|Home | About | Journals | Submit | Contact Us | Français|
Promoting neural regeneration after cerebral infarction has emerged as a potential approach for the treatment of stroke. Insulin-like growth factor 1 (IGF-1) possesses both neurotrophic and angiogenic properties. The aim of this study is to determine whether postischemic gene transfer of IGF-1 enhances neurovascular regeneration in a mouse model of permanent focal cerebral ischemia. Long-term cerebral IGF-1 overexpression was achieved with adeno-associated viral vector (AAV) via stereotaxic injection at 24 h after stroke. AAV-GFP or saline was injected as control. The success of postischemic gene transduction was confirmed by a strong green fluorescent protein signal and by increased IGF-1 protein expression in the peri-infarct region. Postischemic gene transfer of IGF-1 significantly enhanced vascular density at 8 weeks post stroke in the peri-infarct and injection needle tract area compared to AAV-GFP or saline treatment, as shown by immunohistochemical staining with vascular marker lectin. Furthermore, increased vascular density was associated with improved local vascular perfusion. Immunohistochemical staining with neuronal progenitor marker DCX and cell proliferation marker BrdU indicated that AAV-IGF-1 treatment potently increased neurogenesis compared to AAV-GFP injection. These data demonstrate that postischemic treatment of IGF-1 effectively promoted neural and vascular regeneration in the chronic stage of cerebral infarction.
There is great enthusiasm to repair the brain after stroke injury because attenuating the acute ischemic damage that often becomes irreversible within a few hours of ischemic onset is difficult in clinical applications. Repairing the damaged brain and promoting functional restoration during the chronic phase of the disease therefore represent attractive alternative approaches. Within this paradigm, insulin-like growth factor 1 (IGF-1) stands out among many growth factors for its wide-spectrum trophic activities. IGF-1 possesses both neurotrophic and angiogenic effects, and has been demonstrated to protect the central nervous system from experimental ischemic stroke injury (Guan et al, 2001; Leinninger and Feldman, 2005; Liu et al, 2001; Loddick et al, 1998; Schabitz et al, 2001), as well as cultured primary neurons from various types of insults (Cheng and Mattson, 1992; Dore et al, 1997). IGF-1 has also been implicated in playing a critical role in cerebral angiogenesis during development and in adulthood (Conti et al, 2004; Lopez-Lopez et al, 2004). Stroke patients with poor outcome are associated with low levels of IGF-1, suggesting that endogenous IGF-1 level impacts the evolution of cerebral infarction (Denti et al, 2004).
We have reported that AAV mediated IGF-1 overexpression before ischemic stroke enhanced neurovascular remodeling, and improved functional outcome in a mouse model of permanent focal cerebral ischemia (Zhu et al, 2008). AAV-IGF-1 was injected before MCAO to allow maximum expression of IGF-1. This experimental paradigm minimizes the use of viral vector mediated gene transfer in clinical applications. Delivery of trophic growth factors after injury is more relevant to clinical settings, and has therapeutic potential. In this study, we performed postischemic gene transfer of IGF-1 to assess the neural restorative effects of IGF-1.
The study was approved by the University of California, San Francisco Committee of Animal Research and conformed to the NIH Guidelines for use of animals in research. Adult CD-1 mice weighing 30-35 g were subjected to permanent focal ischemia by distal middle cerebral artery occlusion (MCAO) (Zhu et al, 2008). The left middle cerebral artery (MCA) was occluded by electrical coagulation just proximal to the pyriform branch. Body temperature was maintained at 37 ± 0.5°C by using a thermal blanket throughout the surgical procedure. Surface cerebral blood flow (sCBF) was monitored during MCAO using a laser Doppler flowmeter (Vasamedics Inc.). Mice with sCBF that was more than 15% of the baseline were excluded from the experiment.
Viral transduction was performed as described (Zhu et al, 2008). Briefly, 24 h after MCAO, mice were placed in a stereotactic frame (Kopf, Tujunga, CA) under anesthesia, and a burr hole was drilled 2.5 mm lateral to the sagittal suture and 1 mm posterior to the coronal suture. A 10μl Hamilton syringe was slowly inserted into the left caudate nucleus (3.0 mm deep from the dura). Four μl viral suspension (AAV-IGF-1 or AAV-GFP) containing 2 × 1010 vector genomes were injected into the left hemisphere (injection sites are depicted in Figure 1A). Recombinant AAV serotype 2 (rAAV2) containing human IGF-1 or GFP was provided by Ceregene Inc. (San Diego, CA). AAV serotype 2 has been shown to transfect only post-mitotic neurons (Burger et al, 2005b). Systemic co-administration of mannitol has been demonstrated to profoundly amplify rAAV2 transduction efficiency (Burger et al, 2005b). Thus, in our experiments, the mice were injected intraperitoneally with 3 ml of sterile 25% mannitol in 0.9% saline per 100 g body weight 15 min before intracerebral vector injection.
BrdU, a thymidine analogue incorporated into the DNA of dividing cells, was used to track proliferating cells. Before sacrifice, mice were i.p. injected twice daily with BrdU (50mg/kg, Sigma) for 7 consecutive days.
A laser-Doppler flowmetry monitor equipped with a small-caliber probe 0.7 mm in diameter was used to measure surface CBF. The laser Doppler probe was in contact with the surface of the mice’s skull bone during measurement. Blood flow was recorded in the ischemic penumbra area, which is 1.0 mm lateral to the sagittal suture and 1 mm posterior to the coronal suture. Baseline blood flow was recorded 5 min before MCAO. Changes in CBF were measured immediately, and then at 2, 4, and 8 weeks after MCAO. Blood flow values were calculated and expressed as percentages of baseline values (Shen et al, 2006).
The protein concentration was determined using the BCA protein assay kits (Pierce, IL). Equal amounts of proteins were loaded on 10% acrylamide gel for electrophoresis, and were electroblotted onto a PVDF membrane. The membranes were then probed with mouse anti-IGF-1 antibody (Upstate), 1:500, which reacts specifically with IGF-1 originating from human. Following the probing, the membranes were then incubated with horseradish-peroxidase (HRP)—conjugated sheep anti-mouse IgG (Bio-Rad Laboratories). Protein expression was detected with an enhanced chemiluminescence detection system (Amersham Pharmacia Biotech Inc). The image was scanned with a Canon imaging densitometer, and protein band densities were quantified with the NIH Image J program. IGF-1 band intensities were normalized to the band intensities of β-actin, which was used as a loading control.
Immunohistochemical staining was performed as described (Shen et al, 2006). Briefly, sections were incubated with primary antibodies at the following concentrations: mouse anti-IGF-1, 1:100 (Upstate); rat anti-CD31, 1:200 (BD bioscience); mouse anti-BrdU, 1:1000 (Sigma); rabbit anti-doublecortin (DCX), 1:200 (Cell signaling); rabbit anti-Tuj-1, 1:2000 (Covance). After incubating at 4°C overnight and washing, the sections were incubated with biotinylated secondary antibody (Vector Laboratories) at 1:5000 dilution. The sections were treated with the ABC streptavidin detection system. For dual fluorescent staining, after incubating with primary antibodies, sections were incubated with Alexa Fluor 594-conjugated or Alexa Fluor 488-conjugated IgG (Molecular probes) at 1:500 dilution. Negative controls were performed by omitting the primary antibodies during the immunostaining.
Microvessel counting was performed as described (Shen et al, 2006). Briefly, four brain coronal sections from the lectin-stained brain sections adjacent to the needle track were chosen. Three areas of microvessels, immediately to the left, right, and bottom of the needle track, were photographed using a 10× objective. Three random areas in the perifocal region were also photographed. Microvessel counting and cell counting (BrdU and CD31 dual labeled cells) were performed on these photographs. Vessels with a diameter between 3 and 8 μm were counted. The number of microvessels was calculated as the mean of the vascular counts obtained from three pictures; the number of cells was calculated in the same manner. The counting was conducted in a blinded fashion.
Quantification of DCX, Tuj-1, or BrdU positive cells was performed as described (Chen et al, 2005; Zhu et al, 2008). Briefly, DCX, Tuj-1, or BrdU positive cells in the ipsilateral subventricular zone were digitized under a 20× objective with a spot camera (Leica, Germany) equipped with NIH image J software. To clearly differentiate positively stained cells from the background, the digitalized images were contrast-enhanced and a threshold parameter was established to assess the proportion of immunoreactive region within a fixed field of view. The thresholds were selected with a “set color threshold” feature in the Image J software, which permits the user to select pixel regions that are considered positive. After establishing these indices, the same parameter was applied to all images acquired under the same magnification and light intensities on slides that were processed identically. The signals were counted using 4 sections from each mouse and the numbers were averaged. The data are presented as numbers of positive cells per microscopic field. Cell counting was performed by an investigator masked to the experimental groups.
Data are presented as mean ± standard deviation. Parametric data from the AAV-IGF-1, AAV-GFP and saline-treated groups were compared using an one-way analysis of variance (ANOVA), followed by Fisher’s protected least significant difference (PLSD) test, as appropriate. A p value < 0.05 is considered statistically significant.
We first examined the levels of green fluorescent protein (GFP) expression after intraparenchymal AAV-GFP injection to determine whether postischemic gene transduction was successful. A strong GFP signal was observed adjacent and distal to the injection tract, as well as in the peri-infarct region at 8 weeks after MCAO (Figure 1B and 1C). We observed positive GFP signal as early as 2 weeks after AAV transduction (data not shown). GFP signal was not noted in the contralateral noninjected hemisphere (Figure 1D). Western blot analysis showed that postischemic AAV-IGF-1 tranduction resulted in increased IGF-1 protein levels compared to AAV-GFP-administrated mice (Figure 1E and 1F, P < 0.05) at 8 weeks after MCAO. Immunohistochemical staining further showed strong IGF-1 immunoreactivity in IGF-1-injected mice, but not in GFP-transduced mice (Figure 1G). The IGF-1 antibody used in our study specifically reacts with IGF-1 of human origin, and thus we did not detect endogenous IGF-1 protein expressed after the ischemia in AAV-GFP injected mice. Together, our results show that IGF-1 gene was effectively transduced via AAV after ischemia to a large area of the ischemic peri-focal area with sustained expression.
IGF-1 has been shown to be a potent angiogenic factor required for cerebral angiogenesis (Lopez-Lopez et al, 2004). We determined whether IGF-1 overexpression would induce neovascularization. Immunostaining with vascular marker lectin showed increased vascular density in the penumbra of AAV-IGF-1 treated hemispheres (73 ± 14, number of microvessels / field) compared to AAV-GFP (45 ± 15) or saline-injected hemispheres (43 ± 13) (Figure 2A and 2B, P<0.05). IGF-1 gene transduction also increased the number of microvessels in the needle tract region (93 ± 38, P < 0.05) compared to GFP gene transduction (48 ± 9) or saline treatment (51 ± 12).
Double immunofluorescent staining showed that BrdU positive cells co-localized with endothelial cell marker CD31, with higher numbers of BrdU and CD31 double-labeled cells in IGF-1 administrated groups compared with AAV-GFP treatment (Figure 3A and 3B, 3.2 ± 2.6 vs 1.4 ± 1.1, P < 0.05, cells / field). The data indicate that IGF-1 delivery enhanced neovessel formation. To determine whether the newly formed vessels are functional, we measured cerebral blood flow with laser-Doppler flowmetry. IGF-1 gene transfer significantly enhanced vascular perfusion at 8 weeks after MCAO (21 ± 5) compared with AAV-GFP (10 ± 7) treatment (Table 1, percentages of baseline values, P < 0.01). Blood flow was similar between IGF-1 and GFP-treated groups measured immediately, and at 2 and 4 weeks after MCAO.
To determine whether IGF-1 gene transduction promotes neurogenesis, we conducted immunohistochemical analysis with BrdU (a cell proliferation marker), and DCX (a specific marker for neuronal progenitor cells). As shown in Figure 4A and 4B, IGF-1 overexpression resulted in a marked increase of DCX positive cells in the SVZ compared with AAV-GFP administration (24 ± 13 vs 7 ± 6, P < 0.05, cells / field). Increased DCX positive cells were also found in the subcortical white matter region in AAV-IGF-1-treated mice (9 ± 5 vs 4 ± 2, P < 0.05, cells / field), suggesting that IGF-1 not only increased neuronal stem cell proliferation, but also promoted their migration.
In SVZ, there was a significant increase in BrdU positive cells in AAV-IGF-1 injected mice compared with those treated with AAV-GFP (Figure 4C and 4D, 56 ± 19 vs 22 ± 9, P < 0.05, cells / field). Both BrdU and DCX positive cells were present in the AAV-GFP-transduced mice, indicating endogenous neurogenesis induced by ischemic stroke injury.
Using double-fluorescent staining, cells positive for both BrdU and DCX were considerably increased in AAV-IGF-1-administarted mice compared with AAV-GFP-injected mice in the SVZ (Figure 5A and 5B, 17 ± 7 vs 5 ± 3, P < 0.05, cells / field) and in the sub-cortical white matter region (8 ± 3 vs 3 ± 2, P < 0.05, cells / field). DCX positive cells reflect a pool of immature neurons that are derived from proliferated progenitor cells, and not all DCX positive immature neurons will turn into mature neurons. We further performed double fluorescent staining with Tuj-1 (a marker for neuron-specific class III —tubulin) and BrdU. As shown in Figure 6, compared to AAV-GFP treatment, AAV-IGF-1 transduction significantly increased the number of cells that are double positive for Tuj-1 and BrdU in the SVZ area and the subcortical white matter region (11 ± 4 vs 4 ± 2, P < 0.05, cells / field). These results indicate that IGF-1 administration enhanced endogenous neurogenesis.
Our current study demonstrates that postischemic IGF-1 gene transfer enhanced angiogenesis, and promoted neurogenesis. We used AAV to deliver IGF-1 to the ischemic brain. The advantage of gene transfer is that a single injection of vector can lead to efficient production of proteins over a large target area for a sustained period of time. Protein expression following AAV mediated gene delivery follows a well-characterized kinetic pattern that consistently shows a gradual ‘ramp up’ of expression, with relatively low levels for the first several days to 2 weeks following administration and gradually reaching an asymptote at somewhere between 3 and 6 weeks (Burger et al, 2005a). Compared to adenoviral vector, AAV vector possesses several advantages, such as low inflammatory potential and the ability to mediate long-term transgene expression (Burger et al, 2005a). Our Western blot assay and immunohistochemical staining showed that IGF-1 overexpression was maintained for at least 8 weeks after AAV transfer. Based on extensive prior work with AAV vectors and the CAG promoter, we expect this vector construct to provide steady expression of IGF-1 for years. While we did not measure protein levels long-term in this study, our prior studies with the same vector and promoter demonstrate that maximum levels and volumes of expression are reached at about 1 month, and remain steady thereafter for up to two years (Gasmi et al, 2007; Herzog et al, 2008). Whether continuous release of IGF-1 will lead to aberrant vascularization/regeneration is a topic for future consideration via a formal safety/toxicity program, before translating this work to clinical practice.
The acute neuroprotective action of IGF-1 has been well demonstrated (Dluzniewska et al, 2005; Mackay et al, 2003). We have reported previously that IGF-1 gene transfer before ischemic stroke promoted neurovascular regeneration and improved neurological functional recovery (Zhu et al, 2008). With pre-treatment, the improved neurological functions could also be contributed by the neuroprotective effects of IGF-1 in the acute phase of stroke. In the current study, we focused our effort on assessing the restorative effects of IGF-1 by employing postischemic gene transfer. Post-treatment is relevant to clinical settings, and has therapeutic use. We demonstrated here that postischemic IGF-1 gene transduction, compared with GFP injection, efficiently enhanced neurovascular remodeling assessed at 8 weeks after MCAO.
Our results show that IGF-1 overexpression resulted in increased vascular density in the peri-infarct region. In addition, the neovascularization was associated with increased local blood flow. Improved vascular perfusion could lead to long-term functional recovery by supporting neural cell survival, promoting neurogenesis, and facilitating the removal of necrotic debris. Consistent with this view, several reports have demonstrated that enhanced vascular perfusion by neovessel formation leads to functional improvement after stroke (Gertz et al, 2006; Sun et al, 2003; Wei et al, 2001).
Ischemic injury and various growth factors have been reported to enhance neurogenesis (Gross, 2000). IGF-1 protein infusion via osmotic minipump has been reported to increase stroke-induced progenitor cell proliferation in hypertensive rats (Dempsey et al, 2003). Consistent with this finding, we demonstrated here that IGF-1 gene transfer enhanced the number of neuronal progenitor cells in the SVZ area of infarcted hemispheres compared with ischemic injured hemispheres with GFP treatment. Furthermore, IGF-1 gene transfer promoted the migration of neuronal stem cells away from SVZ, as increased DCX positive cells were observed in the subcortical white matter area. Our data suggest that IGF-1 may promote the healing of the ischemic brain by amplifying endogenous neurogenesis. Angiogenesis and neurogenesis are temporally- and spatially-coupled processes (del Zoppo, 2006; Ohab et al, 2006). Blood vessels and neuronal fibers develop side-by-side and guide each other to migrate to the target area. Angiogenic vascular cells synthesize and secret trophic factors such as VEGF or SDF-1 to promote neural progenitor cells to proliferate and migrate to the ischemic-injured area (Ohab et al, 2006).
Taken together, our data demonstrate that postischemic AAV-IGF-1 treatment promotes neurovascular regeneration. We employed a permanent focal ischemia model by distal middle cerebral artery occlusion (MCAO) for establishing AAV-IGF-1 potential efficacy. This stroke model has been shown to induce reproducible cortical infarcts with a perifocal penumbra zone with inadequate perfusion (Chan et al, 1993; Chen et al, 1986; Matsumori et al, 2006). The perifocal area is the ideal therapeutic target for enhancing vascular perfusion, and preventing infarction process. Because clinical strokes are heterogeneous, testing this concept in additional models of stroke is a worthwhile endeavor. Furthermore, whether post-stroke treatment with AAV-IGF-1 can improve neurological functions warrants further investigation, as IGF-1 overexpression via AAV-mediated delivery has the potential to be developed into a therapeutic modality for the restoration of the ischemic-injured brain.
The authors thank Voltaire Gungab for editorial assistance and the staff of the Center for Cerebrovascular Research (http://avm.ucsf.edu/) for their collaborative support.
Sources of Support: This work was supported by AHA 0865114F (YC), NIH grants R21 NS053943 (YC) and P01 NS44155 (WLY, GYY, TH). AAV-IGF-1 and AAV-GFP were provided by Ceregene Incorporated.
Disclosure The contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.