Animal and experimental group

All experiments were approved by the Committee for Laboratory Animal Care and Use at Kumamoto University. Male Wistar rats weighing 293.3–346.7 g (9–10 weeks old) were used. All rats were group-housed with two to three animals per cage and reared in a temperature-controlled (20 ± 2 ℃) and humidity-controlled (50%–60%) room under a 12-h light/dark cycle (8:00/20:00). All rats were subjected to BICAO prior to the indirect bypass procedure and divided into the following three experimental groups:

(1)

Control group (BICAO without indirect bypass; n = 6).

(2)

EMS group (BICAO with indirect bypass using the temporalis muscle; n = 7).

(3)

Dura group (BICAO with indirect bypass using DuraGen; n = 6).

Bilateral internal carotid artery occlusion and indirect bypass

All surgical procedures were performed under anesthesia with 2% isoflurane. BICAO was performed as previously described [13]. Briefly, bilateral internal carotid arteries were exposed via a ventral midline cervical incision and double ligated with 4–0 silk sutures. After BICAO, the rats were placed on a stereotactic head holder with the top of the skull positioned horizontally. A parieto-occipital midline skin incision was made, and the galea was peeled off the skull. A 7-mm2 craniectomy was performed carefully by drilling behind the coronal suture, 1 mm right lateral to the midline (Fig. 1A and B), as described in a previous study [13]. The dura was removed using microsurgical forceps and a microscope to avoid brain injury. In the EMS group, the brain surface was covered by temporal muscles (Fig. 1C). In contrast, in the Dura group, DuraGen was secured to the surrounding craniotomy using 4–0 nylon sutures to cover the brain surface (Fig. 1D). In the control group, to prevent angiogenesis due to inflammatory mechanisms, craniectomy was not performed. All rats were observed for 6 weeks after BICAO, with or without indirect bypass, and were then sacrificed.

Fig. 1

Illustration of craniotomy (A), surgical image after craniotomy (B), indirect bypass with the temporalis muscle (C), and indirect bypass with DuraGen (D)

Histology

The rats were sacrificed 6 weeks after the indirect bypass, and brain tissues were collected as described previously [13]. Briefly, all rats were anesthetized with isoflurane. Subsequently, the rats were transcardially perfused with phosphate-buffered saline (PBS), followed by perfusion with 4% paraformaldehyde in PBS. The brain tissue with indirect bypass was carefully removed. Coronal sections with the indirect bypass tissue, measuring 5 mm, were obtained from each brain, fixed in 4% paraformaldehyde for 48 h, and embedded in paraffin. Coronal sections were sliced to 4-μm thickness. The sections were stained with hematoxylin and eosin for histological evaluation. Sections were photographed at 40 × magnification. The remaining sections were prepared and subjected to immunofluorescence staining.

Sections were immunostained with platelet-derived growth factor receptor alpha (PDGFR-α, 1:100, Santa Cruz Biotechnology, Inc, Dallas, TX, USA) for 1 h to detect fibroblasts between the indirect bypass tissue and brain cortex. Positive staining was detected using a detection kit (Histofine; Nichirei, Tokyo, Japan) by incubating the sections with 3,3′-diaminobenzidine chromogen (Dako North America, Inc., Carpinteria, CA, USA).

CD31 and glucose transporter type 1 (Glut-1) were used as markers of vascular endothelial cells and were evaluated through immunofluorescence staining. CD31 was incubated overnight with the primary antibody (1:50, Abcam 28,364, Abcam, Cambridge, UK) at 4 °C, and Glut-1 (1:10,000, Sigma-Aldrich Co., Burlington, MA, USA) was incubated for 1 h at 24 °C. CD31 and Glut-1 were then incubated with goat polyclonal anti-rabbit immunoglobulin G secondary antibody (Alexa Fluor 488, Abcam) for 1 h to identify cell immunoreactivity. After counterstaining with 4,6-diamidino-2-phenylindole for 24 h at 24 °C, three areas of the brain cortex under the indirect bypass and the contralateral cortex were visualized using fluorescent microscopy (BZ9000, Keyence Co., Osaka, Japan) at 200 × magnification. The number of CD31- or Glut-1–positive vascular endothelial cells was automatically measured. The effect of indirect bypass on angiogenesis was expressed as the ratio of the average number of vascular endothelial cells on the operative side to those on the non-operative side. The ratios were then compared among the three groups. Statistical analysis was performed using one-way analysis of variance.