Reduction of infarct volume and apoptosis by grafting of encapsulated basic fibroblast growth factor—secreting cells in a model of middle cerebral artery occlusion in rats

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Object. This study was conducted to evaluate the effects of grafting encapsulated basic fibroblast growth factor (bFGF)—secreting cells in rat brains subjected to ischemic injury.

Methods. Two cell lines were used for encapsulated grafting in this experiment, namely, a bFGF-secreting cell line established by genetic manipulation of baby hamster kidney (BHK) cells, and a naive BHK cell line. Forty-seven Sprague—Dawley rats were used in this experiment. The animals were divided into the following three groups: those receiving grafts of encapsulated bFGF-secreting cells (BHK-bFGF group); those with grafts of encapsulated naive BHK cells (naive BHK group); and those with no grafts (control group). The authors implanted encapsulated cells into the right striatum of host rats in the BHK-bFGF and naive BHK groups. Six days after grafting, the host and control animals underwent permanent right middle cerebral artery occlusion (MCAO) with an intraluminal suture procedure. The infarct volume was evaluated using 2,3,5-triphenyltetrazolium chloride staining and computerized image analysis 24 hours after MCAO. Fragmentations of DNA in the host brains were analyzed using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling 12 hours after MCAO.

The authors found that the infarct volume in the BHK-bFGF group was reduced by approximately 30% compared with that in the naive BHK and control groups. In the ischemic penumbral area, the number of apoptotic cells in the BHK-bFGF group was significantly decreased compared with that in the other groups.

Conclusions. The grafting of encapsulated BHK bFGF-secreting cells protected the brain from ischemic injury. Encapsulation and grafting of genetically engineered cells such as bFGF-secreting cells is thus thought to be a useful method for protection against cerebral ischemia.

Article Information

Address reprint requests to: Kenjiro Fujiwara, M.D., Department of Neurological Surgery, Kosei General Hospital, 3–3–28 Minamicho, Mihara City 723–8686, Japan. email: kenjiro@gb3.so-net.ne.jp.

© AANS, except where prohibited by US copyright law.

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Figures

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    Bar graph showing production of bFGF from encapsulated BHK-bFGF cells. The BHK-bFGF capsules produced bFGF in a continuous and stable manner for at least 2 months. The amounts of bFGF were approximately 15 to 30 ng per capsule per day, and there was no significant difference in output at any time point examined. Secretion of bFGF was not detected from encapsulated naive BHK cells.

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    Photographs of TTC-stained coronal brain slices showing cerebral infarcts after MCAO. Left: Brain slices obtained in the BHK-bFGF group. Right: Brain slices obtained in the control group. The infarct area in the brains in the left panel is significantly smaller than in those in the right panel.

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    Bar graph showing the infarct volume in each group at 24 hours after MCAO. Infarct volume in the BHK-bFGF group (nine rats, *p < 0.05) was significantly smaller than that in the naive BHK (six rats) and control groups (nine rats).

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    Schematic drawings showing the location of cerebral infarcts and regions sampled for TUNEL staining. Representative cases from the BHK-bFGF (left) and control (right) groups are featured. The infarct area is shaded gray. Region A is the dorsolateral cortex in the ipsilateral (ischemic) hemisphere. This region was typically spared from infarction in the BHK-bFGF group. On the other hand, most of this region was included in the infarct area in the control group. This region is considered to be part of the ischemic penumbra. Region B is the lateral striatum in the ipsilateral (ischemic) hemisphere. This region was commonly involved in the infarct area in both groups. It is considered to be the ischemic core region.

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    Representative photomicrographs showing TUNEL staining. a: Dorsolateral cortex (Region A) in the BHK-bFGF group. b: Dorsolateral cortex (Region A) in the control group. In the dorsolateral cortex, the number of TUNEL-positive cells (arrows) in the BHK-bFGF group was significantly smaller than that in the control group. c: Striatum (Region B) in the BHK-bFGF group. d: Striatum (Region B) in the control group. In the striatum, a large number of TUNEL-positive cells (arrows) were observed in both groups. Bar = 40 µm.

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    Light microscopy photomicrographs of the area surrounding the site of capsule implantation, which was perfused with India ink. The panels show representative cases in the BHK-bFGF (left), naive BHK (center), and control (right) groups. A significant increase in vascular density appears in the area around the graft in the BHK-bFGF group compared with that in the naive BHK and control groups. Bar = 40 µm.

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    Bar graph showing vascular density in the area surrounding the site of capsule implantation in rats 6 days after implantation and in control animals. The vessel/area ratios of the BHK-bFGF, naive BHK, and control groups were 5.142 ± 0.433%, 3.229 ± 0.268%, and 2.187 ± 0.277%, respectively (mean ± SEM). *p < 0.05; **p < 0.001; and ***p < 0.0001.

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    Upper: Photomicrograph showing histological features of cells in a retrieved capsule. A large number of viable BHK-bFGF cells were observed within the full length of the capsule. H & E, original magnification × 200. Lower: Bar graph showing secretion of bFGF from BHK-bFGF capsules before implantation and after retrieval. The mean secretion values are 20 ± 4.44 ng/day (preimplantation) and 25 ± 5.64 ng/day (postretrieval). There was no significant difference between values in capsules before and after implantation. Data are expressed as the means ± SEM.

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