Temporal changes of neurogenesis in the mouse

Temporal changes of neurogenesis in the mouse hippocampus after experimental subarachnoid hemorrhage Masaki Mino*, Hideyuki Kamii*, Miki Fujimura*, Takeo Kondo*, Shin Takasawa{, Hiroshi Okamoto{ and Takashi Yoshimoto* *Department of Neurosurgery, { Department of Biochemistry Tohoku University Graduate School of Medicine, Sendai, Japan ???please indicate which dept each author is in???

Recent studies indicate the existence of progenitor cells and their potential for neurogenesis in the subventricular zone (SVZ) and the hippocampus dentate gyrus (DG) of normal adult mammalian brain. Increased neurogenesis has been shown following cerebral ischemia and traumatic brain injury; however, the involvement of neurogenesis in subarachnoid hemorrhage (SAH) has not been examined. Adult male CD-1 mice were subjected to SAH by endovascular perforation of the left anterior cerebral artery. Mice received intraperitoneal injections of the cell proliferation-specic marker 5 0 -bromodeoxyuridine (BrdU) after SAH induction. BrdU incorporation was examined from 1 to 30 days after SAH by immunohistochemistry. The BrdU-positive cells were detected in SVZ and DG of normal control brain, and were signicantly decreased in both areas three days after SAH. The number of these cells had recovered to its control level seven days after SAH. Double staining with BrdU and NeuN indicated that the majority of the BrdU-positive cells migrating into the granular cell layer of the DG became NeuN-positive 30 days after SAH. In conclusion, temporal changes of the neurogenesis as shown in the present study suggest that neurogenesis in the hippocampus may affect functional outcome after SAH. The induction of the neurogenesis can provide therapeutic value against SAH. [Neurol Res 2003; 25: 839–845] Keywords: Subarachnoid hemorrhage; hippocampus; dentate gyrus; neurogenesis; progenitor cells; mice

INTRODUCTION Recent studies indicate the existence of progenitor cells and their potential for neurogenesis in the subventricular zone (SVZ) and the hippocampus dentate gyrus (DG) of normal adult mammalian brain1 ,2 . Neurogenesis, characterized by proliferation, migration, and differentiation of neuronal progenitor cells, is reported to participate in various insults including cerebral ischemia3– 11 and traumatic brain injury12– 14 . These data raise the possibility of repairing damaged tissue by recruiting their latent regenerative potential. The involvement of neurogenesis in subarachnoid hemorrhage (SAH), however, has not been examined previously. SAH is a life-threatening central nervous system disorder that can result in a signicant mortality and morbidity rate due to primary brain damage and cerebral vasospasm. The biochemical cascades involved in primary brain damage15,16 and cerebral vasospasm1 7,18 have been claried recently; however, the underlying mechanism of cognitive and/or memory disturbances and their recovery following SAH were undetermined. In the present study, we investigated whether the proliferation of neural progenitor cells participates in SAH by detecting their incorporation of 50 -bromodeoxyuridine (BrdU). We further sought to clarify the temporal Correspondence and reprint requests to: Miki Fujimura, MD, PhD, Department of Neurosurgery, Tohoku University Graduate School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan. [[email protected]]Accepted for publication May 2003.

# 2003 Forefront Publishing Group 0161–6412/03/080839–07

and anatomical changes of the progenitor cells following SAH. MATERIALS AND METHODS Induction of SAH Adult male CD-1 mice (35–40 g; n ˆ 44) were subjected to SAH by endovascular perforation of the left anterior cerebral artery as previously described17 ,18 . The mice were anesthetized with 0.5% halothane in 30% oxygen and 70% nitrous oxide with the use of facemask after an intraperitoneal injection of xylazine (10 mg kg¡1 ). The rectal temperature was controlled at 37° C with a Small Animal Warmer & Thermometer (Bio Research Center, Nagoya, Japan). The left common carotid artery was exposed, and the external carotid artery and its branches were isolated and coagulated. A 5-0 monolament nylon suture, blunted at the tip, was introduced into the internal carotid artery through the external carotid artery stump up to the left anterior cerebral artery near the anterior communicating artery, where resistance was encountered, as in a mouse ischemia model used in our previous studies19 . The nylon suture was then advanced 5 mm further to perforate the artery and was immediately withdrawn through the internal carotid artery into the external carotid artery, allowing reperfusion and producing SAH. Sham-operated animals were treated identically, except that the anterior cerebral artery was not perforated. Neurological Research, 2003, Volume 25, December 839

Neurogenesis after subarachnoid hemorrhage: Masaki Mino et al.

BrdU labeling After the induction of SAH, the thymidine analog 50 bromodeoxyuridine (BrdU, Sigma Chemicals, St. Louis, MO, USA), dissolved in saline, was administered intraperitoneally (50 mg kg¡1 ). Two patterns of injection were used in this study. Firstly, we administered a single dose of BrdU (50 mg kg¡1 ) one day prior to the brain sampling. The animals were then killed 1, 3, 7 and 10 days after SAH (n ˆ 7, each) for the purpose of counting the number of cells that incorporated BrdU during a 24 h period and investigating the rate of cell proliferation at a specic time point after SAH. Control mice (n ˆ 7) had received BrdU injection of the same dose and were killed the next day after sham-operation. Secondly, we gave injections of BrdU once daily for seven days after the induction of SAH. Then the animals were killed 14 or 30 days after SAH (n ˆ 3, each) for the purpose of investigating the migration pattern of newly proliferating cells and their characterization. Sham-operated mice (n ˆ 3) had received BrdU injection in the same protocol and were killed 13 or 29 days after the injection. Tissue preparations and immunohistochemistry The mice were perfused with 10 U ml¡1 heparin and subsequently with 4% paraformaldehyde (PFA). The brains were removed, post-xed for 2 h in 4% PFA, and placed in 0.88 mol l¡1 sucrose for 48 h. Twenty-ve micrometer coronal sections were cut on a cryostat at ¡20° C, and the slices were stored in phosphate buffered saline (PBS). Free-oating brain sections were incubated in 0.3% Triton X-100/1£PBS for 2 h and then incubated in 3% H2 O2 /1£PBS for 10 min and then washed in PBS. Sections were placed on silane-coated slides, and processed to immunohistochemical detection of BrdUlabeled nuclei. DNA was denatured to expose the antigen. Sections were pre-treated with 50% formamide/ 2£SSC at 65° C for 2 h, incubated in 2 N HCl at 37° C for 30 min, and rinsed in 0.1 mol l¡1 boric acid (pH 8.5) at room temperature for 10 min. Sections were incubated in blocking solution (5% normal rabbit serum/0.1% Triton X-100/1£PBS) for 30 min and incubated with rat monoclonal anti-BrdU antibody (1 : 400, Abcam). Sections were washed with PBS incubated with biotinylated rabbit anti-rat secondary antibody (1 : 200, Vector Laboratories, Burlingame, CA, USA) for 30 min. Immunoreactivities were developed in horseradish peroxidase/streptavidin/biotin complex solution (Vectastain ABC Kit, Vector Laboratories) for 30 min, and the peroxidase reaction was detected with 0.01% diaminobenzidine (DAB) and 0.002% H2 O2 . Processing was stopped with H2 O; sections were dehydrated through graded alcohols, cleared in lemosol, and coverslipped in permanent mounting medium (Vector Laboratories). Double immunouorescence For the double immunouorescence detection of BrdU and NeuN, sections were pre-treated to denature DNA and were incubated overnight with rat monoclonal anti-BrdU antibody (1 : 400, Abcam) and then for 30 min in biotinylated rabbit anti-rat antibody (1 : 200, Vector 840 Neurological Research, 2003, Volume 25, December

Laboratories). Sections were incubated with ExtrAvidin– FITC conjugate (1 : 200, Vector Laboratories) for 30 min. After PBS washes, they were incubated with mouse monoclonal anti-NeuN antibody (1 : 400, Chemicon), which was followed by incubation for 2 h with TexasRed-labeled horse anti-mouse antibody (1 : 200, Vector Laboratories). For the double immunouorescence detection of BrdU and GFAP, sections were pretreated to denature DNA and were incubated overnight with mouse monoclonal anti-BrdU antibody (1 : 400, Roche) and then for 30 min in biotinylated horse antimouse antibody (1 : 200, Vector Laboratories). Sections were incubated with ExtrAvidin–FITC conjugate (1 : 200, Vector Laboratories) for 30 min. After PBS washes, they were incubated with rabbit polyclonal anti-GFAP antibody (1 : 400, DAKO) overnight, which was followed by incubation for 2 h with TexasRed-labeled goat antirabbit antibody (1 : 200, Vector Laboratories). These sections were washed in PBS and then mounted with coverslips on glass slides with Vectashield (Vector Laboratories). Fluorescence was detected using a Leica QFluoro imaging system equipped with a Leica DMRXA microscope. Quantication and statistical analysis The number of BrdU-positive cells in DG and SVZ on the right hemisphere, the contralateral side of the endovascular perforation, was counted in each three coronal sections (25 mm, spaced 50 mm apart) per animal in a high power eld on a Nikon E600 microscope with digital camera DXM1200. The images were displayed on a computer monitor with an imaging system (ACT-1 version 2.0, Nikon). In hippocampal sections, the number of BrdU immunoreactive nuclei was counted in the area of DG, including the hilus, subgranular zone, and granule cell layer. Results were expressed as the average number of BrdU-positive cells per section and reported as the mean § SE. In ventricular sections, optical dissectors sized at 50£50 mm were randomly sampled within the upper third of the lateral side of the ventricular wall on a computer monitor, and the number of BrdU immunoreactive nuclei in each dissector was counted. The density of BrdU immunoreactive cells in the investigated region was calculated by dividing the total number of BrdU-positive cells by the size of the optical dissectors. Results were expressed as the average number of BrdU-positive cells per square millimeter and reported as the mean § SE. Differences between means were determined by Student’s t test, with p < 0.05 considerd signicant. RESULTS SAH were evident mainly in the basal cistern, especially around the left anterior cerebral artery in all animals. The mortality rate within 72 h was 29%. These observations were completely in accordance with our previous reports1 7,18 . Immunohistochemistry of BrdU demonstrated that the signicant amount of BrdU positive cells was seen in DG and in SVZ before and after SAH (Figure 1). The


Figure 1: Immunohistochemistry for BrdU in A–D: the dentate gyrus and E–H: the subventricular zone A,E: without SAH or B,F: 3, C,G: 7 and D,H: 10 days after SAH. B,F: The number of BrdU-positive cells are decreased both in the dentate gyrus and the subventricular zone three days after SAH, and C,G: recovered to its control level seven days after SAH. Scale bar ˆ 100 mm

BrdU-positive cells were found in DG without SAH, including hilus but mainly on the surface of the granular cell layer (GCL) (Figure 1A), and the mean number of BrdU-positive cells was 10.57 § 0.99/section (mean § SE). Sham-operation did not change the amount of BrdU-positive cells. The number of BrdU-positive cells in DG three days after SAH (4.62 § 0.94/section) was signicantly decreased compared to that in the control mouse (p < 0.01), to the extent of 44% (Figures 1A,B, 2A). The number of BrdU-positive cells in DG recovered to its control level 7 and 10 days after SAH (7.54 § 1.58 and 12.76 § 2.13/section) (Figures 1C,D, 2A). Similar results were obtained in the SVZ. The mean density of

BrdU-positive cells in the SVZ of the control mouse was 5510 § 323/mm2 , and it decreased signicantly one day after SAH (3086 § 620/mm2 ) (p < 0.01) and sustained at low level three days after SAH (2171 § 241/mm2 , p < 0.01 compared to control) (Figures 1E,F, 2B). Seven days after SAH, the number of BrdU-positive cells in SVZ (5425 § 605/mm2 ) increased and recovered to its control level (p < 0.01 compared to day 3), but did not further increase 10 days after SAH (4457 § 257/mm2 ) (Figures 1G,H, 2B). To investigate the fate of the newly proliferated cells that incorporated BrdU in DG, the animals were killed 14 and 30 days after SAH and subjected to double Neurological Research, 2003, Volume 25, December 841


Figure 2: Temporal changes of the number of BrdU-labeled cells in A: the dentate gyrus and B: the subventricular zone from 1–10 days after subarachnoid hemorrhage. *p < 0.01 compared with control specimen

staining with BrdU and NeuN, or with BrdU and GFAP (Figure 3). In single immunohistochemical staining of BrdU, the number of BrdU-positive cells was 32.75 § 10.35/section in 14 days, and 14.45 § 1.84/ section in 30 days after SAH. The double immunouorescence for BrdU and NeuN 14 days after SAH revealed no remarkable co-localization in DG (Figure 3A). Double immunouorescence for BrdU and GFAP failed to reveal double positive cells (Figure 3B). While 30 days after SAH, about 70% of the BrdU-positive cells were labeled with NeuN in the GCL (Figure 3C). No colocalization of GFAP and BrdU was observed within the GCL 30 days after SAH (Figure 3D). Compared to the sham-operated mice which received BrdU injections without SAH, apparent migration of the BrdU-positive cells into the GCL was observed after SAH (Figure 4B), whereas in control mice BrdU-positive cells remain on the inner surface of the GCL (Figure 4A). These data suggest the differentiation of the neural progenitor cells into neurons of the GCL on day 30 after SAH. In SVZ, the number of BrdU-positive cells markedly decreased 14 days after SAH (Figure 5B ), and further decreased 30 days after SAH (Figure 5C ), indicating their migration along the rostral migratory stream to the olfactory bulb20 . 842 Neurological Research, 2003, Volume 25, December

DISCUSSION The present study demonstrated, for the rst time, that the temporal changes of neurogenesis occurred following SAH. This observation derived from our following ndings. Firstly, neural progenitor cells as shown by BrdU labeling were detected in SVZ and DG of normal control brain, which were signicantly decreased three days after SAH and then recovered to their control level at 7 days (Figures 1 and 2). Secondly, double staining with BrdU and neuronal marker NeuN indicated that the majority of the BrdU-positive cells in the DG became NeuN-positive 30 days after SAH (Figure 3). Finally, marked migration of the newly proliferated cells, most of which differentiated into neuron, was observed 30 days after SAH (Figure 4). Taken together, induction of SAH had an inhibitory effect on neurogenesis at three days, while it ultimately resulted in the migration of newborn neuron into granular cell layer of DG. Neurogenesis has been reported to be involved in a variety of central nervous system disorders including cerebral ischemia3–1 1 and traumatic brain injury12– 14 . The increased neurogenesis at hippocampal GCL was observed with its peak at 11 days after transient global cerebral ischemia in gerbil3 . Neurogenesis at DG is also reported to occur nine days after focal cerebral ischemia2 1 . The progenitor cells that began to proliferate on the inner surface of subgranular zone and then migrate into the GCL were reported to express the immature neuronal marker polysialylated neural cell adhesion molecule (PSA–NCAM), and then differentiate into neurons7,9,11 . In traumatic brain injury, the increased neurogenesis is observed as early as three days after injury12 . The newly proliferated progenitors partly differentiated into astrocytes and contributed to the astroglial scar formation, and some progenitors differentiated into neurons in DG of the contralateral hemisphere after traumatic brain injury13 . Contrary to these observations on cerebral ischemia and traumatic brain injury, SAH as shown in our present study resulted in the inhibition of neurogenesis from one to three days after the onset, subsequent recovery of the amount of neurogenesis at seven days, and the induction of the migration of the newborn neuron into GCL 30 days after SAH. These ndings are apparently distinct from cerebral ischemia and traumatic brain injury. The exact mechanism of the decreased neurogenesis following SAH is undetermined, but the possible explanation could be as follows. Firstly, since initial onset of SAH is known to cause primary brain damage including apoptotic cell death15 ,1 6,22,23 , neural progenitor cells may also be damaged by the initial attack of SAH, and then the survived progenitor cells may proliferate subsequently which may result in the recovery of the BrdU-positive cells at seven days. Secondly, cerebral ischemia caused by the sudden increase of the intracranial pressure or by cerebral vasospasm may alter the microvascular environments around progenitor cells and then affect the neurogenesis, because neurogenesis is reported to be accompanied by microvascular angiogenesis24– 28 . Thirdly, the up-regulation of the inammatory molecules such as interleukin-


Figure 3: Double immunouorescence for BrdU, shown in green, and A,C: NeuN or B,D: GFAP, shown in red, in the dentate gyrus A,B: 14 and C,D: 30 days after SAH. C: The majority of the BrdU-positive cells were NeuN-positive (arrows) 30 days after SAH. A: No co-localization of NeuN and BrdU was shown 14 days after SAH. B,D: GFAP and BrdU were not colocalized even at 30 days after SAH. Scale bar ˆ 50 mm

Figure 4: Double immunouorescence for BrdU, shown in green, and NeuN, shown in red, in the dentate gyrus A: without SAH and B: 30 days after SAH. A: In the mouse without SAH, BrdU-positive cells remain on the inner surface of the granular cell layer (arrows), whereas B: apparent migration of the BrdU-positive cells into the granule cell layer was seen in the SAH-induced mouse (arrows). Scale bar ˆ 20 mm Neurological Research, 2003, Volume 25, December 843


Figure 5: Immunohistochemistry for BrdU in the subventricular zone A: 7, B: 14, and C: 30 days after SAH. B: The number of BrdU-positive cells decreased remarkably 14 days after SAH, and C: only a few BrdU-positive cells were seen 30 days after SAH. Scale bar ˆ 100 mm

6, which are known to reduce the amount of the neurogenesis2 9 , may inhibit the proliferation of neural progenitor cells three days after SAH. Alternatively, the down-regulation of neurotrophic factors including broblast growth factor-2 (FGF-2), which increases the neurogenesis2 1,30 , may contribute to the decreased neurogenesis. Finally, the adrenocorticosteroid, which may increase following SAH, could contribute to the inhibition of the proliferation of the neural progenitors31 . Further examinations using the inhibitors or genetic engineering animals of these molecules on SAH model are warranted to address this important issue. CONCLUSION SAH is a life-threatening central nervous system disorder that can result in a signicant mortality and morbidity rate due to primary brain damage and cerebral vasospasm. Several behavioral decits are also reported in the animal model studies3 2 . Because recent evidence suggests that the inhibition of neurogenesis in DG results in the cognitive dysfunctions27 ,3 3–3 5 , our results raise the concern that the inhibition of the neurogenesis as shown in the present study may contribute to cognitive dysfunctions after SAH. Therefore, the prevention of the decreased neurogenesis or the induction of the neurogenesis in hippocampus after SAH may be benecial to relieve functional deterioration. In fact, it is known that transplanted neural stem/progenitor cells have benets for the improvement of neurological functions following cerebral ischemia or traumatic brain injury36 –3 8 . Furthermore, the induction of the neurotrophic molecules such as FGF-2, insulin-like growth factor-1 (IGF-1), or vascular endothelial growth factor (VEGF), is known to promote neurogenesis in the hippocampus2 1,28 ,3 9 and may contribute to the recovery from the neurological dysfunction. These issues regarding SAH remain to be elucidated in the future study. Temporal changes of the neurogenesis as shown in the present study suggest that neurogenesis in the hippocampus may affect functinal outcome after SAH. 844 Neurological Research, 2003, Volume 25, December

The induction of the neurogenesis can provide therapeutic value against SAH.

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