGTGAAGCCCGATAA-3ʹ [forward primer] and 5ʹ-ACACCTACAGACCACCAAT-3ʹ [reverse primer]; glyceraldehyde 3-phosphate dehydrogenase [GAPDH], 5ʹ-ATGATTCTACCCACGGCAAG-3ʹ [forward primer] and 5ʹ-CTGGAAGATGGTGATGGGTT-3ʹ [reverse primer]) were purchased from the Beijing Genomics Institute (Beijing, China). ROCK2 mRNA levels were compared with those of control rats (value in control rats set as 1.0).

2.6 ROCK2 protein expression in the perihematomal region

Brain tissues around the lesion sites in right caudate nucleus of rats were removed and homogenized with 1 mL animal tissue active protein extraction kit (Shenggong, Shanghai, China). Total protein was determined using a bicinchoninic acid protein assay kit (Beyotime, Shanghai, China) and ROCK2 levels were determined using a commercially available ELISA kit (LifeSpan BioSciences, Inc., Seattle, Washington, USA). In each case, the assay kit was used according to the manufacturer’s protocol. ROCK2 protein levels were calculated as a percentage of total protein.

2.7 Assessment of cerebral edema

Brain water content was used to evaluate cerebral edema on days 0, 1, 3, 7, and 14. The rats were killed by decapitation, and their brains were excised. A coronal incision was made to evaluate hematoma formation, and two pieces of tissues (∼2 mm²) were removed from the area around the hematoma. The tissues were weighed immediately to obtain wet weights and again after drying at 100°C for 24 h to obtain dry weights. The brain water content (%) in the perihematomal areas was calculated as follows:

2.8 Hematoxylin and eosin staining

The rats were exsanguinated via the right atrium and perfused with saline (200 mL) and then with 4% paraformaldehyde (200 mL) to fix the brain tissues in vivo. The rats were decapitated, and the brain tissues were fixed by plunging into 10% formaldehyde solution. The tissues were then dehydrated and embedded in paraffin and the sections (4 μm) were prepared for hematoxylin and eosin (H&E) staining. Hematoma was observed in the basal ganglia on days 1, 3, 7, and 14 after the onset of ICH. Inflammatory infiltration into perihematomal tissue was observed using a light microscope, and the data were analyzed using Motic Digital Medical Image software (Motic China Group Co., Ltd, Xiamen, China). Neutrophils were counted in four independent fields of view (×400).

2.9 Nissl staining

For Nissl staining, the brain tissues were fixed in 10% formaldehyde solution, dehydrated, embedded in paraffin using standard procedures, and cut into 4 μm sections using a rotary microtome (Leica, Solms, Hessen, Germany). The paraffin sections were dewaxed and rehydrated, stained using toluidine blue solution (0.5%), differentiated in 95% alcohol, dried, and cleared using xylene. Morphological changes in the neurons were observed using a light microscope, and the data were analyzed using Motic Digital Medical Image software. Surviving neurons were counted in four independent fields of view (×400).

2.10 Statistical analysis

Statistical analysis was performed using SPSS statistical software 20.0 (SPSS Inc., Chicago, IL, USA). All data were expressed as mean ± SD. If the comparison between two independent samples meets the normal distribution, the independent sample t test is used, otherwise the nonparametric Mann–Whitney U test is used. The data were analyzed by one-way analysis of variance if the comparison between three or more samples conforms to normal distribution and has homogeneous variance; the pairwise comparison was performed using the least significant difference (LSD) method. Otherwise the nonparametric Kruskal–Wallis H test was used. P values <0.05 were considered to be statistically significant.

3.1 Animal survival rate

The survival rate of the 117 rats was 83.76% (98/117), including death after anesthesia (four rats in the ICH group and three rats in the sham-operated group); after modeling, eight rats died before the corresponding time point (three rats died on day 1 and five rats died on day 3); the death time of the experimental animals was usually 1–3 days after the model was prepared, and the death rate was as high as 22%. The rats with Zea Longa score 0 (four rats in the ICH group and one rat in the ICH/HF group) and Zea Longa score 4 (five rats in the ICH group and five rats in the ICH/HF group) were excluded. The success rate of the ICH model was 79.17% (57/72).

3.2 Assessment of the ICH model

The ICH model was assessed using Zea Longa scores. A score of 1 indicates that the rat was unable to completely extend its left forelimb (Figure 1A), a score of 2 indicates that the rat circled to the left when walking (Figure 1B), and a score of 3 indicates that the rat fell to the left (Figure 1C). A gross brain specimen of a rat following ICH is shown in Figure 1D. Hematoma was present following ICH (Figure 1E) but not in the sham-operated rats (Figure 1F).

Figure 1

Scoring of intracerebral hemorrhage model and hematoma formation. (A–C) Rats with Zea Longa scores of 1, 2, and 3, respectively; (D) gross brain specimen after autologous blood infusion; (E) hematoma following ICH; and (F) no hematoma in the sham-operated rats.

3.3 HF treatment reduces serum IL-6 and TNF-α levels in a time-dependent manner

Serum IL-6 and TNF-α levels were determined by ELISA on days 0, 1, 3, 7, and 14 following ICH. Compared with the normal and sham-operated groups, rats in the ICH group exhibited significantly higher levels of IL-6 and TNF-α (Figure 2A and B, n = 6, P < 0.05). Treatment with HF significantly decreased the expression of IL-6 and TNF-α at the corresponding time points (Figure 2A and B, n = 6, P < 0.05). IL-6 and TNF-α levels in the ICH and ICH/HF groups peaked 3 days after the onset of ICH (P < 0.05). IL-6 levels in the ICH group at 14 days were not significantly different from those in the ICH/HF and sham-operated groups (P > 0.05).

Figure 2

Brain water content and expression of inflammatory factors and ROCK2 on days 1, 3, 7, and 14. (A and B) Levels of serum IL-6 and TNF-α (measured using ELISA, n = 6); (C) levels of ROCK2 mRNA (measured using qRT-PCR, n = 5); (D) levels of ROCK2 protein in perihematomal area (measured using ELISA, n = 5); (E) brain water content (assessed using wet/dry weight method, n = 5); data are presented as mean ± SD; **P < 0.05 compared with the normal and sham-operated groups; ^##P < 0.05 compared with the ICH group.

3.4 HF treatment inhibits the expression of ROCK2 at different time points

qRT-PCR and ELISA were used to measure the expression of ROCK2 mRNA and protein, respectively, in the perihematomal area. Compared with the normal and sham-operated groups, the levels of ROCK2 mRNA in the ICH group were significantly increased on days 1, 3, and 7 following ICH, peaking on day 3 (Figure 2C, n = 5, P < 0.05). The expression of ROCK2 mRNA in the ICH/HF group was significantly lower at the corresponding time points (Figure 2C, n = 5, P < 0.05). The expression of ROCK2 protein essentially aligned with the mRNA expression (Figure 2C and D), except that the expression of ROCK2 protein in the ICH/HF group is not significantly lower than that in the ICH group on days 1 and 7 (Figure 2D, P > 0.05).

3.5 HF treatment attenuates brain edema from day 1 to day 14 following ICH

Compared with the normal and sham-operated groups, the brain water content was significantly higher in the ICH group at all time points (Figure 2E, n = 5, P < 0.05). The brain water content in the ICH/HF group was significantly lower than that in the ICH group (Figure 2E, n = 5, P < 0.05). The brain water content in the ICH group began to increase on day 1 and reached a peak on day 3 (Figure 2E, n = 5, P < 0.05), suggesting a time-dependent increase in the brain water content following ICH.

3.6 HF treatment reduces neutrophil infiltration into the perihematomal areas following ICH

H&E staining showed no hematoma formation in rats in the normal (Figure 3A) and sham-operated (Figure 3B) groups, indicating no brain injury. Rats in the ICH and ICH/HF groups, however, showed hematomas at all time points following ICH (Figure 3C–J). H&E staining also revealed inflammatory infiltration around the hematoma in the ICH and ICH/HF groups (Figure 4). At early time points (days 1 and 3), the predominant inflammatory cells in the ICH group were neutrophils, and the number of neutrophils was significantly higher on day 1 than on day 3 (Figure 4, n = 4, P < 0.05). In comparison with the ICH group, the ICH/HF group showed markedly fewer neutrophils on day 1 after ICH (Figure 4, n = 4, P < 0.05). A small number of lymphocytes and macrophages were present in the perihematomal area on days 1 and 3.

Figure 3

H&E staining showing hematoma formation (×40). (A) Normal and (B) sham-operated groups had normal brain tissue in the basal ganglia; (C–J) ICH and ICH/HF groups had hematomas at each time point.

Figure 4

H&E staining of brain sections in the ICH and ICH/HF groups on days 1, 3, 7, and 14 after ICH (×400). Arrows indicate neutrophils (N), lymphocytes (L), macrophages (M), microglia (Mi), neuronal cells (Nc), and erythrocytes (E). In the early stages of ICH (days 1 and 3), a large number of neutrophils were observed in the perihematomal area, with fewer lymphocytes and macrophages. In the later stages of ICH (days 7 and 14), neuronal cells and hemosiderin were present. ^#P < 0.05 compared with day 1; *P < 0.05 compared with the ICH group (n = 4).

3.7 HF treatment increases neuronal survival following ICH

An assessment of morphological changes in neurons in the perihematomal area using Nissl staining indicated that neurons of rats in the normal (Figure 5A) and sham-operated (Figure 5B) groups were normal, without any brain damage or change in the neuronal morphology. The injured neurons in the perihematomal area during days 1–14 following ICH in rats were characterized by nuclear pyknosis, nuclear fragmentation, decreased or disappeared Nissl bodies, and cyton swelling, or Nissl staining became shallow and unclear. Nissl staining showed more dead/dying neurons (Figure 5C–J). The quantitative results showed that administration of HF significantly increased the number of surviving neurons in the perihematomal area after ICH induction at all time points (Figure 5K, n = 4, P < 0.05).

Figure 5

Nissl staining of brain sections from rats in the normal, sham-operated, ICH, and ICH/HF groups (×400). (A) Normal and (B) sham-operated rats had normal neuronal cells (black arrows). (C–J) Damaged neurons (white arrows,) were present in injured brains after the onset of ICH. Damaged neurons were characterized by nuclear pyknosis, nuclear fragmentation, decreased or disappeared Nissl bodies, and cyton swelling, or Nissl staining became shallow and unclear. (K) Quantitative analysis of the number of surviving neurons from ICH and ICH/HF rat brains in the perihematomal area at each time point. Data were collected from four independent fields of view (×400) and are presented as mean ± SD; compared with the ICH group, the ICH/HF group had more surviving neurons at each time point (n = 4, ^##P < 0.05).

4.1 Limitations

This study has several limitations. First, in this study, non-anticoagulant autologous arterial blood was injected into the right caudate nucleus to prepare the ICH model, which may be different from the mechanism of spontaneous ICH. Second, the data for hemorrhage volumes at each time point and analyses of differences between groups were not provided in this study. Although the aim of this study was to evaluate the neuroprotective effects of HF in rats with ICH, the activity of Rho-kinase was not measured. Additional research may be required to define the role of Rho-kinase activation in ICH-induced secondary brain injury: for example, measurements of changes in the Rho-kinase activity in the perihematomal brain tissue and blood cells after ICH and determination of the inhibitory effect of HF on Rho-kinase activation. Moreover, we did not study the ICH-triggered apoptosis or neuron necrosis, which is also a limitation of this study. We will further explore this in the future research.