Long-Lasting Regeneration After Ischemia in the Cerebral Cortex
Background and Purpose— Because fibroblast growth factor 2 is a mitogen for central nervous system stem cells, we explored whether long-term fibroblast growth factor 2 delivery to the brain can improve functional outcome and induce cortical neurogenesis after ischemia.
Methods— Rats underwent permanent distal middle cerebral artery occlusion resulting in an ischemic injury limited to the cortex. We used an adeno-associated virus transfection system to induce long-term fibroblast growth factor 2 expression and monitored behavioral and histological changes.
Results— Treatment increased the number of proliferating cells and improved motor behavior. Neurogenesis continued throughout 90 days after the ischemia, and the occurrence of newly generated cells with characteristics of neural precursors and immature neurons was most evident 90 days after treatment.
Conclusions— Focal cortical ischemia elicits an ongoing neurogenic response that can be enhanced with fibroblast growth factor 2 leading to improved functional outcome.
Cell therapy has been proposed in ischemia but is complex because it requires harvesting appropriate cells, their expansion in vitro and transplantation.1 Previous studies showed that endogenous precursors can generate new neurons in the ischemic striatum but not in the cortex, despite a large lesion burden at this site.2,3 However, newer studies show that repair mechanisms may allow cortical recovery as well,4–6 and thus cortical regeneration after ischemia remains controversial. Our study was prompted by data showing a slow and growth factor–dependent recovery from transient global ischemia.7 Fibroblast growth factor 2 (FGF2) is a major mitogen for neural stem cells (NSC).8 An increase in infarct volume in mice lacking FGF2 and amelioration of focal deficits by FGF2 confirm the importance of this signaling pathway in the ischemic cortex.9 We therefore used an adeno-associated viral (AAV) vector10 to deliver FGF2 over long periods of time after ischemic cortical ischemia and searched for the effects on behavioral recovery and neurogenesis.
Materials and Methods
AAV helper plasmids were generously provided by Drs Rabinowitz and Samulski (University of North Carolina). Recombinant AAV vectors (Figure 1) were generated and purified as previously described11 (for details please refer to supplemental Materials and Methods, available online at http://stroke.ahajournals.org).
Viruses were injected into 2 separate locations bordering the infarct immediately after induction of cerebral ischemia (Figure 1) using a convection system to enhance viral distribution. Overall, 40×109 viral particles were injected into each site.
Focal Ischemia in Rats
Spontaneously hypertensive rats underwent permanent distal middle cerebral artery occlusion (PMCAO) by craniotomy and electrocoagulation.12 Animals were divided into 3 groups: A: sham PMCAO+AAV-FGF2 (n=3); B: PMCAO+AAV (n=28); and C: PMCAO+AAV-FGF2 (n=30). Animals were perfused 7 (n=2 in C), 21 (n=3 in B and C), 30 (n=21 in B and C, and n=3 in A) or 90 days after the procedure (n=4 in B and C). All evaluation listed below were performed blinded to the treatment status.
Animals received IP injection of Bromo-deoxy-Uridine (BrdU; 50 mg/kg q, 12 hours) on days 1 to 5 post-op to label dividing cells.
We used ICV Cytosine Arabinosine (Ara-C) injection via mini-osmotic pumps in rats immediately after PMCAO to selectively target dividing cells in the subventricular zone (SVZ) and eliminate their contribution to recovery.7 Rats were treated with ARA-C (2% in 0.9% NaCl) administered at a total dose of 0.5 mL into the lateral ventricle via alzet mini-osmotic pumps (model 1007d). Animals were divided into groups similarly to groups B and C described above and were given Ara-C or vehicle (n=3/group) for 7 days.
Proliferative History Experiments
We used a double-labeling strategy to determine proliferative history in dividing cells after ischemia. PMCAO rats (n=3) were injected with Chloro-deoxy-Uridine (CldU) on days 1 to 5 and Iodo-deoxy-Uridine on days 42 to 46 as described.13 Animals were perfused 60 days postischemia and their brains were evaluated.13
All animals were examined with a standardized disability scale (maximal deficit=10 points; no deficit=0) that incorporates motor function, limb placement tests and locomotion as previously described14 (for details please refer to supplemental Materials and Methods).
Animals were killed 30 days postischemia. Brain slices 200 μm apart were stained with Geimsa stain. Measurement of the lesioned area was done with image-analysis software in 10 rats killed at 30 days in each group.
Brains were frozen-sectioned at 16 μm and double- or triple-stained for immunohistochemistry evaluation using fate-specific antibodies (see supplemental Materials and Methods). Confocal images were taken under Zeiss LSM-510 microscope and multiple z-sections were obtained to ensure colocalization.
Immunopositive cells were counted in a blinded fashion using a nonbiased system. Cells were enumerated on slides obtained from homologous coronal slices containing the infarct between +0.2 and −3.8 mm from bregma. Cells were counted in 4 regions of interest (ROI): SVZ on the infarct side, corpus callosum and white matter on both sides and the peri-infarct cortex (Figure 1). In each slide 7 high-power fields (x630) separated by 150 μm were counted in each ROI. At least 5 slides were counted for each animal.
Analysis was performed with the SigmaStat package (SPSS Inc). Data are presented as mean±SD and comparisons between all groups (sham and the experimental groups) were performed using analysis of variance (ANOVA) with Bonferroni correction.
FGF2 Expression in the Brain
AAV vectors delivered the transgene to a large region of the injured hemisphere with prolonged expression time. FGF2 expressing cells were seen throughout the infarcted hemisphere and were not limited to the injection sites. The results show that many double-positive cells for FGF2 and green fluorescent protein (GFP) were present in the injected hemisphere 30 days postischemia in the AAV-FGF group (>65% of cells) but not in the AAV controls (Figure 1A and 1B) and that many of the infected cells were neurons (>85%; Figure 1C and 1D, and supplemental Figure I, available online at http://stroke.ahajournals.org). FGF2 was also expressed by astrocytes in the ischemic hemisphere (supplemental Figure I) but no GFP/glial fibrillary acidic protein (GFAP)+ cells were observed, suggesting that this FGF2 expression represents a response to injury and does not result from AAV transfection.
Distal PMCAO resulted in lesions occupying the frontolateral cortex but left intact the vascular supply to the SVZ.15 Infarct size tended to be larger in the FGF2 group but the intergroup difference was not statistically significant (by 1-way ANOVA with 2 degrees of freedom, F=2.3; P=0.31; Figure 2A).
Neurogenesis at 7 Days After Ischemia
At 7 days postischemia a robust increase in the number of newborn BrdU+ cells was observed in the SVZ, the corpus callosum and in the peri-infarct area. This increment was larger in FGF2-treated rats (Figure 2B). About 10% of these BrdU+ cells colabeled with microglia markers (see below). When Ara-C was injected into the lateral ventricle for 7 days,7 the proliferative response observed in the peri-infarct cortex was significantly reduced but not completely abolished (Figure 2B). Most cells that expressed BrdU in the cortex (≈85%) after 7 days of treatment with ARA-C were also positive for the microglial markers ED1 and CD11b. These results show that cell proliferation occurs in the injured cortex and suggest that most newborn cells with a nonmicroglial phenotype in the cortex originate in the SVZ. None of the few nonmicroglial BrdU+ cells seen in the cortex after application of Ara-C expressed NeuN or GFAP and most expressed nestin.
Neurogenesis at 30 to 90 Days After Ischemia
In sham-operated rats killed 30 days after the surgery, 97% of BrdU+ cells were seen in the SVZ and another 3% in the white matter. Extremely rare BrdU+ cells were observed in the cortex (4 cells in 60 high-power fields). The number of SVZ BrdU+ cells in sham-operated animals was significantly smaller than their number in rats with stroke (2-fold less). Most of these cells were nestin+ (78%) at the SVZ. None of the BrdU+ cells in these animals coexpressed neuronal or glial markers in the white matter or cortex. In ischemic rats BrdU+ cells were seen in the SVZ, the white and the gray matter surrounding the infarct at days 30 and 90 after focal ischemia in both groups (Figure 2C and 2D). Surprisingly, only a few newborn cells were present in the striatum. A milder increase in the number of newborn cells was also observed in the contralateral brain. A significant reduction in the number of newborn cells was observed in the peri-infarct area between day 7 and 30 postischemia (Figure 2B and 2C). This cell loss suggests that most cells born early after the ischemia do not survive in the intermediate-term.
The number of BrdU+ cells in the SVZ remained constant between day 30 and 90 postischemia (Figure 2C and 2D). In marked contrast, the number of BrdU+ cells in the peri-infarct cortex increased in all groups at 90 days after the ischemia, suggesting newborn cell accumulation at this area over time. The late peak in the number of BrdU+ cells was more robust in the AAV-FGF group (Figure 2D).
At 7 days after ischemia, 10% of the BrdU+ cells in the cortex coexpressed the macrophage/microglia markers ED1 (supplemental Figure II, available online at http://stroke.ahajournals.org), cd11b or IBA-1 (data not shown). The percentage of BrdU+ cells coexpressing microglia markers remained high for 30 days postischemia in all groups but <1% of the BrdU+ cells expressed microglia antigens 90 days postischemia (supplemental Figure II). Similar inflammatory responses were observed in both groups as judged by expression of leukocyte and microglia markers. These results suggest that an increase in noninflammatory BrdU+ cells in the cortex occurs only after the inflammatory response declines.
Timing of Neurogenesis
To further evaluate whether cells continue to divide at the peri-infarct area or divide only when at SVZ, we used a sequential labeling protocol recently described.13 Briefly, cells were labeled first with CldU on days 1 to 5 postischemia and then with IdU 42 to 46 days after the ischemia. The results (Figure 3) demonstrate that most cells continue to divide at the infarct boundary as many double-labeled cells were seen at this location. In contrast, most dividing cells at the SVZ on day 60 were only labeled with IdU. These results imply that progenitors first divide at the SVZ and on migration to the infarct zone continue to divide over a long period of time, whereas cells labeled at the SVZ only divide there for a limited time.
Taken together these results suggest that neurogenesis is an ongoing process that continues over long periods of time and that this process can be significantly enhanced with FGF2 stimulation.
Newborn Cell Identity
The cellular changes that follow ischemia were further analyzed by use of antibodies that identify central nervous system stem cells, precursors and mature cells (Figure 4).
At 7 days postischemia only rare BrdU+ cells in the cortex coexpressed neuronal or glial markers (0.17% BrdU+/NeuN+, 1.8% BrdU+/GFAP+).5,16 Most of these cells expressed markers for immature NSC including SOX2 and nestin and some of these cells (≈60%) also expressed vascular endothelial growth factor (VEGF).
At 30 days postischemia, SOX2+ NSC17 were abundant in the ventricular region and at the infarct boundary zone where they were particularly prevalent in the subcortical white matter (Figure 4B). VEGF is expressed in the precursor cells of the ventricular zone and later in immature neurons and subsequently in astrocytes.18 VEGF expression was analyzed because this secreted protein promotes vascular development and may also act as a neural survival signal.18 The majority of SOX2+ cells coexpressed VEGF (Figure 4B’). The number of SOX2+ cells was increased in the AAV-FGF group almost 2-fold (Figure 4C). Many neural progenitor cells (NPC) expressing the proneural transcription factors MASH1 and PAX619 were seen around the infarct (peri-infarct zone; Figure 4D through 4E). Treatment with AAV-FGF2 significantly increased the numbers of NPC (Figure 4F). In AAV-FGF-treated animals, 66.8±8.6% of the BrdU+ cells were also PAX6+ (versus 15.5±4.5% in AAV-controls; by 1-way ANOVA with 1 degree of freedom, F=23.07; P<0.001). Importantly, the absolute numbers of SOX2+ cells at the SVZ (Figure 4A), as well as the number of PAX6+ or MASH1+ cells in the peri-infarct area (Figure 4B and 4C) were much larger than the numbers of BrdU+ cells indicating that labeling with BrdU greatly underestimates the number of progenitors. At 30 days postischemia we also observed some BrdU+ cells that coexpressed the migrating neuroblast marker doublecortin16 in the peri-infarct area (Figure 4G). Moreover, 12.9±2.9% of the BrdU+ cells coexpressed the immature neuronal antigen HU16 in the AAV-FGF group (versus 4.03±1.2% in AAV-controls; P=0.027 by 1-way ANOVA with 1 degree of freedom, F=4.92; Figure 4H). At this time coexpression of BrdU and GFAP was commonly observed in AAV-controls (≈65% of BrdU+, Figure 4H) and less often in AAV-FGF treated rats (≈21% of BrdU+; P<0.001 by 1-way ANOVA with 1 degree of freedom, F=96.4).
At 90 days after ischemia, expression of the immature markers SOX2 and nestin was still present in most of the BrdU+ cells in the white matter and the region of cortex immediately adjacent to the infarct border (Figure 5A and 5B). In regions of cortex farther from the lesion, BrdU+ cells expressing doublecortin (Figure 5C) and the mature neuronal marker NeuN were present (Figure 5D–D′). Immature and mature newborn neurons were more frequent in the AAV-FGF group (NeuN 29.78±3.4% and Hu 22.1+5.8% versus 12.05±3.5% and 1.6±0.6%, respectively; P<0.001 for both by 1-way ANOVA with 1 degree of freedom, F=12.87; Figure 5E). In contrast, newborn cells coexpressing GFAP were significantly less frequent in the AAV-FGF group (P<0.001 by 1-way ANOVA with 1 degree of freedom, F=13.05; Figure 5E).
Collectively, these results show that by 30 and 90 days after the ischemia, FGF2 treatment results in a robust increase in the number of NSC and NPC at the SVZ and white matter and of NPC and immature neurons in the peri-infarct cortex. Importantly, newborn immature neurons could only be observed in the peri-infarct cortex located a few cell layers away from the immediate infarct border where only more primitive SOX2+/nestin+ cells were seen.
Impact of Newborn Cells on Behavioral Changes
Animals treated with AAV-FGF2 had significantly better improvement in behavior (Figure 5F). Differences between groups became apparent starting 20 days postischemia and became more robust with time as animals treated with FGF2 continued to improve.
The data presented here suggest that: (1) partial behavioral recovery can occur after a focal ischemic injury restricted to the cortex; (2) newborn cells are first found in the SVZ, subsequently migrate through white matter, and accumulate in the cortex and subcortical white matter areas surrounding the infarct forming a regenerative zone with many primitive neural precursor cells immediately adjacent to the injured tissue; (3) this accumulation is an ongoing process that occurs over long periods of time; and (4) that AAV-FGF2 promotes the expression of genes characteristic of telencephalic precursors and immature neurons in the cortical regions surrounding the infarct. The data suggest that therapy based on endogenous repair would target distinct cells with features of neural precursors or immature neurons at specific times after ischemia.
Reports of an endogenous neurogenic response restricted to the striatum after a large focal ischemia in the forebrain3,6 and of a slow endogenous repair process in the hippocampus after global ischemia7 have been published before. Cellular regeneration in the striatum and hippocampus might be a consequence of their known proximity to proliferative zones that are known to generate neurons in the adult. However, the presence of cortical regeneration is still debatable.2 Although cortical neurogenesis is controversial, very localized lesions of neurons in this area have been shown to generate cell replacement with newly generated neurons surviving for long periods and extending axons to appropriate distant target sites.4 Occluding the MCA at the pial surface results in ischemic injury restricted to the cerebral cortex, rapid activation of proliferation in the SVZ and migration of newborn cells into the parenchyma.5,15 Newborn cells were recently shown to continuously form after ischemic injury involving the striatum.6 In that set of experiments BrdU was administered either within the first 2 weeks after ischemic onset or at weeks 7 to 8 after ischemia. BrdU/DCX double-positive cells were found to be formed throughout the experiment, and the SVZ was found to be of increased size over time and to continuously produce DCX+ cells. The data presented here extends these reports by suggesting that persistent stimulation with FGF2 promotes long-lasting precursors in the ventricular region that migrate to the peri-infarcted area where they continue to divide over time, resulting in a continuous accumulation of neural precursors in the cortex. Our study is limited because we did not perform a direct lineage analysis to ensure that all progenitors emerge from the SVZ. Future experiments with labeling of NPC at the SVZ with retroviruses expressing GFP and tracking these cells at intervals until at the cortex should give the complete answer to whether all NPCs emerge from the SVZ or whether at least some emerge from other locations including the cortex, where they may reside in a quiescent form. In agreement with 2 recent articles that could not detect newborn neurons at the immediate infarct border,5,20 our results show that newborn progenitors are constantly present in the area immediately bordering the lesion and that a second domain of newly generated cells with the properties of immature neurons exists only a few cell-layers deeper to the lesion border. The origin of these 2 distinct zones is unknown. It is possible that factors such as inflammation, metabolic stress or local inhibitory components within the white matter surrounding the infarct prevent terminal differentiation of newborn cells into neurons. Importantly, newborn cells expressing neuronal antigens could be seen far more often in the group treated with FGF2 compared with AAV-controls.
It is also important to note that newborn progenitors only began to accumulate in significant numbers at timepoints after the major microglial inflammatory response declined. Thus, early inflammatory changes may hamper initial efforts for neurogenesis after ischemia and probably negatively influence functional outcome.
The lack of spontaneous behavioral recovery in the AAV group is somewhat surprising because some degree of recovery is expected in the stroke model used and was indeed seen when we compared the results to historical controls at our laboratory. However, this may be attributable to the proinflammatory effects of AAV (however minor) which may have compounded on the ischemic damage. At least part of this proinflammatory damage which was similar in the AAV- FGF group was probably reversed by excess neurogenesis observed in the latter group.
The exact mechanisms responsible for behavioral improvements observed in AAV-FGF animals remain unclear because the generation of newborn neurons appears to be a lengthy process and newborn neurons were only observed in significant numbers relatively late after lesioning. FGF2 may exert neuroprotective effects in ischemia,9 but the lack of significant differences in infarct volumes, as well as the late improvement in functional deficits, argues against a protective effect of FGF2 in our experimental settings. Our results indicate that the mere increase in the numbers of newborn cells is associated with better functional outcomes after cortical ischemia. The results also show that young, undifferentiated SOX2+/nestin+ cells secrete trophic factors such as VEGF which has been shown to salvage motor neurons.21 Furthermore, VEGF-induced angiogenesis in the peri-infarct area,18 as well as the presence of young cells that are capable of metabolizing and neutralizing excitatory amino acids and free radicals, may also contribute to improved metabolism and function of surviving neurons.
Importantly, FGF2 treatment resulted in increased numbers of progenitors in the cortex only in the presence of ischemia. This implies that factors related to the presence of ischemic injury (eg, stromal growth factor 1α) are necessary to further increase neurogenesis and induce migration of newborn cells to the cortex.
Taken together, our results suggest that in ischemic lesions restricted to the cortex, long-term stimulation with FGF2 can stimulate regenerative processes that induce partial behavioral recovery. These preliminary data encourage studies to determine the regenerative consequences of these long-term cellular responses to further define appropriate ligands that can promote recovery.
- Received July 23, 2006.
- Revision received August 23, 2006.
- Accepted September 10, 2006.
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Leker RR, Teichner A, Grigoriadis N, Ovadia H, Brenneman DE, Fridkin M, Giladi E, Romano J, Gozes I. Nap, a femtomolar-acting peptide, protects the brain against ischemic injury by reducing apoptotic death. Stroke. 2002; 33: 1085–1092.
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Supplemental Materials and Methods
Cells and Plasmids
Two hundred and ninety-three HEK cells (ATCC) were cultured in Dulbecco modified Eagle medium (Invitrogen) and maintained at 5% CO2 and 37°C. Media was supplemented with 10% fetal bovine serum and penicillin-streptomycin. Mouse FGF-2 cDNA (ATCC #M30644) was obtained by HindIII/SpeI digest and ligated directly into p-TR-CIG to generate pTR-cFGF2-IG. The plasmid pTR-CMVeGFP is a modification of p-TR-UF2 where the 5′ Not I site was changed to HindIII. This allowed for directed cloning of the CMV promoter in place of the NSE promoter at the EcoRI/HindIII site of pTR-UF41,2 (generously provided by N. Muzyczka, University of Florida, Gainesville, FL) to generate, pTR-CIG.
Virus was purified from 293 HEK cells at 60 hours post-transfection, cells and media were collected, spun down at 3000 RPM. Cell pellet was resuspended in 5 mL, 10 mmol/L sodium phosphate, 150 mmol/L NaCl, 1 mmol/L MgCl, transferred to a fresh 50-mL centrifuge tube and 10% DOC was added to a final concentration of0.5%.
Benonase was added to a final concentration of 50 U/mL and incubated at 37°C for 60 minutes. The lysate was cleared by centrifugation at 3500 RPM and then purified by iodixanol density gradient centrifugation. Peak fractions were determined by dot blot hybridization, probed with a GFP fragment. Peak fractions were extensively dialyzed against PBS, concentrated using Centricon 100K filtering devices (Amicon). Vectors were stored at −80C° in small aliquots.
Adult male spontaneously hypertensive rats weighing >300 g were housed in accordance to NIH guidelines. All surgical procedures were approved by the Institutional Animal Care and Use committee.
The following stereotaxic coordinates were used: First site; +4.2 mm anterior-posterior, −1 mm lateral-medial, −2 mm dorsal-ventral from bregma; Second site: −4.8 mm anterior-posterior, −5.5 mm lateral-medial, −8 mm dorsal-ventral from bregma.
The pumps (ALZET mini-osmotic pump model 1007D delivering at a rate of 0.5 μL/hour for 7 days) were implanted immediately after AAV injection and an intracerebral cannula was inserted into the right lateral ventricle (stereotaxic coordinates from bregma: anterior-posterior +0 mm, lateral −1.5 mm, dorso-ventral 3.3 mm). The animals were perfused transcardially with ice-cold 50 mL physiological saline followed by 4% paraformaldehide 8 days after stroke onset. The brains were removed and prepared for immunohistological studies.
Motor Disability Score
Animals are scored 1 point for each of the following parameters: flexion of the forelimb contralateral to the stroke when momentarily hung by the tail, extension of the contralateral hind limb when pulled from the table and rotation to the paretic side against resistance. Additionally, 1 point was scored for circling motion to the paretic side when attempting to walk, 1 point for failure to walk out of a circle 50 cm in diameter within 10 seconds, 2 points for failure to leave the circle within 20 seconds and 3 points for inability to exit the circle within 60 seconds. Additionally, 1 point each was scored for inability to extend the paretic forepaw when gently pushed against the table from above, laterally and sideways. Thus, an animal with a maximal deficit scored 10 points and an animal with no deficit scored 0 points.
Antibodies used include: rat anti-BrdU (1:200 Accurate), rabbit anti-nestin (1:5000), rabbit anti-GFAP (DAKO 1:200), mouse anti-Cd11b and mouse anti-ED1 (Serotec 1:100), rabbit anti-IBA (Wako 1:2000), Guinea pig anti-doublecortin (1:2000), mouse anti-PSA-NCAM (1:200), mouse anti-SOX2 (R&D systems 1:10), rabbit anti-SOX1 and SOX2 (1:400), rabbit anti-MASH1 (1:200), rabbit anti-PAX6 (1:500), mouse anti-NeuN (1:200), mouse anti-CNPase (1:100), all from Chemicon, Temecula, Calif. Double staining was visualized with Alexa Fluor 488- and Alexa Fluor 546-conjugated secondary antibodies (Molecular Probe), and nuclei were visualized with DAPI (Sigma).
For BrdU-staining, mounted frozen sections were postfixed for 15 minutes in 4% paraformaldehide and then washed in PBSX1 3 times. Slides were incubated in 2N HCL at 37°C for 30 minutes and then washed in PBSX1 3 times. Slides were then blocked with 5% normal goat serum (except for instances where the primary antibody for double immunohistochemistry was prepared in goat in which case donkey serum was used) containing 0.01% triton and incubated with primary antibodies (anti BrdU from Accurate at 1:200 and another fate specific antibody) overnight at 4°C. Slides were then washed and incubated with secondary antibodies at room temperature. Nuclei were then stained with DAPI.