Role of a Burr Hole and Calvarial Bone Marrow-Derived Stem Cells in the Ischemic Rat Brain: A Possible Mechanism for the Efficacy of Multiple Burr Hole Surgery in Moyamoya Disease

Abstract

Objective: This study investigates the role of a burr hole and calvarial bone marrow-derived stem cells (BMSCs) in a transient ischemic brain injury model in the rat and postulates a possible mechanism for the efficacy of multiple cranial burr hole (MCBH) surgery in moyamoya disease (MMD).

Methods: Twenty Sprague-Dawley rats (250 g, male) were divided into four groups : normal control group (n=5), burr hole group (n=5), ischemia group (n=5), and ischemia+burr hole group (n=5). Focal ischemia was induced by the transient middle cerebral artery occlusion (MCAO). At one week after the ischemic injury, a 2 mm-sized cranial burr hole with small cortical incision was made on the ipsilateral (left) parietal area. Bromodeoxyuridine (BrdU, 50 mg/kg) was injected intraperitoneally, 2 times a day for 6 days after the burr hole trephination. At one week after the burr hole trephination, brains were harvested. Immunohistochemical stainings for BrdU, CD34, VEGF, and Doublecortin and Nestin were done.

Results: In the ischemia+burr hole group, BrdU (+), CD34 (+), and Doublecortin (+) cells were found in the cortical incision site below the burr hole. A number of cells with Nestin (+) or VEGF (+) were found in the cerebral parenchyma around the cortical incision site. In the other groups, BrdU (+), CD34 (+), Doublecortin (+), and Nestin (+) cells were not detected in the corresponding area. These findings suggest that BrdU (+) and CD34 (+) cells are bone marrow-derived stem cells, which may be derived from the calvarial bone marrow through the burr hole. The existence of CD34 (+) and VEGF (+) cells indicates increased angiogenesis, while the existence of Doublecortin (+), Nestin (+) cells indicates increased neurogenesis.

Conclusion: Based on these findings, the BMSCs through burr holes seem to play an important role for the therapeutic effect of the MCBH surgery in MMD.

Keywords: Angiogenesis; Bone marrow; Burr hole; Moyamoya disease; Neurogenesis; Stem cell.

Fig. 1. Possible treatment mechanism for multiple cranial burr hole surgery. In moyamoya disease, calvarial bone marrow-derived stem cells migrate into the ischemic cerebral cortex through multiple cranial burr holes and play a role in angiogenesis and neurogenesis.

Fig. 2. The site of burr hole trephination. A 2-mm-diameter burr hole trephination was made 3 mm lateral and 3 mm posterior to the bregma.

Fig. 3. Schematic flow sheet for this study. BrdU : 5-bromodeoxyuridine, IP : intraperitoneal.

Fig. 4. Immunohistochemical stain with BrdU. A : Normal control group. B : Burr hole group. C : Ischemia group. D : Ischemia+burr hole group. In the ischemia+burr hole group, BrdU (+) cells (arrow) were found at the cortical injury site beneath the burr hole in the coronal section. In the burr hole group, a few BrdU (+) cells were detected. Original magnification ×200. BrdU : 5-bromodeoxyuridine.

Fig. 5. Immunohistochemical stain with CD34 in the ischemia+burr-hole group. CD34 (+) cells (arrow) were detected at the site of the burr hole. A : Original magnification ×100. B : Black rectangle in A, original magnification ×400.

Fig. 6. Immunohistochemical stain with VEGF in the ischemia+burr hole group. Many VEGF (+) cells (arrow) were found under the burr hole. A : Original magnification ×100. VEGF (+) cells markedly increased at the cerebral parenchyma adjacent to the cortical incision site below the burr hole. B : Black rectangle in A, original magnification ×400. VEGF : vascular endothelial growth factor.

Fig. 7. Immunohistochemical stain with Doblecortin (DCX). In the ischemia +burr hole group, many DCX (+) cells (arrow) are observed at the burr hole site. A : Original magnification ×100. B : Black rectangle in A, original magnification ×400.

Fig. 8. Immunohistochemical stain with Nestin in the ischemia+burr hole group. Numerous Nestin (+) cells (arrow) were present at the cerebral parenchyma adjacent to the cortical incision site below the burr hole. A : Original magnification ×100. B : Black rectangle in A, original magnification ×400.