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Cerebral cortex lesions decrease the number of bromodeoxyuridine-positive subventricular zone cells in mice
Neuroscience Letters 329 (2002) 161–164 www.elsevier.com/locate/neulet
Cerebral cortex lesions decrease the number of bromodeoxyuridinepositive subventricular zone cells in mice Gwendolyn E. Goings, Bernadeta L. Wibisono, Francis G. Szele* CMIER Neurobiology Program, Children’s Memorial Hospital, Department of Pediatrics, Feinberg School of Medicine, Northwestern University, 2430 N. Halsted, No. 209, Chicago, IL 60614-3394, USA Received 2 May 2002; received in revised form 4 June 2002; accepted 6 June 2002
Abstract We previously showed that cortical lesions in rats increase the number of subventricular zone (SVZ) cells. Here, we examined the response of the SVZ to cortical lesions in mice from 6 h to 35 days later. Whereas the total number of cells did not change, the number of cells in S-phase (bromodeoxyuridine-positive) decreased in a biphasic manner (from 6 h to day 3, and again at days 25–35). In addition, there was a delayed (days 25–35) increase in immunoreactivity for polysialylated neural cell adhesion molecule, a marker of neuroblasts. The results suggest that in mice there are rapid as well as delayed responses in the SVZ to injury of the overlying cerebral cortex. They also show that the SVZ of different mammalian species can exhibit widely divergent responses to the same brain injury. q 2002 Published by Elsevier Science Ireland Ltd. Keywords: Repair; Plasticity; Neurogenesis; Polysialylated neural cell adhesion molecule; Subventricular zone; CD-1; Bromodeoxyuridine
Though mitosis in the brain diminishes greatly after development, it persists in the subventricular zone (SVZ) [2,22]. The SVZ consists of astrocyte-like cells, progenitor cells, and neuroblasts which migrate to the olfactory bulbs to become interneurons [5,6]. All three cell types (except for a subpopulation of astrocytes) divide and mitosis continues indefinitely in vitro in the presence of epidermal growth factor and fibroblast growth factor-2 [6,15,20]. Removal of growth factors, and adhesion, promotes differentiation into the three major neural lineages [15,19]. These experiments showing self-renewal and broad fate potential indicate that the adult SVZ is a major repository of stem cells in the adult central nervous system (CNS). As such, it remains a potential source of cells for neuronal replacement after injury or degenerative illness [2]. In order to potentially attain this goal, the in situ biological response of the SVZ to a variety of brain insults must be examined. The SVZ has been shown to respond to models of injury/illness with decreases [3,11,13] or increases [4,16,18] in the number of cells going through mitosis. Whereas separation of the SVZ from the olfactory bulb caused decreases in bromo-
* Corresponding author. Tel.: 11-773-880-3791; fax: 11-773868-8066. E-mail address: [email protected] (F.G. Szele).
deoxyuridine (BrdU) incorporation in mice [11,13], it caused increases in rat [1], suggesting that it is hazardous to extrapolate similar responses of the SVZ to a given injury across species. We reported before that cortical lesions cause significant increases in cell numbers and polysialylated neural cell adhesion molecule (PSA-NCAM) immunoreactivity in the SVZ of rats, suggesting that the number of neuroblasts increased [23]. Whereas the number of cells increased two-fold after cortical lesion, the number cells going through S-phase did not change consistently [23]. As we plan to use genetically engineered mice to examine the response of the SVZ to brain injury, we began by measuring cell numbers, BrdU incorporation, and PSANCAM immunoreactivity in the SVZ of adult male CD-1 mice after cortical injury. Aspiration cortical lesions (Fig. 1A) were conducted similar to Szele and Chesselet [23]. Adult (.25 g), male CD-1 mice were anesthetized with Ketamine (100 mg/kg)/ Xylazine (10 mg/kg), and the cerebral cortex was removed from the Bregma to 1.2 mm anterior to Bregma. The lesion was restricted to the primary motor cortex, the forelimb somatosensory cortex, and the rostral-most aspect of the hindlimb somatosensory cortex. Removal of this region may result in slight impairment of specific forelimb placement tasks, but does not impair normal feeding or grooming.
0304-3940/02/$ - see front matter q 2002 Published by Elsevier Science Ireland Ltd. PII: S03 04 - 394 0( 0 2) 00 61 1- 0
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Fig. 1. (A) Schematic coronal section showing the SVZ in red. In some analyses, the SVZ was subdivided into its dorsolateral component (dl svz, green rectangle) and its striatal component (str svz, blue rectangle). (B) DAPI staining in striatal SVZ. Nuclei were stained with DAPI (Sigma, St. Louis, MO; 40 mg/ml) for 10 min. Note the high density of cells in the SVZ compared with the striatum, which allows easy microscopic distinction of the two areas for quantification. (C) BrdU immunohistochemistry of dividing cells (arrow shows typical example) in the dl svz. (D) PSA-NCAM immunohistochemistry in the dl svz showing typical labeling (arrow) and example of area quantified (outline). Control animals in B–D. al, area lesioned; spt, septum; str, striatum.
The lesions spared the secondary motor cortex, the cingulate cortex, the somatosensory barrel fields, and the primary somatosensory cortex. Mice were allowed to survive from 6 h to 35 days after cortical injury and then were perfused with 4% paraformaldehyde. In all quantifications, 20 mm free-floating sections were cut on a freezing cryostat and stored in cryoprotectant at 220 8C. Every sixth section from the crossing of the corpus callosum in the (anterior striatum) to the decussation of the anterior commissure (posterior striatum) was collected and quantified. The number of 4 0 , 6-diamidino-2-phenylindole dihydrochloride hydrate (DAPI)-stained cells (Fig. 2), BrdU-positive cells (Fig. 3), or surface area of PSA-NCAM immunoreactivity (Fig. 4) per section was averaged and then these values averaged across animals. All statistical comparisons between non-lesioned controls and lesioned animals were made using unpaired two-tailed Student’s t-tests. Significance was considered to be P , 0:05. All values are expressed as averages ^ SEM. An initial cohort of mice was sacrificed at 2 (N ¼ 3), 3 (N ¼ 5), 6 (N ¼ 5) and 15 (N ¼ 5) days after cortical lesion and compared with non-lesioned controls (N ¼ 5). The controls were anesthetized only, in parallel with the lesioned mice, and sacrificed in parallel 3 (N ¼ 1), 6 (N ¼ 2), and 15 (N ¼ 2) days later. In this, and in subsequent experiments, we counted the total number of cells in the dorsolateral SVZ and in the striatal SVZ (Fig. 1A; Szele and Chesselet [23])
Fig. 2. The total number of cells per section in the SVZ. DAPIstained nuclei were counted at 40 £ using the Neurolucida image analysis system (Microbrightfield, Williston, VT); cells were visualized on a computer monitor and confirmed by focusing through the cell while visualizing it through the microscope eyepiece. Lineage relationships amongst cells in the walls of the lateral ventricles remain uncertain and there is a formal possibility that both ependymal cells (which line the ventricle) and SVZ cells behave like stem cells in this region [7,12]. Therefore, for total cell counts, we counted ependymal cells together with SVZ cells. Error bars show standard errors, and significance was tested for using unpaired, two-tailed, Student’s t-tests.
separately but found very similar results in the two subdivisions and therefore combined numbers. There was no significant change in the total SVZ cell numbers after lesion compared with controls, (unpaired two-tailed Student’s ttest, not shown). We then counted BrdU-positive cells and found decreases at 2 days (109 ^ 11 vs. 76 ^ 14; P ¼ not significant (n.s.)) and statistically significant decreases at 3 days (109 ^ 11 vs. 80 ^ 4; P ¼ 0:03) after cortical lesion.
Fig. 3. The number of BrdU-positive cells per section in the SVZ. BrdU, incorporated into the cell nucleus during S-phase of the cell cycle, is an indirect marker of mitosis. Single intraperitoneal injections of BrdU (200 mg/kg) were given 2 h prior to sacrifice, sections were incubated in 0.1 M HCl for 20 min at 60 8C, and then in antiBrdU (1:1000, polyclonal, Fitzgerald, MA), and biotinylated secondaries (1:200, Jackson Immunoresearch, PA). BrdU-positive cells in the dorsolateral SVZ and striatal SVZ were counted visually with a 40 £ objective at the same time. *P ¼ 0:05–0:001; **P , 0:001. Error bars show standard errors, and significance was tested for using unpaired, two-tailed, Student’s t-tests.
G.E. Goings et al. / Neuroscience Letters 329 (2002) 161–164
Fig. 4. The area per section occupied by positive PSA-NCAM immunoreactivity in the dorsolateral component of the SVZ (dl svz). Sections were exposed to anti-PSA-NCAM [8] (1:5000, monoclonal, G. Rougon, Marseilles, France) and then biotinylated secondaries (1:200, Jackson Immunoresearch, PA). Images of the dl svz were captured with a Polaroid DMC digital camera (Polaroid, Boston, MA). Using NIH Image (Shareware) the dl svz was outlined (see Fig. 1) and the percentage surface area above a threshold quantified. The threshold was determined using positive PSA-NCAM immunoreactivity in non-lesioned controls and was kept constant for all sections. *P ¼ 0:05–0:001. Error bars show standard errors, and significance was tested for using unpaired, two-tailed, Student’s t-tests.
To begin to assess the response to brain injury of specific cell subtypes in the SVZ, we performed immunohistochemistry with the Men-B antibody [8] to quantify levels of PSANCAM which is selectively expressed by the neuroblasts of the SVZ [21]. PSA-NCAM immunoreactivity was transiently decreased at 2 days after cortical lesion (5.6 ^ 0.9 vs. 1.7 ^ 0.4 mm 2; P ¼ 0:02), but by 15 days, had risen above control values (5.6 ^ 0.9 vs. 9.0 ^ 2.4 mm 2; P ¼ n.s.) To confirm these findings, and to look for earlier and later changes, we sacrificed a second cohort of mice at 6 h (N ¼ 6), and at 1 (N ¼ 6), 2 (N ¼ 5), 3 (N ¼ 5), 6 (N ¼ 6), 15 (N ¼ 6), 25 (N ¼ 7) and 35 days (N ¼ 6) after cortical lesions and compared them with non-lesioned controls (N ¼ 9). The controls in the second cohort were anesthetized only, in parallel with the lesioned mice, and sacrificed in parallel 6 h (N ¼ 3), and 2 days later (N ¼ 6). As in the preliminary group, we found no changes in total cell numbers in the SVZ after lesion (Fig. 2). The number of BrdU-positive cells in the non-lesioned control groups was not significantly different (100 ^ 8 vs. 102 ^ 6; P ¼ 0:85). A biphasic decrease in numbers of BrdU-positive cells was found in the SVZ after cortical lesions (Fig. 3). These decreases reached statistical significance already at 6 h (101 ^ 4 vs. 84 ^ 7; P ¼ 0:04), and at 1 (101 ^ 4 vs. 70 ^ 6; P ¼ 0:001), 2 (101 ^ 4 vs. 62 ^ 6; P ¼ 0:0001), 3 (101 ^ 4 vs. 67 ^ 6; P ¼ 0:0004), 25 (101 ^ 4 vs. 73 ^ 8; P ¼ 0:004), and 35 days (101 ^ 4 vs. 70 ^ 5; P ¼ 0:0006) after lesion. The numbers of BrdU-positive cells at 6 (86 ^ 7) and 15 days (86 ^ 10) were also fewer
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than controls (101 ^ 4), however, these differences did not reach statistical significance. PSA-NCAM in this cohort of animals was again transiently decreased, although this did not reach significance. Also, the decrease was earlier than in our first study (6 h vs. 2 days). PSA-NCAM then increased at days 25 (15.0 ^ 2.8 vs. 33.3 ^ 5.6 mm 2; P ¼ 0:007) and 35 (15.0 ^ 2.8 vs. 46.0 ^ 11.2 mm 2; P ¼ 0:005; Fig. 4). These results show that the SVZ of different species can respond dissimilarly to the same insult. In rat, we had shown increases in total cell number but no changes in BrdU incorporation, whereas here we show no change in total cell number but decreases in the number of cells incorporating BrdU. In addition, we show here that the response of the SVZ to cortical injury in mice is: (1), rapid; (2), can last approximately up to one month; and (3), shows a biphasic pattern. Others have also found delayed changes in numbers of BrdU-positive cells in the SVZ after models of injury or disease [4,13]. Cell numbers in the SVZ can be regulated by a combination of cell proliferation, migration, and apoptosis. Thus, the decreases in cell division may have been offset by decreases in migration to the olfactory bulb or alternatively decreases in cell death. Either scenario could have resulted in the total cell number not changing significantly. In considering what molecular mechanisms cause decreases in the number of BrdU-positive cells, we have focused on serotonin. A serotonergic neurotoxin (5,7-dihydroxytryptamine) and an inhibitor of serotonin synthesis (pchlorophenylalanine) both caused decreases in BrdU incorporation [3]. Interestingly, olfactory bulbectomy, which also affects BrdU incorporation in the SVZ [1,11,13], causes changes in serotonergic neurotransmission [10]. It is unknown whether cortical lesions influence the serotonergic system in rats or mice, so we will test the hypotheses that the serotonergic innervation of the SVZ changes after cortical lesion in mouse and that this decreases the number of BrdUpositive cells. Interestingly, in rats, changes in cell numbers were only associated with changes in PSA-NCAM, but not with markers of SVZ astrocytes or progenitor cells [23]. Similarly, in mouse, changes in numbers of dividing cells were temporally correlated with early decreases and then late increases in PSA-NCAM. We looked for reactive astrocytosis in the SVZ of mice after cortical lesions and although we found robust glial fibrillary acidic protein (GFAP) and vimentin immunoreactivity surrounding the lesion, GFAP and vimentin levels were not changed in the SVZ (data not shown), similar to our studies in the rat [23]. PSA-NCAM can also be expressed in vitro by oligodendrocyte precursors isolated from P1 rats [9], and in vivo by oligodendrocytes during remyelination of the spinal cord [17] as well as in reactive astrocytes in the spinal cord and hippocampus [14,17]. While under normal conditions SVZ neuroblasts express PSA-NCAM [6,15], the formal possibility remains that after cortical injury, other SVZ cell subtypes may express this marker. In light of the results presented in this paper, it will be very important and interesting to exam-
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ine the response of human SVZ to various injuries and CNS diseases. This research was supported in part by National Institute of Neurological Disorders and Stroke grant RO1 NS42253 (F.G.S). The authors thank Genevieve Rougon for the gift of the PSA-NCAM antibody. [1] Alonso, G., Prieto, M. and Chauvet, N., Tangential migration of young neurons arising from the subventricular zone of adult rats is impaired by surgical lesions passing through their natural migratory pathway, J. Comp. Neurol., 405 (1999) 508–528. [2] Alvarez-Buylla, A., Herrera, D.G. and Wichterle, H., The subventricular zone: source of neuronal precursors for brain repair, Prog. Brain Res., 127 (2000) 1–11. [3] Brezun, J.M. and Daszuta, A., Depletion in serotonin decreases neurogenesis in the dentate gyrus and the subventricular zone of adult rats, Neuroscience, 89 (1999) 999–1002. [4] Calza, L., Giardino, L., Pozza, M., Bettelli, C., Micera, A. and Aloe, L., Proliferation and phenotype regulation in the subventricular zone during experimental allergic encephalomyelitis: in vivo evidence of a role for nerve growth factor, Proc. Natl. Acad. Sci. USA, 95 (1998) 3209–3214. [5] Corotto, F.S., Henegar, J.A. and Maruniak, J.A., Neurogenesis persists in the subependymal layer of the adult mouse brain, Neurosci. Lett., 149 (1993) 111–114. [6] Doetsch, F., Garcia-Verdugo, J.M. and Alvarez-Buylla, A., Cellular composition and three-dimensional organization of the subventricular germinal zone in the adult mammalian brain, J. Neurosci., 17 (1997) 5046–5061. [7] Doetsch, F., Caille, I., Lim, D.A., Garcia-Verdugo, J.M. and Alvarez-Buylla, A., Subventricular zone astrocytes are neural stem cells in the adult mammalian brain, Cell, 97 (1999) 703–716. [8] Dubois, C., Okandze, A., Figarella-Branger, D., Rampini, C. and Rougon, G., A monoclonal antibody against Meningococcus group B polysaccharides used to immunocapture and quantify polysialylated NCAM in tissues and biological fluids, J. Immunol. Methods, 181 (1995) 125–135. [9] Grinspan, J.B. and Franceschini, B., Platelet-derived growth factor is a survival factor for PSA-NCAM 1 oligodendrocyte pre-progenitor cells, J. Neurosci. Res., 41 (1995) 540–551. [10] Huether, G., Acute regulation and long-term modulation of presynaptic serotonin output, Adv. Exp. Med. Biol., 467 (1999) 1–10. [11] Jankovski, A., Garcia, C., Soriano, E. and Sotelo, C., Proliferation, migration and differentiation of neuronal progenitor cells in the adult mouse subventricular zone surgically
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separated from its olfactory bulb, Eur. J. Neurosci., 10 (1998) 3853–3868. Johansson, C.B., Momma, S., Clarke, D.L., Risling, M., Lendahl, U. and Frisen, J., Identification of a neural stem cell in the adult mammalian central nervous system, Cell, 96 (1999) 25–34. Kirschenbaum, B., Doetsch, F., Lois, C. and Alvarez-Buylla, A., Adult subventricular zone neuronal precursors continue to proliferate and migrate in the absence of the olfactory bulb, J. Neurosci., 19 (1999) 2171–2180. Le Gal La Salle, G., Rougon, G. and Valin, A., The embryonic form of neural cell surface molecule (E-NCAM) in the rat hippocampus and its reexpression on glial cells following kainic acid-induced status epilepticus, J. Neurosci., 12 (1992) 872–882. Lois, C. and Alvarez-Buylla, A., Proliferating subventricular zone cells in the adult mammalian forebrain can differentiate into neurons and glia, Proc. Natl. Acad. Sci. USA, 90 (1993) 2074–2077. Nait-Oumesmar, B., Decker, L., Lachapelle, F., AvellanaAdalid, V., Bachelin, C. and Van Evercooren, A.B., Progenitor cells of the adult mouse subventricular zone proliferate, migrate and differentiate into oligodendrocytes after demyelination, Eur. J. Neurosci., 11 (1999) 4357–4366. Oumesmar, B.N., Vignais, L., Duhamel-Clerin, E., AvellanaAdalid, V., Rougon, G. and Baron-Van Evercooren, A., Expression of the highly polysialylated neural cell adhesion molecule during postnatal myelination and following chemically induced demyelination of the adult mouse spinal cord, Eur. J. Neurosci., 7 (1995) 480–491. Parent, J.M., Valentin, V.V. and Lowenstein, D.H., Prolonged seizures increase proliferating neuroblasts in the adult rat subventricular zone-olfactory bulb pathway, J. Neurosci., 22 (2002) 3174–3188. Reynolds, B.A. and Weiss, S., Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system (See comments), Science, 255 (1992) 1707–1710. Richards, L.J., Kilpatrick, T.J. and Bartlett, P.F., De novo generation of neuronal cells from the adult mouse brain, Proc. Natl. Acad. Sci. USA, 89 (1992) 8591–8595. Rousselot, P., Lois, C. and Alvarez-Buylla, A., Embryonic (PSA) N-CAM reveals chains of migrating neuroblasts between the lateral ventricle and the olfactory bulb of adult mice, J. Comp. Neurol., 351 (1995) 51–61. Smart, I., The subependymal layer of the mouse brain and its cell production as shown by radioautography after thymidine-H3 injection, J. Comp. Neurol., 116 (1961) 325–338. Szele, F.G. and Chesselet, M.F., Cortical lesions induce an increase in cell number and PSA-NCAM expression in the subventricular zone of adult rats, J. Comp. Neurol., 368 (1996) 439–454.
Cerebral cortex lesions decrease the number of bromodeoxyuridinepositive subventricular zone cells in mice Gwendolyn E. Goings, Bernadeta L. Wibisono, Francis G. Szele* CMIER Neurobiology Program, Children’s Memorial Hospital, Department of Pediatrics, Feinberg School of Medicine, Northwestern University, 2430 N. Halsted, No. 209, Chicago, IL 60614-3394, USA Received 2 May 2002; received in revised form 4 June 2002; accepted 6 June 2002
Abstract We previously showed that cortical lesions in rats increase the number of subventricular zone (SVZ) cells. Here, we examined the response of the SVZ to cortical lesions in mice from 6 h to 35 days later. Whereas the total number of cells did not change, the number of cells in S-phase (bromodeoxyuridine-positive) decreased in a biphasic manner (from 6 h to day 3, and again at days 25–35). In addition, there was a delayed (days 25–35) increase in immunoreactivity for polysialylated neural cell adhesion molecule, a marker of neuroblasts. The results suggest that in mice there are rapid as well as delayed responses in the SVZ to injury of the overlying cerebral cortex. They also show that the SVZ of different mammalian species can exhibit widely divergent responses to the same brain injury. q 2002 Published by Elsevier Science Ireland Ltd. Keywords: Repair; Plasticity; Neurogenesis; Polysialylated neural cell adhesion molecule; Subventricular zone; CD-1; Bromodeoxyuridine
Though mitosis in the brain diminishes greatly after development, it persists in the subventricular zone (SVZ) [2,22]. The SVZ consists of astrocyte-like cells, progenitor cells, and neuroblasts which migrate to the olfactory bulbs to become interneurons [5,6]. All three cell types (except for a subpopulation of astrocytes) divide and mitosis continues indefinitely in vitro in the presence of epidermal growth factor and fibroblast growth factor-2 [6,15,20]. Removal of growth factors, and adhesion, promotes differentiation into the three major neural lineages [15,19]. These experiments showing self-renewal and broad fate potential indicate that the adult SVZ is a major repository of stem cells in the adult central nervous system (CNS). As such, it remains a potential source of cells for neuronal replacement after injury or degenerative illness [2]. In order to potentially attain this goal, the in situ biological response of the SVZ to a variety of brain insults must be examined. The SVZ has been shown to respond to models of injury/illness with decreases [3,11,13] or increases [4,16,18] in the number of cells going through mitosis. Whereas separation of the SVZ from the olfactory bulb caused decreases in bromo-
* Corresponding author. Tel.: 11-773-880-3791; fax: 11-773868-8066. E-mail address: [email protected] (F.G. Szele).
deoxyuridine (BrdU) incorporation in mice [11,13], it caused increases in rat [1], suggesting that it is hazardous to extrapolate similar responses of the SVZ to a given injury across species. We reported before that cortical lesions cause significant increases in cell numbers and polysialylated neural cell adhesion molecule (PSA-NCAM) immunoreactivity in the SVZ of rats, suggesting that the number of neuroblasts increased [23]. Whereas the number of cells increased two-fold after cortical lesion, the number cells going through S-phase did not change consistently [23]. As we plan to use genetically engineered mice to examine the response of the SVZ to brain injury, we began by measuring cell numbers, BrdU incorporation, and PSANCAM immunoreactivity in the SVZ of adult male CD-1 mice after cortical injury. Aspiration cortical lesions (Fig. 1A) were conducted similar to Szele and Chesselet [23]. Adult (.25 g), male CD-1 mice were anesthetized with Ketamine (100 mg/kg)/ Xylazine (10 mg/kg), and the cerebral cortex was removed from the Bregma to 1.2 mm anterior to Bregma. The lesion was restricted to the primary motor cortex, the forelimb somatosensory cortex, and the rostral-most aspect of the hindlimb somatosensory cortex. Removal of this region may result in slight impairment of specific forelimb placement tasks, but does not impair normal feeding or grooming.
0304-3940/02/$ - see front matter q 2002 Published by Elsevier Science Ireland Ltd. PII: S03 04 - 394 0( 0 2) 00 61 1- 0
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Fig. 1. (A) Schematic coronal section showing the SVZ in red. In some analyses, the SVZ was subdivided into its dorsolateral component (dl svz, green rectangle) and its striatal component (str svz, blue rectangle). (B) DAPI staining in striatal SVZ. Nuclei were stained with DAPI (Sigma, St. Louis, MO; 40 mg/ml) for 10 min. Note the high density of cells in the SVZ compared with the striatum, which allows easy microscopic distinction of the two areas for quantification. (C) BrdU immunohistochemistry of dividing cells (arrow shows typical example) in the dl svz. (D) PSA-NCAM immunohistochemistry in the dl svz showing typical labeling (arrow) and example of area quantified (outline). Control animals in B–D. al, area lesioned; spt, septum; str, striatum.
The lesions spared the secondary motor cortex, the cingulate cortex, the somatosensory barrel fields, and the primary somatosensory cortex. Mice were allowed to survive from 6 h to 35 days after cortical injury and then were perfused with 4% paraformaldehyde. In all quantifications, 20 mm free-floating sections were cut on a freezing cryostat and stored in cryoprotectant at 220 8C. Every sixth section from the crossing of the corpus callosum in the (anterior striatum) to the decussation of the anterior commissure (posterior striatum) was collected and quantified. The number of 4 0 , 6-diamidino-2-phenylindole dihydrochloride hydrate (DAPI)-stained cells (Fig. 2), BrdU-positive cells (Fig. 3), or surface area of PSA-NCAM immunoreactivity (Fig. 4) per section was averaged and then these values averaged across animals. All statistical comparisons between non-lesioned controls and lesioned animals were made using unpaired two-tailed Student’s t-tests. Significance was considered to be P , 0:05. All values are expressed as averages ^ SEM. An initial cohort of mice was sacrificed at 2 (N ¼ 3), 3 (N ¼ 5), 6 (N ¼ 5) and 15 (N ¼ 5) days after cortical lesion and compared with non-lesioned controls (N ¼ 5). The controls were anesthetized only, in parallel with the lesioned mice, and sacrificed in parallel 3 (N ¼ 1), 6 (N ¼ 2), and 15 (N ¼ 2) days later. In this, and in subsequent experiments, we counted the total number of cells in the dorsolateral SVZ and in the striatal SVZ (Fig. 1A; Szele and Chesselet [23])
Fig. 2. The total number of cells per section in the SVZ. DAPIstained nuclei were counted at 40 £ using the Neurolucida image analysis system (Microbrightfield, Williston, VT); cells were visualized on a computer monitor and confirmed by focusing through the cell while visualizing it through the microscope eyepiece. Lineage relationships amongst cells in the walls of the lateral ventricles remain uncertain and there is a formal possibility that both ependymal cells (which line the ventricle) and SVZ cells behave like stem cells in this region [7,12]. Therefore, for total cell counts, we counted ependymal cells together with SVZ cells. Error bars show standard errors, and significance was tested for using unpaired, two-tailed, Student’s t-tests.
separately but found very similar results in the two subdivisions and therefore combined numbers. There was no significant change in the total SVZ cell numbers after lesion compared with controls, (unpaired two-tailed Student’s ttest, not shown). We then counted BrdU-positive cells and found decreases at 2 days (109 ^ 11 vs. 76 ^ 14; P ¼ not significant (n.s.)) and statistically significant decreases at 3 days (109 ^ 11 vs. 80 ^ 4; P ¼ 0:03) after cortical lesion.
Fig. 3. The number of BrdU-positive cells per section in the SVZ. BrdU, incorporated into the cell nucleus during S-phase of the cell cycle, is an indirect marker of mitosis. Single intraperitoneal injections of BrdU (200 mg/kg) were given 2 h prior to sacrifice, sections were incubated in 0.1 M HCl for 20 min at 60 8C, and then in antiBrdU (1:1000, polyclonal, Fitzgerald, MA), and biotinylated secondaries (1:200, Jackson Immunoresearch, PA). BrdU-positive cells in the dorsolateral SVZ and striatal SVZ were counted visually with a 40 £ objective at the same time. *P ¼ 0:05–0:001; **P , 0:001. Error bars show standard errors, and significance was tested for using unpaired, two-tailed, Student’s t-tests.
G.E. Goings et al. / Neuroscience Letters 329 (2002) 161–164
Fig. 4. The area per section occupied by positive PSA-NCAM immunoreactivity in the dorsolateral component of the SVZ (dl svz). Sections were exposed to anti-PSA-NCAM [8] (1:5000, monoclonal, G. Rougon, Marseilles, France) and then biotinylated secondaries (1:200, Jackson Immunoresearch, PA). Images of the dl svz were captured with a Polaroid DMC digital camera (Polaroid, Boston, MA). Using NIH Image (Shareware) the dl svz was outlined (see Fig. 1) and the percentage surface area above a threshold quantified. The threshold was determined using positive PSA-NCAM immunoreactivity in non-lesioned controls and was kept constant for all sections. *P ¼ 0:05–0:001. Error bars show standard errors, and significance was tested for using unpaired, two-tailed, Student’s t-tests.
To begin to assess the response to brain injury of specific cell subtypes in the SVZ, we performed immunohistochemistry with the Men-B antibody [8] to quantify levels of PSANCAM which is selectively expressed by the neuroblasts of the SVZ [21]. PSA-NCAM immunoreactivity was transiently decreased at 2 days after cortical lesion (5.6 ^ 0.9 vs. 1.7 ^ 0.4 mm 2; P ¼ 0:02), but by 15 days, had risen above control values (5.6 ^ 0.9 vs. 9.0 ^ 2.4 mm 2; P ¼ n.s.) To confirm these findings, and to look for earlier and later changes, we sacrificed a second cohort of mice at 6 h (N ¼ 6), and at 1 (N ¼ 6), 2 (N ¼ 5), 3 (N ¼ 5), 6 (N ¼ 6), 15 (N ¼ 6), 25 (N ¼ 7) and 35 days (N ¼ 6) after cortical lesions and compared them with non-lesioned controls (N ¼ 9). The controls in the second cohort were anesthetized only, in parallel with the lesioned mice, and sacrificed in parallel 6 h (N ¼ 3), and 2 days later (N ¼ 6). As in the preliminary group, we found no changes in total cell numbers in the SVZ after lesion (Fig. 2). The number of BrdU-positive cells in the non-lesioned control groups was not significantly different (100 ^ 8 vs. 102 ^ 6; P ¼ 0:85). A biphasic decrease in numbers of BrdU-positive cells was found in the SVZ after cortical lesions (Fig. 3). These decreases reached statistical significance already at 6 h (101 ^ 4 vs. 84 ^ 7; P ¼ 0:04), and at 1 (101 ^ 4 vs. 70 ^ 6; P ¼ 0:001), 2 (101 ^ 4 vs. 62 ^ 6; P ¼ 0:0001), 3 (101 ^ 4 vs. 67 ^ 6; P ¼ 0:0004), 25 (101 ^ 4 vs. 73 ^ 8; P ¼ 0:004), and 35 days (101 ^ 4 vs. 70 ^ 5; P ¼ 0:0006) after lesion. The numbers of BrdU-positive cells at 6 (86 ^ 7) and 15 days (86 ^ 10) were also fewer
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than controls (101 ^ 4), however, these differences did not reach statistical significance. PSA-NCAM in this cohort of animals was again transiently decreased, although this did not reach significance. Also, the decrease was earlier than in our first study (6 h vs. 2 days). PSA-NCAM then increased at days 25 (15.0 ^ 2.8 vs. 33.3 ^ 5.6 mm 2; P ¼ 0:007) and 35 (15.0 ^ 2.8 vs. 46.0 ^ 11.2 mm 2; P ¼ 0:005; Fig. 4). These results show that the SVZ of different species can respond dissimilarly to the same insult. In rat, we had shown increases in total cell number but no changes in BrdU incorporation, whereas here we show no change in total cell number but decreases in the number of cells incorporating BrdU. In addition, we show here that the response of the SVZ to cortical injury in mice is: (1), rapid; (2), can last approximately up to one month; and (3), shows a biphasic pattern. Others have also found delayed changes in numbers of BrdU-positive cells in the SVZ after models of injury or disease [4,13]. Cell numbers in the SVZ can be regulated by a combination of cell proliferation, migration, and apoptosis. Thus, the decreases in cell division may have been offset by decreases in migration to the olfactory bulb or alternatively decreases in cell death. Either scenario could have resulted in the total cell number not changing significantly. In considering what molecular mechanisms cause decreases in the number of BrdU-positive cells, we have focused on serotonin. A serotonergic neurotoxin (5,7-dihydroxytryptamine) and an inhibitor of serotonin synthesis (pchlorophenylalanine) both caused decreases in BrdU incorporation [3]. Interestingly, olfactory bulbectomy, which also affects BrdU incorporation in the SVZ [1,11,13], causes changes in serotonergic neurotransmission [10]. It is unknown whether cortical lesions influence the serotonergic system in rats or mice, so we will test the hypotheses that the serotonergic innervation of the SVZ changes after cortical lesion in mouse and that this decreases the number of BrdUpositive cells. Interestingly, in rats, changes in cell numbers were only associated with changes in PSA-NCAM, but not with markers of SVZ astrocytes or progenitor cells [23]. Similarly, in mouse, changes in numbers of dividing cells were temporally correlated with early decreases and then late increases in PSA-NCAM. We looked for reactive astrocytosis in the SVZ of mice after cortical lesions and although we found robust glial fibrillary acidic protein (GFAP) and vimentin immunoreactivity surrounding the lesion, GFAP and vimentin levels were not changed in the SVZ (data not shown), similar to our studies in the rat [23]. PSA-NCAM can also be expressed in vitro by oligodendrocyte precursors isolated from P1 rats [9], and in vivo by oligodendrocytes during remyelination of the spinal cord [17] as well as in reactive astrocytes in the spinal cord and hippocampus [14,17]. While under normal conditions SVZ neuroblasts express PSA-NCAM [6,15], the formal possibility remains that after cortical injury, other SVZ cell subtypes may express this marker. In light of the results presented in this paper, it will be very important and interesting to exam-
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