- Email: [email protected]
Vascular endothelial growth factor improves recovery of sensorimotor and cognitive deficits after focal cerebral ischemia in the rat
BR A I N R ES E A RC H 1 1 1 5 ( 2 00 6 ) 1 8 6 –19 3
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
Vascular endothelial growth factor improves recovery of sensorimotor and cognitive deficits after focal cerebral ischemia in the rat Yaoming Wang, Veronica Galvan, Olivia Gorostiza, Marina Ataie, Kunlin Jin, David A. Greenberg⁎ Buck Institute for Age Research, 8001 Redwood Boulevard, Novato, CA 94945, USA
A R T I C LE I N FO
AB S T R A C T
Article history:
Vascular endothelial growth factor (VEGF) is an angiogenesis factor with neurotrophic,
Accepted 20 July 2006
neuroprotective and neuroproliferative effects. Depending on the dose, route and time of
Available online 22 August 2006
administration in relation to focal cerebral ischemia, VEGF can improve histological outcome and sensorimotor function in rodents. However, VEGF also increases vascular
Keywords:
permeability, which can lead to brain edema and exacerbate ischemic brain injury. Thus,
VEGF
although VEGF is a candidate therapeutic for stroke and other ischemic disorders, its benefit
Stroke
relative to risk is uncertain. Considering that functional rather than histological measures of
Memory
outcome are probably most relevant to therapeutic prospects for human stroke, we investigated the effects of VEGF after middle cerebral artery occlusion in rats using a series
Abbreviations:
of behavioral tests. We report that VEGF improves functional outcome in ischemic rats,
aCSF, artificial cerebrospinal fluid
including both sensorimotor and cognitive deficiencies.
EBST, elevated body swing test
© 2006 Elsevier B.V. All rights reserved.
ECA, external carotid artery ICV, intracerebroventricular MCA, middle cerebral artery MCAO, middle cerebral artery occlusion VEGF, vascular endothelial growth factor
1.
Introduction
Vascular endothelial growth factor (VEGF) is an angiogenic and vascular permeability factor (Marti and Risau, 1999), which also has neurotrophic, neuroprotective and neuroproliferative effects (Greenberg and Jin, 2005). The ability of VEGF to elicit both angiogenesis and neuroprotection has led to
interest in its possible therapeutic application in stroke. Several studies have documented that, depending on the dose, route and timing of administration, VEGF can improve histological outcome from experimental stroke (Hayashi et al., 1998; Kaya et al., 2005; Manoonkitiwongsa et al., 2004; Sun et al., 2003; Wang et al., 2005; Zhang et al., 2000), which appears to result from direct neuroprotective action. Adverse effects of
⁎ Corresponding author. Fax: +1 415 209 2230. E-mail address: [email protected] (D.A. Greenberg). 0006-8993/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2006.07.060
187
BR A I N R ES E A RC H 1 1 1 5 ( 2 00 6 ) 1 8 6 –1 93
VEGF, especially its propensity to increase vascular leakage (Fischer et al., 2004) and, therefore, brain edema, can also worsen ischemic brain injury (Van Bruggen et al., 1999; Zhang et al., 2000). Less attention has been given to detailed evaluation of the effects of VEGF on post-stroke behavioral performance. Zhang et al. (2000) found that intravenous VEGF, given for 4 h beginning 48 h after embolic occlusion of the middle cerebral artery (MCA) in rats, improved motor performance measured by the accelerating rotarod test at 7, 14 and 28 days, and somatosensory deficits evaluated with the adhesive removal test at 28 days, post-stroke. We reported that intraventricular VEGF, administered continuously from 24 to 96 h after suture-induced MCA occlusion in rats, enhanced motor and proprioceptive function at 3, 7, 14 and 28 days (Sun et al., 2003). Zhu et al. (2005) transplanted neurosphere-derived neural stem cells, some of which were transfected with VEGF, into the striatum of rats 3 days after suture occlusion of the MCA, and showed improvement in the neurological severity score, which reflects motor, sensory, reflex and balance function, at 4, 6, 8, 10 and 12 weeks. Finally, Kaya et al. (2005) demonstrated that a single intraventricular dose of VEGF, given 1 or 3 h after MCA occlusion (MCAO) in mice, reduced the severity of contralateral limb paralysis 24 h later. Because the relative risk to benefit of VEGF in cerebral ischemia is controversial and since the outcome measure most relevant to clinical efficacy is neurobehavioral function (rather than, for example, infarct size), we examined the effects of VEGF administration after experimental stroke in rats using a broad-based panel of behavioral tests. The results indicate that VEGF improves functional outcome in rats with brain ischemia, including basic and complex sensorimotor as well as cognitive deficiencies assessed by a behavioral testing battery. Thus, under appropriate conditions, VEGF seems capable of conferring long-term functional benefit in the aftermath of acute ischemic stroke.
2.
Results
2.1.
Physiological measures
Table 1 – Physiologic measures in rats undergoing MCAO PaCO2 MABP PaO2 (mm Hg) (mm Hg) (mm Hg) Before ischemia Sham109 ± 11 operated MCAO 116 ± 8 MCAO+ 114 ± 8 VEGF After ischemia Sham114 ± 6 operated MCAO 115 ± 9 MCAO+ 115 ± 7 VEGF
2.2.
Elevated body swing test
The elevated body swing test was used to detect asymmetry in motor behavior. MCAO caused an increase in turning bias throughout the 8-week reperfusion period (Fig. 1), from near 0% in sham-operated controls to 80–95%. Thus, postischemic rats showed a strong and persistent tendency to turn their upper bodies to the side opposite the ischemic hemisphere. VEGF administration decreased the turning bias after MCAO to 55–70% at 1–8 weeks.
Glucose (mg/ml)
85 ± 6
58 ± 4
7.38 ± 1.0
178 ± 16
86 ± 5 87 ± 4
56 ± 5 58 ± 4
7.40 ± 1.1 7.39 ± 0.9
176 ± 12 177 ± 8
83 ± 5
57 ± 4
7.37 ± 1.2
177 ± 10
85 ± 5 85 ± 5
58 ± 5 57 ± 4
7.36 ± 1.3 7.40 ± 0.9
179 ± 12 178 ± 7
Data shown are mean values ± SD from 10 rats per condition. MABP, mean arterial blood pressure; PaO2, partial pressure of O2 in arterial blood; PaCO2, partial pressure of CO2 in arterial blood; pH, arterial blood pH; Glucose, plasma concentration of glucose. There were no significant differences across treatment groups for any of the variables examined.
2.3.
Forelimb and hindlimb placing tests
Rats were also tested for limb placing in response to visual, vibrissae, tactile and proprioceptive stimulation, which requires sensorimotor integration. Sham-operated rats showed consistently high (normal = 12) forelimb and hindlimb placement scores over the 8-week postischemic period (Fig. 2). MCAO reduced scores at all time points examined, but VEGF treatment restored scores towards normal beginning at 1 week.
2.4.
Cylinder test
Sham-operated rats used the forelimbs symmetrically in the cylinder test (∼50% use of ipsilateral limb in Fig. 3). However, in ischemic rats at 15 and 25 days post-MCAO, the unaffected (ipsilateral) limb was used preferentially. Limb use asymmetry following MCAO was reduced in rats given VEGF, and this effect was greater at 25 than at 15 days.
2.5.
Physiological measures, including mean blood pressure, blood pH, blood gases, hematocrit and blood glucose, were recorded before and after MCAO and were within normal ranges (Table 1).
pH
Morris water maze
To investigate whether VEGF treatment influences postischemic cognitive deficits, we first measured spatial learning and memory performance in an open-field water maze, 15 and 25 days after MCAO. All rats performed similarly in the pretraining sessions, with decreasing escape latencies in successive trials both in the cued (Fig. 4A) and visible (Fig. 4B) tasks, consistent with normal learning. Probe trials, in which the escape platform was removed from the tank, were performed after the first (probe 1) and last sessions (probe 2) of the spatial task. These revealed increased target quadrant occupancy during probe 2 compared to probe 1, indicating normal improvement in recall of the former location of the platform for all rats (Fig. 4C). Compared to sham-operated controls, rats subjected to MCAO showed longer platform-finding latencies at 15 and 25 days, and these latencies were reduced by VEGF treatment
188
BR A I N R ES E A RC H 1 1 1 5 ( 2 00 6 ) 1 8 6 –19 3
Fig. 1 – Elevated body swing test. Turning bias after sham surgery and after MCAO followed by intraventricular infusion of aCSF or VEGF. *p < 0.05 compared to MCAO followed by intraventricular infusion of aCSF.
(Fig. 5A). MCAO also reduced the amount of time spent in the target (platform-containing) quadrant of the water maze, and time spent in the target quadrant at 25 days was increased towards normal by VEGF (Fig. 5B).
3.
Discussion
Our results indicate that VEGF can reverse both sensorimotor and cognitive deficits following MCAO in the rat and that its
Fig. 2 – Forelimb and hindlimb placing tests. (A) Forelimb and (B) hindlimb placing after sham surgery and after MCAO followed by intraventricular infusion of aCSF or VEGF. *p < 0.05, **p < 0.01 compared to MCAO followed by intraventricular infusion of aCSF.
Fig. 3 – Cylinder test. Average percent bias favoring the use of the limb ipsilateral to (unaffected by) MCAO was assessed at 15 (left) and 25 (right) days after sham surgery and after MCAO followed by intraventricular infusion of aCSF or VEGF. *p < 0.05 compared to both sham surgery (Con) and MCAO + VEGF (ANOVA and Bonferroni t test).
beneficial effect can last for at least 8 weeks. We reported previously that VEGF, given ICV as in the present study, reduced infarct size and improved motor and proprioceptive function for up to 4 weeks after MCAO (Sun et al., 2003), and others have reported similar effects with intravenous VEGF (Zhang et al., 2000) or intracerebral transplantation of neural stem cells transfected with VEGF (Zhu et al., 2005). In this study, we have extended these studies to incorporate a more extensive analysis of the postischemic behavioral effects of VEGF, including its effect on spatial learning and memory performance. In rodents as in humans, MCAO produces a syndrome characterized predominantly by hemiparesis and hemisensory loss. Accordingly, we employed a battery of sensorimotor tests tailored specifically to assess long-term functional outcome from MCAO (Roof et al., 2001). Thus, the elevated body swing test and cylinder test detect asymmetrical motor behavior such as that resulting from hemiparesis due to an ischemic cortical lesion (Borlongan and Sanberg, 1995; Schallert et al., 1986), and the forelimb and hindlimb placing tests depend on integration of visual, tactile and proprioceptive sensory stimulation to elicit appropriate motor responses (De Ryck et al., 1989; Roof et al., 2001). We observed that VEGF treatment improved performance on the elevated body swing and forelimb and hindlimb placing tests throughout the 8week period of postischemic recovery. In the cylinder test, VEGF-treated rats also showed superior performance when tested 25 days after MCAO. These findings are consistent with sustained enhancement of sensorimotor recovery by VEGF. How does VEGF improve sensorimotor outcome after MCAO? Prior evidence suggests that a direct neuroprotective effect of VEGF is more likely responsible for improved histological and functional sensorimotor outcome from stroke than is VEGF-mediated angiogenesis or neurogenesis (Sun et al., 2003). This is because a reduction in infarct size and better neurological function can be seen as early as 3 days post-stroke in VEGF-treated rats, whereas VEGF-induced angiogenesis and neurogenesis proceed more slowly. In the present study, VEGF-treated rats showed a reduction in
BR A I N R ES E A RC H 1 1 1 5 ( 2 00 6 ) 1 8 6 –1 93
189
Fig. 4 – Water maze training before MCAO. All experimental animals were trained in the Morris water maze, as described in Experimental procedures. Average latencies to the escape platform for all experimental animals, before surgery, during the cued (A) and hidden (B) tasks. (C) Average time spent in the target quadrant (former platform location) by each experimental animal in the first (probe 1) and second (probe 2) probe trials. The performance of each animal in each probe trial is plotted individually. Higher values of target quadrant occupancy correspond to an increasingly accurate recall of the former platform location.
turning bias and improved limb placing as early as 1 week, by which time neither angiogenesis nor neurogenesis is sufficiently advanced to account for these effects (Sun et al., 2003). Of course, this does not exclude a possible role for angiogenesis or neurogenesis in the maintenance of improvement nor in enhancement of recovery that becomes evident only at longer intervals. Although we did not investigate the molecular mechanism through which VEGF improved functional outcome in this study, our prior studies implicate activation of VEGFR2/Flk1 receptors, phosphatidylinositol 3′-kinase/Akt and NF-κB (Jin et al., 2000a,b) and reduced cleavage of caspase-3 (Jin et al., 2001). We also found that postischemic defects in the Morris water maze were less severe after VEGF treatment. This task measures spatial learning, which requires formation of navigation strategies, place learning, memory and visionguided behavior. Specifically, VEGF reduced latency to target at 15 days and 25 days and increased target quadrant occupancy at 25 days post-MCAO. This pattern of improvement suggests that the potential benefit of VEGF in stroke may not be restricted to primary sensorimotor function but may
extend to more complex and highly integrated cortical activities. This is important because, although memory deficits are not prominently associated with MCA-distribution strokes, defects in language (aphasia), spatial awareness (agnosia) and performance of previously learned tasks (apraxia) are. How does VEGF improve memory and learning after MCAO? MCAO in the rat, performed as in the present study, affects the striatum and cerebral cortex, but not the hippocampus, which is required for spatial memory (Moosmang et al., 2005). Therefore, memory impairment following MCAO in the rat must reflect the involvement of another brain region. In fact, the dorsal striatum and nucleus accumbens (De Leonibus et al., 2005; Gengler et al., 2005), as well as prefrontal cortex (Delatour and Gisquet-Verrier, 1996; Floresco et al., 1997), have also been implicated in spatial memory and associated tasks in rodents, and memory for some spatial representations appears to be independent of the hippocampus (Winocur et al., 2005). Memory disturbance is not a prominent finding in patients with MCAO (perhaps partly because it is difficult to identify in the face of other deficits),
190
BR A I N R ES E A RC H 1 1 1 5 ( 2 00 6 ) 1 8 6 –19 3
Fig. 5 – Water maze task at 15 and 25 days after MCAO. (A) Average escapes latencies during two consecutive hidden task sessions, performed 15 or 25 days after MCAO followed by intraventricular infusion of aCSF (MCAO) or VEGF (MCAO + VEGF) as described in Experimental procedures. *p < 0.05 compared to MCAO. Values for sham-operated rats (black bar on y axis) were 25 ± 7 s (n = 16). (B) Average time spent in the target quadrant (former platform location) during the probe trial 15 or 25 days after MCAO followed by intraventricular infusion of aCSF (MCAO) or VEGF (MCAO + VEGF). *p < 0.05 compared to MCAO. Values for sham-operated rats (black bar on y axis) were 33 ± 3 s (n = 16).
but impaired topographic memory and disorders of spatial localization are well documented following MCA strokes that involve the nondominant hemisphere (Landis et al., 1986; Meerwaldt and Van Harskamp, 1982). Because VEGF stimulates neurogenesis in the adult rodent brain (Jin et al., 2002) and since hippocampal neurogenesis has been implicated in memory function (Shors et al., 2001; Snyder et al., 2005), it is conceivable that VEGF-induced neurogenesis contributed to the memory improvement that we observed. For example, stimulation of hippocampal neurogenesis by VEGF appears to be responsible for the ability of environmental enrichment to enhance cognitive performance in rats (Cao et al., 2004). In addition, gerbils that received cranial irradiation to ablate neuronal progenitors and, thereby, inhibit neurogenesis, prior to transient global cerebral ischemia, showed more severe impairment in the Morris water maze than did unirradiated gerbils (Raber et al., 2004). This seems to suggest that postischemic memory function can be modified by ongoing neurogenesis. VEGF couples hypoxia to angiogenesis in the ischemic brain (Marti et al., 2000). VEGF expression in the brain is increased by ischemia (Hayashi et al., 1997; Kovacs et al., 1996; Lennmyr et al., 1998; Plate et al., 1999; Zhang et al., 2002), but the effect of endogenous VEGF on outcome in this setting is controversial. For example, an anti-VEGF antibody given ICV increased infarct size, implying a protective role for endogenous VEGF (Bao et al., 1999), but a VEGF receptor (VEGFR1/Flt1) fusion protein that sequesters VEGF (Van Bruggen et al., 1999) or a neutralizing antibody against VEGF (Kimura et al., 2005) reduced brain edema and infarct size, consistent with a deleterious effect of VEGF. Some human studies also suggest that endogenous VEGF may adversely affect outcome from stroke. For example, VEGF levels are increased in hypercho-
lesterolemia (Xi et al., in press), which is also associated with better prognosis following stroke (Devuyst et al., 2003; Vauthey et al., 2000). Depending on the dose, route and timing of administration, exogenous VEGF may exacerbate postischemic brain edema or hemorrhage (Manoonkitiwongsa et al., 2004; Zhang et al., 2000). Specifically, early post-stroke administration of VEGF by the IV route has been associated with clinical worsening, whereas delayed or ICV administration has shown benefit. IV VEGF, given 1 h after the onset of ischemia, increased blood– brain barrier leakage, hemorrhagic transformation and infarct volume, but when treatment was delayed until 48 h poststroke, IV VEGF enhanced functional recovery (Zhang et al., 2000). Another study showed that a single ICV dose of VEGF, given 1 h or 3 h after MCAO, decreased infarct volume and vascular leakage (Kaya et al., 2005). We chose to administer VEGF ICV at 24 h because our previous results showed a reduction in infarct size with this regimen (Sun et al., 2003). However, the manner in which VEGF might affect sensorimotor or cognitive outcome after MCAO if given according to a different scheme is impossible to predict. Our results bolster the evidence that VEGF or smallmolecule drugs that promote its effects might have therapeutic applications in stroke. With judicious selection of the dose, route and timing of administration, VEGF can reduce infarct size after MCAO in rodents (Hayashi et al., 1998; Kaya et al., 2005; Manoonkitiwongsa et al., 2004; Sun et al., 2003; Wang et al., 2005; Zhang et al., 2000), improve postischemic sensorimotor function (Kaya et al., 2005; Zhang et al., 2000; Zhu et al., 2005) and enhance learning and memory as assessed using the Morris water maze (this study). Its propensity for side effects related to increased vascular permeability or hemorrhagic diathesis might be modified by the concurrent administration of other factors, such as angiopoietins (Thurston et al., 2000), or by substitution of VEGFB, which is similarly neuroprotective in ischemia (Sun et al., 2004) and also stimulates neurogenesis (Sun et al., 2006) but has less tendency to increase vessel permeability (Mould et al., 2005).
4.
Experimental procedures
4.1.
Focal cerebral ischemia
All procedures were approved by local committee review and conducted according to the NIH Guide for the Care and Use of Laboratory Animals, with an effort to minimize suffering and reduce animal numbers. Transient focal cerebral ischemia was induced by the suture occlusion technique (Longa et al., 1989). Male Sprague–Dawley rats weighing 280–310 g were anesthetized with 4% isoflurane in 70% N2O and 30% O2, using a mask. A midline incision was made in the neck, the right external carotid artery (ECA) was carefully exposed and dissected and a 3-0 monofilament nylon suture was inserted from the ECA into the right internal carotid artery to occlude the right MCA at its origin. After 90 min, the suture was removed to allow reperfusion and the wound was closed. Sham-operated rats underwent identical surgery except that the suture was not inserted. Rectal temperature was maintained at 37.0 ± 0.5 °C
BR A I N R ES E A RC H 1 1 1 5 ( 2 00 6 ) 1 8 6 –1 93
using a heating pad and heating lamp. Blood pressure and blood glucose concentration were monitored.
4.2.
VEGF administration
VEGF was administered by the intracerebroventricular (ICV) route as previously described (Sun et al., 2003). One day after ischemia, rats were anesthetized with 4% isoflurane in 70% N2O/30% O2 and implanted with an osmotic minipump (Alzet 1003D; Alza Scientific Products, Mountain View, California, USA). The cannula was placed in the left lateral ventricle 4.0 mm deep as measured from the pial surface, 0.8 mm anterior to the bregma and 1.3 mm lateral to the midline. VEGF (10 μg/ml) was administered at 1 μl/h ICV for 3 days, in artificial cerebrospinal fluid (aCSF) consisting of 128 mM NaCl, 2.5 mM KCl, 0.95 mM CaCl2 and 1.99 mM MgCl2. Control rats received aCSF without VEGF for the same amount of time.
4.3.
Behavioral testing
Postischemic and sham-operated rats treated with VEGF or vehicle underwent a series of behavioral tests designed to measure long-term functional outcome from MCAO (Roof et al., 2001). The tests used were (1) sensorimotor tests including (a) elevated body swing test (EBST) and (b) forelimb and hindlimb placing tests, both conducted weekly for 8 weeks; and (c) limb use asymmetry (cylinder) test, on days 15 and 25; and (2) cognitive testing (Morris water maze), conducted before and 15 and 25 days after stroke. All behavioral testing was done during the rats' light cycle. The experimenter conducting and scoring behavioral tests was blind to the experimental condition.
4.3.1.
Elevated body swing test
In this test of asymmetric motor behavior, rats held by the base of the tail were raised ∼10 cm above the testing surface (Borlongan and Sanberg, 1995). The initial direction of body swing, constituting a turn of the upper body of >10° to either side, was recorded in three sets of ten trials, performed over 5 min. The number of turns in each (left or right) direction was recorded, and the percentage of turns made to the side contralateral to the ischemic hemisphere (percent left-biased swing) was calculated. For each rat, average scores for each week were determined. Percent turning bias in the EBST was combined with percent asymmetry in wall movements for the cylinder test (see below) to provide an overall asymmetry score.
4.3.2.
Forelimb and hindlimb placing tests
Rats were held by supporting the underside of the torso, allowing the forelimbs and hindlimbs to hang freely (De Ryck et al., 1989; Roof et al., 2001). Placement of the left or right forelimb onto a tabletop was then recorded as rats were moved slowly toward the edge of a tabletop, either (i, ii) stopping short of touching the vibrissae as the rats moved forward or laterally (forward or lateral vision-induced placing), (iii) touching the vibrissae (vibrissae-induced placing), (iv, v) making light contact with the dorsal or lateral aspect of the forepaw to the edge of the tabletop (dorsal or lateral tactile-
191
induced placing) or (vi) pressing the forepaws to the edge of the table (proprioceptive placing). Hindlimb placing was tested in an analogous manner. Each of the six tests listed above was scored as: 0, no placing; 1, incomplete or delayed placing; 2, complete, immediate placing. Average placing scores were calculated for each animal, with a total score of 12 (2 on each of six tests) considered normal.
4.3.3.
Cylinder test
Asymmetric use of the forelimbs (forelimb-use bias) was analyzed by observing the rat's movements in a transparent, 18-cm wide, 30-cm high Plexiglas cylinder, over 3-minute intervals (Schallert et al., 1986). The cylinder was sufficiently large to permit movement while not permitting the rat to reach the upper edge, yet small enough to promote rearing and wall exploration. A mirror behind the cylinder made it possible to observe and record forelimb movements with the rat facing away from the examiner. Following an episode of rearing and wall exploration, a landing was scored for the first limb to contact the ground or for both limbs if they made simultaneous contact. Percent-use scores were calculated for (i) movements using the unimpaired limb and (ii) movements using the impaired limb, relative to the total number of movements. Percentage use of the impaired was subtracted from percentage use of the unimpaired limb to yield an overall limb-bias score. Wall exploration and landing movements were analyzed separately. Average cylinder test scores were calculated for each animal and for each week of testing, which consisted of two sessions per week.
4.3.4.
Morris water maze
Cognitive deficits were assessed using the Morris water maze (Morris, 1984). Before and at different reperfusion times after stroke, rats were given a series of six trials, spaced 1 h apart, in a dark-colored, 160-cm-diameter tank filled with opaque white water at a temperature of 22.0 ± 1.0 °C. To test nonspatial learning, rats were trained to find a 12 cm × 12 cm platform, submerged 1 cm below the water's surface, which was marked with a colored pole as a landmark, and placed in different quadrants of the tank. Rats were lowered into the tank, facing the wall, and released at different locations in each trial. Each rat had up to 90 s to find the submerged platform; if the platform was not found during this time, the rat was guided to it. After staying on the platform for 20 s, the rat was relocated to a dry cage. One hour later, each rat was retested, using an identical release site and platform position, to assess retention of platform location. This was repeated six times for each rat, at 30-min to 60-min intervals. To test spatial learning, the tank was surrounded by opaque dark panels placed ∼ 90 cm from its edge. Four rectangular drawings with geometric designs in black and white were evenly spaced on the panels to serve as distal cues. The rats were trained to find the submerged platform by swimming six times every day for 2 days. These six trials were followed by a probe trial, during which the platform was removed from the pool, and the rat was permitted to swim for 60 s. The percent time and portion of the swim path that each rat spent in the quadrant where the platform had been were used to measure retention of the platform's location. This procedure was repeated 15 and 25 days later. Swim paths were recorded using a computer-
192
BR A I N R ES E A RC H 1 1 1 5 ( 2 00 6 ) 1 8 6 –19 3
interfaced camera tracking system (Water2020, HVS Image, UK). Parameters measured in each trial included: path length and latency to platform (reflecting the ability to learn and remember platform location), swimming velocity (to detect the effect of possible differences in swimming ability on results), percentage of swimming path confined to the outer 15 cm of the tank rather than searching for the platform, percentage of path in a 36-cm-diameter circle surrounding platform, and initial heading error (deviation between actual and correct initial direction of swimming in the first 3 s, indicative of memory for the general location of the platform).
4.4.
Statistical analysis
Results were reported as mean ± SD. The significance of differences between means was assessed by Student's t test (single comparisons) and two-way ANOVA followed by Bonferroni's post hoc or Fisher's PLSD (multiple comparisons), with p < 0.05 considered statistically significant.
Acknowledgments This work was supported by NIH grants NS44921 (D.A.G.) and AG21980 (K.J.).
REFERENCES
Bao, W.L., Lu, S.D., Wang, H., Sun, F.Y., 1999. Intraventricular vascular endothelial growth factor antibody increases infarct volume following transient cerebral ischemia. Chung-Kuo Yao Li Hsueh Pao 20, 313–318. Borlongan, C.V., Sanberg, P.R., 1995. Elevated body swing test: a new behavioral parameter for rats with 6-hydroxydopamine-induced hemiparkinsonism. J. Neurosci. 15, 5372–5378. Cao, L., Jiao, X., Zuzga, D.S., Liu, Y., Fong, D.M., Young, D., During, M.J., 2004. Vegf links hippocampal activity with neurogenesis, learning and memory. Nat. Genet. 36, 827–835. Delatour, B., Gisquet-Verrier, P., 1996. Prelimbic cortex specific lesions disrupt delayed-variable response tasks in the rat. Behav. Neurosci. 110, 1282–1298. De Leonibus, E., Oliverio, A., Mele, A., 2005. A study on the role of the dorsal striatum and the nucleus accumbens in allocentric and egocentric spatial memory consolidation. Learn. Mem. 12, 491–503. De Ryck, M., Van Reempts, J., Borgers, M., Wauquier, A., Janssen, P.A., 1989. Photochemical stroke model: flunarizine prevents sensorimotor deficits after neocortical infarcts in rats. Stroke 20, 1383–1390. Devuyst, G., Karapanayiotides, T., Hottinger, I., Van Melle, G., Bogousslavsky, J., 2003. Prodromal and early epileptic seizures in acute stroke: does higher serum cholesterol protect? Neurology 61, 249–252. Fischer, S., Wiesnet, M., Marti, H.H., Renz, D., Schaper, W., 2004. Simultaneous activation of several second messengers in hypoxia-induced hyperpermeability of brain derived endothelial cells. J. Cell. Physiol. 198, 359–369. Floresco, S.B., Seamans, J.K., Phillips, A.G., 1997. Selective roles for hippocampal, prefrontal cortical, and ventral striatal circuits in radial-arm maze tasks with or without a delay. J. Neurosci. 17, 1880–1890.
Gengler, S., Mallot, H.A., Holscher, C., 2005. Inactivation of the rat dorsal striatum impairs performance in spatial tasks and alters hippocampal theta in the freely moving rat. Behav. Brain Res. 164, 73–82. Greenberg, D.A., Jin, K., 2005. From angiogenesis to neuropathology. Nature 438, 954–959. Hayashi, T., Abe, K., Suzuki, H., Itomaya, Y., 1997. Rapid induction of vascular endothelial growth factor gene expression after transient middle cerebral artery occlusion in rats. Stroke 28, 2039–2044. Hayashi, T., Abe, K., Itoyama, Y., 1998. Reduction of ischemic damage by application of vascular endothelial growth factor in rat brain after transient ischemia. J. Cereb. Blood Flow Metab. 18, 887–895. Jin, K., Mao, X.O., Batteur, S.P., Mceachron, E., Leahy, A., Greenberg, D.A., 2001. Caspase-3 and the regulation of hypoxic neuronal death by vascular endothelial growth factor. Neuroscience 108, 351–358. Jin, K., Zhu, Y., Sun, Y., Mao, X.O., Xie, L., Greenberg, D.A., 2002. Vascular endothelial growth factor (vegf) stimulates neurogenesis in vitro and in vivo. Proc. Natl. Acad. Sci. U. S. A. 99, 11946–11950. Jin, K.L., Mao, X.O., Greenberg, D.A., 2000a. Vascular endothelial growth factor rescues HN33 neural cells from death induced by serum withdrawal. J. Mol. Neurosci. 14, 197–203. Jin, K.L., Mao, X.O., Greenberg, D.A., 2000b. Vascular endothelial growth factor: direct neuroprotective effect in in vitro ischemia. Proc. Natl. Acad. Sci. U. S. A. 97, 10242–10247. Kaya, D., Gursoy-Ozdemir, Y., Yemisci, M., Tuncer, N., Aktan, S., Dalkara, T., 2005. Vegf protects brain against focal ischemia without increasing blood–brain permeability when administered intracerebroventricularly. J. Cereb. Blood Flow Metab. 25, 1111–1118. Kimura, R., Nakase, H., Tamaki, R., Sakaki, T., 2005. Vascular endothelial growth factor antagonist reduces brain edema formation and venous infarction. Stroke 36, 1259–1263. Kovacs, Z., Ikezaki, K., Samoto, K., Inamura, T., Fukui, M., 1996. Vegf and Flt: expression time kinetics in rat brain infarct. Stroke 27, 1865–1873. Landis, T., Cummings, J.L., Benson, D.F., Palmer, E.P., 1986. Loss of topographic familiarity. An environmental agnosia. Arch. Neurol. 43, 132–136. Lennmyr, F., Ata, K.A., Funa, K., Olsson, Y., Terent, A., 1998. Expression of vascular endothelial growth factor (Vegf) and its receptors (Flt-1 and Flk-1) following permanent and transient occlusion of the middle cerebral artery in the rat. J. Neuropathol. Exp. Neurol. 57, 874–882. Longa, E.Z., Weinstein, P.R., Carlson, S., Cummins, R., 1989. Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke 20, 84–91. Manoonkitiwongsa, P.S., Schultz, R.L., Mccreery, D.B., Whitter, E.F., Lyden, P.D., 2004. Neuroprotection of ischemic brain by vascular endothelial growth factor is critically dependent on proper dosage and may be compromised by angiogenesis. J. Cereb. Blood Flow Metab. 24, 693–702. Marti, H.H., Risau, W., 1999. Angiogenesis in ischemic disease. Thromb. Haemost. 82 (Suppl. 1), 44–52. Marti, H.J., Bernaudin, M., Bellail, A., Schoch, H., Euler, M., Petit, E., Risau, W., 2000. Hypoxia-induced vascular endothelial growth factor expression precedes neovascularization after cerebral ischemia. Am. J. Pathol. 156, 965–976. Meerwaldt, J.D., Van Harskamp, F., 1982. Spatial disorientation in right-hemisphere infarction. J. Neurol. Neurosurg. Psychiatry 45, 586–590. Moosmang, S., Haider, N., Klugbauer, N., Adelsberger, H., Langwieser, N., Muller, J., Stiess, M., Marais, E., Schulla, V., Lacinova, L., Goebbels, S., Nave, K.A., Storm, D.R., Hofmann, F.,
BR A I N R ES E A RC H 1 1 1 5 ( 2 00 6 ) 1 8 6 –1 93
Kleppisch, T., 2005. Role of hippocampal Cav1.2 Ca2+ channels in NMDA receptor-independent synaptic plasticity and spatial memory. J. Neurosci. 25, 9883–9892. Morris, R., 1984. Development of a water-maze procedure for studying spatial learning in the rat. J. Neurosci. Methods 11, 47–60. Mould, A.W., Greco, S.A., Cahill, M.M., Tonks, I.D., Bellomo, D., Patterson, C., Zournazi, A., Nash, A., Scotney, P., Hayward, N.K., Kay, G.F., 2005. Transgenic overexpression of vascular endothelial growth factor-B isoforms by endothelial cells potentiates postnatal vessel growth in vivo and in vitro. Circ. Res. 97, E60–E70. Plate, K.H., Beck, H., Danner, S., Allegrini, P.R., Wiessner, C., 1999. Cell type specific upregulation of vascular endothelial growth factor in an MCA-occlusion model of cerebral infarct. J. Neuropathol. Exp. Neurol. 58, 654–666. Raber, J., Fan, Y., Matsumori, Y., Liu, Z., Weinstein, P.R., Fike, J.R., Liu, J., 2004. Irradiation attenuates neurogenesis and exacerbates ischemia-induced deficits. Ann. Neurol. 55, 381–389. Roof, R.L., Schielke, G.P., Ren, X., Hall, E.D., 2001. A comparison of long-term functional outcome after 2 middle cerebral artery occlusion models in rats. Stroke 32, 2648–2657. Schallert, T., Hernandez, T.D., Barth, T.M., 1986. Recovery of function after brain damage: severe and chronic disruption by diazepam. Brain Res. 379, 104–111. Shors, T.J., Miesegaes, G., Beylin, A., Zhao, M., Rydel, T., Gould, E., 2001. Neurogenesis in the adult is involved in the formation of trace memories. Nature 410, 372–376. Snyder, J.S., Hong, N.S., Mcdonald, R.J., Wojtowicz, J.M., 2005. A role for adult neurogenesis in spatial long-term memory. Neuroscience 130, 843–852. Sun, Y., Jin, K., Xie, L., Childs, J., Mao, X.O., Logvinova, A., Greenberg, D.A., 2003. VEGF-induced neuroprotection, neurogenesis, and angiogenesis after focal cerebral ischemia. J. Clin. Invest. 111, 1843–1851. Sun, Y., Jin, K., Childs, J.T., Xie, L., Mao, X.O., Greenberg, D.A., 2004. Increased severity of cerebral ischemic injury in vascular endothelial growth factor-B-deficient mice. J. Cereb. Blood Flow Metab. 24, 1146–1152. Sun, Y., Jin, K., Childs, J.T., Xie, L., Mao, X.O., Greenberg, D.A., 2006. Vascular endothelial growth factor-B (VEGFB) stimulates
193
neurogenesis: evidence from knockout mice and growth factor administration. Dev. Biol. 289, 329–335. Thurston, G., Rudge, J.S., Ioffe, E., Zhou, H., Ross, L., Croll, S.D., Glazer, N., Holash, J., Mcdonald, D.M., Yancopoulos, G.D., 2000. Angiopoietin-1 protects the adult vasculature against plasma leakage. Nat. Med. 6, 460–463. Van Bruggen, N., Thibodeaux, H., Palmer, J.T., Lee, W.P., Fu, L., Cairns, B., Tumas, D., Gerlai, R., Williams, S.P., Van Lookeren Campagne, M., Ferrara, N., 1999. VEGF antagonism reduces edema formation and tissue damage after ischemia/ reperfusion injury in the mouse brain. J. Clin. Invest. 104, 1613–1620. Vauthey, C., De Freitas, G.R., Van Melle, G., Devuyst, G., Bogousslavsky, J., 2000. Better outcome after stroke with higher serum cholesterol levels. Neurology 54, 1944–1949. Wang, Y., Kilic, E., Kilic, U., Weber, B., Bassetti, C.L., Marti, H.H., Hermann, D.M., 2005. VEGF overexpression induces post-ischaemic neuroprotection, but facilitates haemodynamic steal phenomena. Brain 128, 52–63. Winocur, G., Moscovitch, M., Fogel, S., Rosenbaum, R.S., Sekeres, M., 2005. Preserved spatial memory after hippocampal lesions: effects of extensive experience in a complex environment. Nat. Neurosci. 8, 273–275. Xi, L., Ghosh, S., Wang, X., Das, A., Anderson, F.P., Kukreja, R.C., in press. Hypercholesterolemia enhances tolerance to lethal systemic hypoxia in middle-aged mice: possible role of VEGF downregulation in brain. Mol. Cell. Biochem. doi:10.1007/ s11010-006-9194-7. Zhang, Z.G., Zhang, L., Jiang, Q., Zhang, R., Davies, K., Powers, C., Bruggen, N., Chopp, M., 2000. VEGF enhances angiogenesis and promotes blood–brain barrier leakage in the ischemic brain. J. Clin. Invest. 106, 829–838. Zhang, Z.G., Zhang, L., Tsang, W., Soltanian-Zadeh, H., Morris, D., Zhang, R., Goussev, A., Powers, C., Yeich, T., Chopp, M., 2002. Correlation of VEGF and angiopoietin expression with disruption of blood–brain barrier and angiogenesis after focal cerebral ischemia. J. Cereb. Blood Flow Metab. 22, 379–392. Zhu, W., Mao, Y., Zhao, Y., Zhou, L.F., Wang, Y., Zhu, J.H., Zhu, Y., Yang, G.Y., 2005. Transplantation of vascular endothelial growth factor-transfected neural stem cells into the rat brain provides neuroprotection after transient focal cerebral ischemia. Neurosurgery 57, 325–333 (Discussion 325–33).
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
Vascular endothelial growth factor improves recovery of sensorimotor and cognitive deficits after focal cerebral ischemia in the rat Yaoming Wang, Veronica Galvan, Olivia Gorostiza, Marina Ataie, Kunlin Jin, David A. Greenberg⁎ Buck Institute for Age Research, 8001 Redwood Boulevard, Novato, CA 94945, USA
A R T I C LE I N FO
AB S T R A C T
Article history:
Vascular endothelial growth factor (VEGF) is an angiogenesis factor with neurotrophic,
Accepted 20 July 2006
neuroprotective and neuroproliferative effects. Depending on the dose, route and time of
Available online 22 August 2006
administration in relation to focal cerebral ischemia, VEGF can improve histological outcome and sensorimotor function in rodents. However, VEGF also increases vascular
Keywords:
permeability, which can lead to brain edema and exacerbate ischemic brain injury. Thus,
VEGF
although VEGF is a candidate therapeutic for stroke and other ischemic disorders, its benefit
Stroke
relative to risk is uncertain. Considering that functional rather than histological measures of
Memory
outcome are probably most relevant to therapeutic prospects for human stroke, we investigated the effects of VEGF after middle cerebral artery occlusion in rats using a series
Abbreviations:
of behavioral tests. We report that VEGF improves functional outcome in ischemic rats,
aCSF, artificial cerebrospinal fluid
including both sensorimotor and cognitive deficiencies.
EBST, elevated body swing test
© 2006 Elsevier B.V. All rights reserved.
ECA, external carotid artery ICV, intracerebroventricular MCA, middle cerebral artery MCAO, middle cerebral artery occlusion VEGF, vascular endothelial growth factor
1.
Introduction
Vascular endothelial growth factor (VEGF) is an angiogenic and vascular permeability factor (Marti and Risau, 1999), which also has neurotrophic, neuroprotective and neuroproliferative effects (Greenberg and Jin, 2005). The ability of VEGF to elicit both angiogenesis and neuroprotection has led to
interest in its possible therapeutic application in stroke. Several studies have documented that, depending on the dose, route and timing of administration, VEGF can improve histological outcome from experimental stroke (Hayashi et al., 1998; Kaya et al., 2005; Manoonkitiwongsa et al., 2004; Sun et al., 2003; Wang et al., 2005; Zhang et al., 2000), which appears to result from direct neuroprotective action. Adverse effects of
⁎ Corresponding author. Fax: +1 415 209 2230. E-mail address: [email protected] (D.A. Greenberg). 0006-8993/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2006.07.060
187
BR A I N R ES E A RC H 1 1 1 5 ( 2 00 6 ) 1 8 6 –1 93
VEGF, especially its propensity to increase vascular leakage (Fischer et al., 2004) and, therefore, brain edema, can also worsen ischemic brain injury (Van Bruggen et al., 1999; Zhang et al., 2000). Less attention has been given to detailed evaluation of the effects of VEGF on post-stroke behavioral performance. Zhang et al. (2000) found that intravenous VEGF, given for 4 h beginning 48 h after embolic occlusion of the middle cerebral artery (MCA) in rats, improved motor performance measured by the accelerating rotarod test at 7, 14 and 28 days, and somatosensory deficits evaluated with the adhesive removal test at 28 days, post-stroke. We reported that intraventricular VEGF, administered continuously from 24 to 96 h after suture-induced MCA occlusion in rats, enhanced motor and proprioceptive function at 3, 7, 14 and 28 days (Sun et al., 2003). Zhu et al. (2005) transplanted neurosphere-derived neural stem cells, some of which were transfected with VEGF, into the striatum of rats 3 days after suture occlusion of the MCA, and showed improvement in the neurological severity score, which reflects motor, sensory, reflex and balance function, at 4, 6, 8, 10 and 12 weeks. Finally, Kaya et al. (2005) demonstrated that a single intraventricular dose of VEGF, given 1 or 3 h after MCA occlusion (MCAO) in mice, reduced the severity of contralateral limb paralysis 24 h later. Because the relative risk to benefit of VEGF in cerebral ischemia is controversial and since the outcome measure most relevant to clinical efficacy is neurobehavioral function (rather than, for example, infarct size), we examined the effects of VEGF administration after experimental stroke in rats using a broad-based panel of behavioral tests. The results indicate that VEGF improves functional outcome in rats with brain ischemia, including basic and complex sensorimotor as well as cognitive deficiencies assessed by a behavioral testing battery. Thus, under appropriate conditions, VEGF seems capable of conferring long-term functional benefit in the aftermath of acute ischemic stroke.
2.
Results
2.1.
Physiological measures
Table 1 – Physiologic measures in rats undergoing MCAO PaCO2 MABP PaO2 (mm Hg) (mm Hg) (mm Hg) Before ischemia Sham109 ± 11 operated MCAO 116 ± 8 MCAO+ 114 ± 8 VEGF After ischemia Sham114 ± 6 operated MCAO 115 ± 9 MCAO+ 115 ± 7 VEGF
2.2.
Elevated body swing test
The elevated body swing test was used to detect asymmetry in motor behavior. MCAO caused an increase in turning bias throughout the 8-week reperfusion period (Fig. 1), from near 0% in sham-operated controls to 80–95%. Thus, postischemic rats showed a strong and persistent tendency to turn their upper bodies to the side opposite the ischemic hemisphere. VEGF administration decreased the turning bias after MCAO to 55–70% at 1–8 weeks.
Glucose (mg/ml)
85 ± 6
58 ± 4
7.38 ± 1.0
178 ± 16
86 ± 5 87 ± 4
56 ± 5 58 ± 4
7.40 ± 1.1 7.39 ± 0.9
176 ± 12 177 ± 8
83 ± 5
57 ± 4
7.37 ± 1.2
177 ± 10
85 ± 5 85 ± 5
58 ± 5 57 ± 4
7.36 ± 1.3 7.40 ± 0.9
179 ± 12 178 ± 7
Data shown are mean values ± SD from 10 rats per condition. MABP, mean arterial blood pressure; PaO2, partial pressure of O2 in arterial blood; PaCO2, partial pressure of CO2 in arterial blood; pH, arterial blood pH; Glucose, plasma concentration of glucose. There were no significant differences across treatment groups for any of the variables examined.
2.3.
Forelimb and hindlimb placing tests
Rats were also tested for limb placing in response to visual, vibrissae, tactile and proprioceptive stimulation, which requires sensorimotor integration. Sham-operated rats showed consistently high (normal = 12) forelimb and hindlimb placement scores over the 8-week postischemic period (Fig. 2). MCAO reduced scores at all time points examined, but VEGF treatment restored scores towards normal beginning at 1 week.
2.4.
Cylinder test
Sham-operated rats used the forelimbs symmetrically in the cylinder test (∼50% use of ipsilateral limb in Fig. 3). However, in ischemic rats at 15 and 25 days post-MCAO, the unaffected (ipsilateral) limb was used preferentially. Limb use asymmetry following MCAO was reduced in rats given VEGF, and this effect was greater at 25 than at 15 days.
2.5.
Physiological measures, including mean blood pressure, blood pH, blood gases, hematocrit and blood glucose, were recorded before and after MCAO and were within normal ranges (Table 1).
pH
Morris water maze
To investigate whether VEGF treatment influences postischemic cognitive deficits, we first measured spatial learning and memory performance in an open-field water maze, 15 and 25 days after MCAO. All rats performed similarly in the pretraining sessions, with decreasing escape latencies in successive trials both in the cued (Fig. 4A) and visible (Fig. 4B) tasks, consistent with normal learning. Probe trials, in which the escape platform was removed from the tank, were performed after the first (probe 1) and last sessions (probe 2) of the spatial task. These revealed increased target quadrant occupancy during probe 2 compared to probe 1, indicating normal improvement in recall of the former location of the platform for all rats (Fig. 4C). Compared to sham-operated controls, rats subjected to MCAO showed longer platform-finding latencies at 15 and 25 days, and these latencies were reduced by VEGF treatment
188
BR A I N R ES E A RC H 1 1 1 5 ( 2 00 6 ) 1 8 6 –19 3
Fig. 1 – Elevated body swing test. Turning bias after sham surgery and after MCAO followed by intraventricular infusion of aCSF or VEGF. *p < 0.05 compared to MCAO followed by intraventricular infusion of aCSF.
(Fig. 5A). MCAO also reduced the amount of time spent in the target (platform-containing) quadrant of the water maze, and time spent in the target quadrant at 25 days was increased towards normal by VEGF (Fig. 5B).
3.
Discussion
Our results indicate that VEGF can reverse both sensorimotor and cognitive deficits following MCAO in the rat and that its
Fig. 2 – Forelimb and hindlimb placing tests. (A) Forelimb and (B) hindlimb placing after sham surgery and after MCAO followed by intraventricular infusion of aCSF or VEGF. *p < 0.05, **p < 0.01 compared to MCAO followed by intraventricular infusion of aCSF.
Fig. 3 – Cylinder test. Average percent bias favoring the use of the limb ipsilateral to (unaffected by) MCAO was assessed at 15 (left) and 25 (right) days after sham surgery and after MCAO followed by intraventricular infusion of aCSF or VEGF. *p < 0.05 compared to both sham surgery (Con) and MCAO + VEGF (ANOVA and Bonferroni t test).
beneficial effect can last for at least 8 weeks. We reported previously that VEGF, given ICV as in the present study, reduced infarct size and improved motor and proprioceptive function for up to 4 weeks after MCAO (Sun et al., 2003), and others have reported similar effects with intravenous VEGF (Zhang et al., 2000) or intracerebral transplantation of neural stem cells transfected with VEGF (Zhu et al., 2005). In this study, we have extended these studies to incorporate a more extensive analysis of the postischemic behavioral effects of VEGF, including its effect on spatial learning and memory performance. In rodents as in humans, MCAO produces a syndrome characterized predominantly by hemiparesis and hemisensory loss. Accordingly, we employed a battery of sensorimotor tests tailored specifically to assess long-term functional outcome from MCAO (Roof et al., 2001). Thus, the elevated body swing test and cylinder test detect asymmetrical motor behavior such as that resulting from hemiparesis due to an ischemic cortical lesion (Borlongan and Sanberg, 1995; Schallert et al., 1986), and the forelimb and hindlimb placing tests depend on integration of visual, tactile and proprioceptive sensory stimulation to elicit appropriate motor responses (De Ryck et al., 1989; Roof et al., 2001). We observed that VEGF treatment improved performance on the elevated body swing and forelimb and hindlimb placing tests throughout the 8week period of postischemic recovery. In the cylinder test, VEGF-treated rats also showed superior performance when tested 25 days after MCAO. These findings are consistent with sustained enhancement of sensorimotor recovery by VEGF. How does VEGF improve sensorimotor outcome after MCAO? Prior evidence suggests that a direct neuroprotective effect of VEGF is more likely responsible for improved histological and functional sensorimotor outcome from stroke than is VEGF-mediated angiogenesis or neurogenesis (Sun et al., 2003). This is because a reduction in infarct size and better neurological function can be seen as early as 3 days post-stroke in VEGF-treated rats, whereas VEGF-induced angiogenesis and neurogenesis proceed more slowly. In the present study, VEGF-treated rats showed a reduction in
BR A I N R ES E A RC H 1 1 1 5 ( 2 00 6 ) 1 8 6 –1 93
189
Fig. 4 – Water maze training before MCAO. All experimental animals were trained in the Morris water maze, as described in Experimental procedures. Average latencies to the escape platform for all experimental animals, before surgery, during the cued (A) and hidden (B) tasks. (C) Average time spent in the target quadrant (former platform location) by each experimental animal in the first (probe 1) and second (probe 2) probe trials. The performance of each animal in each probe trial is plotted individually. Higher values of target quadrant occupancy correspond to an increasingly accurate recall of the former platform location.
turning bias and improved limb placing as early as 1 week, by which time neither angiogenesis nor neurogenesis is sufficiently advanced to account for these effects (Sun et al., 2003). Of course, this does not exclude a possible role for angiogenesis or neurogenesis in the maintenance of improvement nor in enhancement of recovery that becomes evident only at longer intervals. Although we did not investigate the molecular mechanism through which VEGF improved functional outcome in this study, our prior studies implicate activation of VEGFR2/Flk1 receptors, phosphatidylinositol 3′-kinase/Akt and NF-κB (Jin et al., 2000a,b) and reduced cleavage of caspase-3 (Jin et al., 2001). We also found that postischemic defects in the Morris water maze were less severe after VEGF treatment. This task measures spatial learning, which requires formation of navigation strategies, place learning, memory and visionguided behavior. Specifically, VEGF reduced latency to target at 15 days and 25 days and increased target quadrant occupancy at 25 days post-MCAO. This pattern of improvement suggests that the potential benefit of VEGF in stroke may not be restricted to primary sensorimotor function but may
extend to more complex and highly integrated cortical activities. This is important because, although memory deficits are not prominently associated with MCA-distribution strokes, defects in language (aphasia), spatial awareness (agnosia) and performance of previously learned tasks (apraxia) are. How does VEGF improve memory and learning after MCAO? MCAO in the rat, performed as in the present study, affects the striatum and cerebral cortex, but not the hippocampus, which is required for spatial memory (Moosmang et al., 2005). Therefore, memory impairment following MCAO in the rat must reflect the involvement of another brain region. In fact, the dorsal striatum and nucleus accumbens (De Leonibus et al., 2005; Gengler et al., 2005), as well as prefrontal cortex (Delatour and Gisquet-Verrier, 1996; Floresco et al., 1997), have also been implicated in spatial memory and associated tasks in rodents, and memory for some spatial representations appears to be independent of the hippocampus (Winocur et al., 2005). Memory disturbance is not a prominent finding in patients with MCAO (perhaps partly because it is difficult to identify in the face of other deficits),
190
BR A I N R ES E A RC H 1 1 1 5 ( 2 00 6 ) 1 8 6 –19 3
Fig. 5 – Water maze task at 15 and 25 days after MCAO. (A) Average escapes latencies during two consecutive hidden task sessions, performed 15 or 25 days after MCAO followed by intraventricular infusion of aCSF (MCAO) or VEGF (MCAO + VEGF) as described in Experimental procedures. *p < 0.05 compared to MCAO. Values for sham-operated rats (black bar on y axis) were 25 ± 7 s (n = 16). (B) Average time spent in the target quadrant (former platform location) during the probe trial 15 or 25 days after MCAO followed by intraventricular infusion of aCSF (MCAO) or VEGF (MCAO + VEGF). *p < 0.05 compared to MCAO. Values for sham-operated rats (black bar on y axis) were 33 ± 3 s (n = 16).
but impaired topographic memory and disorders of spatial localization are well documented following MCA strokes that involve the nondominant hemisphere (Landis et al., 1986; Meerwaldt and Van Harskamp, 1982). Because VEGF stimulates neurogenesis in the adult rodent brain (Jin et al., 2002) and since hippocampal neurogenesis has been implicated in memory function (Shors et al., 2001; Snyder et al., 2005), it is conceivable that VEGF-induced neurogenesis contributed to the memory improvement that we observed. For example, stimulation of hippocampal neurogenesis by VEGF appears to be responsible for the ability of environmental enrichment to enhance cognitive performance in rats (Cao et al., 2004). In addition, gerbils that received cranial irradiation to ablate neuronal progenitors and, thereby, inhibit neurogenesis, prior to transient global cerebral ischemia, showed more severe impairment in the Morris water maze than did unirradiated gerbils (Raber et al., 2004). This seems to suggest that postischemic memory function can be modified by ongoing neurogenesis. VEGF couples hypoxia to angiogenesis in the ischemic brain (Marti et al., 2000). VEGF expression in the brain is increased by ischemia (Hayashi et al., 1997; Kovacs et al., 1996; Lennmyr et al., 1998; Plate et al., 1999; Zhang et al., 2002), but the effect of endogenous VEGF on outcome in this setting is controversial. For example, an anti-VEGF antibody given ICV increased infarct size, implying a protective role for endogenous VEGF (Bao et al., 1999), but a VEGF receptor (VEGFR1/Flt1) fusion protein that sequesters VEGF (Van Bruggen et al., 1999) or a neutralizing antibody against VEGF (Kimura et al., 2005) reduced brain edema and infarct size, consistent with a deleterious effect of VEGF. Some human studies also suggest that endogenous VEGF may adversely affect outcome from stroke. For example, VEGF levels are increased in hypercho-
lesterolemia (Xi et al., in press), which is also associated with better prognosis following stroke (Devuyst et al., 2003; Vauthey et al., 2000). Depending on the dose, route and timing of administration, exogenous VEGF may exacerbate postischemic brain edema or hemorrhage (Manoonkitiwongsa et al., 2004; Zhang et al., 2000). Specifically, early post-stroke administration of VEGF by the IV route has been associated with clinical worsening, whereas delayed or ICV administration has shown benefit. IV VEGF, given 1 h after the onset of ischemia, increased blood– brain barrier leakage, hemorrhagic transformation and infarct volume, but when treatment was delayed until 48 h poststroke, IV VEGF enhanced functional recovery (Zhang et al., 2000). Another study showed that a single ICV dose of VEGF, given 1 h or 3 h after MCAO, decreased infarct volume and vascular leakage (Kaya et al., 2005). We chose to administer VEGF ICV at 24 h because our previous results showed a reduction in infarct size with this regimen (Sun et al., 2003). However, the manner in which VEGF might affect sensorimotor or cognitive outcome after MCAO if given according to a different scheme is impossible to predict. Our results bolster the evidence that VEGF or smallmolecule drugs that promote its effects might have therapeutic applications in stroke. With judicious selection of the dose, route and timing of administration, VEGF can reduce infarct size after MCAO in rodents (Hayashi et al., 1998; Kaya et al., 2005; Manoonkitiwongsa et al., 2004; Sun et al., 2003; Wang et al., 2005; Zhang et al., 2000), improve postischemic sensorimotor function (Kaya et al., 2005; Zhang et al., 2000; Zhu et al., 2005) and enhance learning and memory as assessed using the Morris water maze (this study). Its propensity for side effects related to increased vascular permeability or hemorrhagic diathesis might be modified by the concurrent administration of other factors, such as angiopoietins (Thurston et al., 2000), or by substitution of VEGFB, which is similarly neuroprotective in ischemia (Sun et al., 2004) and also stimulates neurogenesis (Sun et al., 2006) but has less tendency to increase vessel permeability (Mould et al., 2005).
4.
Experimental procedures
4.1.
Focal cerebral ischemia
All procedures were approved by local committee review and conducted according to the NIH Guide for the Care and Use of Laboratory Animals, with an effort to minimize suffering and reduce animal numbers. Transient focal cerebral ischemia was induced by the suture occlusion technique (Longa et al., 1989). Male Sprague–Dawley rats weighing 280–310 g were anesthetized with 4% isoflurane in 70% N2O and 30% O2, using a mask. A midline incision was made in the neck, the right external carotid artery (ECA) was carefully exposed and dissected and a 3-0 monofilament nylon suture was inserted from the ECA into the right internal carotid artery to occlude the right MCA at its origin. After 90 min, the suture was removed to allow reperfusion and the wound was closed. Sham-operated rats underwent identical surgery except that the suture was not inserted. Rectal temperature was maintained at 37.0 ± 0.5 °C
BR A I N R ES E A RC H 1 1 1 5 ( 2 00 6 ) 1 8 6 –1 93
using a heating pad and heating lamp. Blood pressure and blood glucose concentration were monitored.
4.2.
VEGF administration
VEGF was administered by the intracerebroventricular (ICV) route as previously described (Sun et al., 2003). One day after ischemia, rats were anesthetized with 4% isoflurane in 70% N2O/30% O2 and implanted with an osmotic minipump (Alzet 1003D; Alza Scientific Products, Mountain View, California, USA). The cannula was placed in the left lateral ventricle 4.0 mm deep as measured from the pial surface, 0.8 mm anterior to the bregma and 1.3 mm lateral to the midline. VEGF (10 μg/ml) was administered at 1 μl/h ICV for 3 days, in artificial cerebrospinal fluid (aCSF) consisting of 128 mM NaCl, 2.5 mM KCl, 0.95 mM CaCl2 and 1.99 mM MgCl2. Control rats received aCSF without VEGF for the same amount of time.
4.3.
Behavioral testing
Postischemic and sham-operated rats treated with VEGF or vehicle underwent a series of behavioral tests designed to measure long-term functional outcome from MCAO (Roof et al., 2001). The tests used were (1) sensorimotor tests including (a) elevated body swing test (EBST) and (b) forelimb and hindlimb placing tests, both conducted weekly for 8 weeks; and (c) limb use asymmetry (cylinder) test, on days 15 and 25; and (2) cognitive testing (Morris water maze), conducted before and 15 and 25 days after stroke. All behavioral testing was done during the rats' light cycle. The experimenter conducting and scoring behavioral tests was blind to the experimental condition.
4.3.1.
Elevated body swing test
In this test of asymmetric motor behavior, rats held by the base of the tail were raised ∼10 cm above the testing surface (Borlongan and Sanberg, 1995). The initial direction of body swing, constituting a turn of the upper body of >10° to either side, was recorded in three sets of ten trials, performed over 5 min. The number of turns in each (left or right) direction was recorded, and the percentage of turns made to the side contralateral to the ischemic hemisphere (percent left-biased swing) was calculated. For each rat, average scores for each week were determined. Percent turning bias in the EBST was combined with percent asymmetry in wall movements for the cylinder test (see below) to provide an overall asymmetry score.
4.3.2.
Forelimb and hindlimb placing tests
Rats were held by supporting the underside of the torso, allowing the forelimbs and hindlimbs to hang freely (De Ryck et al., 1989; Roof et al., 2001). Placement of the left or right forelimb onto a tabletop was then recorded as rats were moved slowly toward the edge of a tabletop, either (i, ii) stopping short of touching the vibrissae as the rats moved forward or laterally (forward or lateral vision-induced placing), (iii) touching the vibrissae (vibrissae-induced placing), (iv, v) making light contact with the dorsal or lateral aspect of the forepaw to the edge of the tabletop (dorsal or lateral tactile-
191
induced placing) or (vi) pressing the forepaws to the edge of the table (proprioceptive placing). Hindlimb placing was tested in an analogous manner. Each of the six tests listed above was scored as: 0, no placing; 1, incomplete or delayed placing; 2, complete, immediate placing. Average placing scores were calculated for each animal, with a total score of 12 (2 on each of six tests) considered normal.
4.3.3.
Cylinder test
Asymmetric use of the forelimbs (forelimb-use bias) was analyzed by observing the rat's movements in a transparent, 18-cm wide, 30-cm high Plexiglas cylinder, over 3-minute intervals (Schallert et al., 1986). The cylinder was sufficiently large to permit movement while not permitting the rat to reach the upper edge, yet small enough to promote rearing and wall exploration. A mirror behind the cylinder made it possible to observe and record forelimb movements with the rat facing away from the examiner. Following an episode of rearing and wall exploration, a landing was scored for the first limb to contact the ground or for both limbs if they made simultaneous contact. Percent-use scores were calculated for (i) movements using the unimpaired limb and (ii) movements using the impaired limb, relative to the total number of movements. Percentage use of the impaired was subtracted from percentage use of the unimpaired limb to yield an overall limb-bias score. Wall exploration and landing movements were analyzed separately. Average cylinder test scores were calculated for each animal and for each week of testing, which consisted of two sessions per week.
4.3.4.
Morris water maze
Cognitive deficits were assessed using the Morris water maze (Morris, 1984). Before and at different reperfusion times after stroke, rats were given a series of six trials, spaced 1 h apart, in a dark-colored, 160-cm-diameter tank filled with opaque white water at a temperature of 22.0 ± 1.0 °C. To test nonspatial learning, rats were trained to find a 12 cm × 12 cm platform, submerged 1 cm below the water's surface, which was marked with a colored pole as a landmark, and placed in different quadrants of the tank. Rats were lowered into the tank, facing the wall, and released at different locations in each trial. Each rat had up to 90 s to find the submerged platform; if the platform was not found during this time, the rat was guided to it. After staying on the platform for 20 s, the rat was relocated to a dry cage. One hour later, each rat was retested, using an identical release site and platform position, to assess retention of platform location. This was repeated six times for each rat, at 30-min to 60-min intervals. To test spatial learning, the tank was surrounded by opaque dark panels placed ∼ 90 cm from its edge. Four rectangular drawings with geometric designs in black and white were evenly spaced on the panels to serve as distal cues. The rats were trained to find the submerged platform by swimming six times every day for 2 days. These six trials were followed by a probe trial, during which the platform was removed from the pool, and the rat was permitted to swim for 60 s. The percent time and portion of the swim path that each rat spent in the quadrant where the platform had been were used to measure retention of the platform's location. This procedure was repeated 15 and 25 days later. Swim paths were recorded using a computer-
192
BR A I N R ES E A RC H 1 1 1 5 ( 2 00 6 ) 1 8 6 –19 3
interfaced camera tracking system (Water2020, HVS Image, UK). Parameters measured in each trial included: path length and latency to platform (reflecting the ability to learn and remember platform location), swimming velocity (to detect the effect of possible differences in swimming ability on results), percentage of swimming path confined to the outer 15 cm of the tank rather than searching for the platform, percentage of path in a 36-cm-diameter circle surrounding platform, and initial heading error (deviation between actual and correct initial direction of swimming in the first 3 s, indicative of memory for the general location of the platform).
4.4.
Statistical analysis
Results were reported as mean ± SD. The significance of differences between means was assessed by Student's t test (single comparisons) and two-way ANOVA followed by Bonferroni's post hoc or Fisher's PLSD (multiple comparisons), with p < 0.05 considered statistically significant.
Acknowledgments This work was supported by NIH grants NS44921 (D.A.G.) and AG21980 (K.J.).
REFERENCES
Bao, W.L., Lu, S.D., Wang, H., Sun, F.Y., 1999. Intraventricular vascular endothelial growth factor antibody increases infarct volume following transient cerebral ischemia. Chung-Kuo Yao Li Hsueh Pao 20, 313–318. Borlongan, C.V., Sanberg, P.R., 1995. Elevated body swing test: a new behavioral parameter for rats with 6-hydroxydopamine-induced hemiparkinsonism. J. Neurosci. 15, 5372–5378. Cao, L., Jiao, X., Zuzga, D.S., Liu, Y., Fong, D.M., Young, D., During, M.J., 2004. Vegf links hippocampal activity with neurogenesis, learning and memory. Nat. Genet. 36, 827–835. Delatour, B., Gisquet-Verrier, P., 1996. Prelimbic cortex specific lesions disrupt delayed-variable response tasks in the rat. Behav. Neurosci. 110, 1282–1298. De Leonibus, E., Oliverio, A., Mele, A., 2005. A study on the role of the dorsal striatum and the nucleus accumbens in allocentric and egocentric spatial memory consolidation. Learn. Mem. 12, 491–503. De Ryck, M., Van Reempts, J., Borgers, M., Wauquier, A., Janssen, P.A., 1989. Photochemical stroke model: flunarizine prevents sensorimotor deficits after neocortical infarcts in rats. Stroke 20, 1383–1390. Devuyst, G., Karapanayiotides, T., Hottinger, I., Van Melle, G., Bogousslavsky, J., 2003. Prodromal and early epileptic seizures in acute stroke: does higher serum cholesterol protect? Neurology 61, 249–252. Fischer, S., Wiesnet, M., Marti, H.H., Renz, D., Schaper, W., 2004. Simultaneous activation of several second messengers in hypoxia-induced hyperpermeability of brain derived endothelial cells. J. Cell. Physiol. 198, 359–369. Floresco, S.B., Seamans, J.K., Phillips, A.G., 1997. Selective roles for hippocampal, prefrontal cortical, and ventral striatal circuits in radial-arm maze tasks with or without a delay. J. Neurosci. 17, 1880–1890.
Gengler, S., Mallot, H.A., Holscher, C., 2005. Inactivation of the rat dorsal striatum impairs performance in spatial tasks and alters hippocampal theta in the freely moving rat. Behav. Brain Res. 164, 73–82. Greenberg, D.A., Jin, K., 2005. From angiogenesis to neuropathology. Nature 438, 954–959. Hayashi, T., Abe, K., Suzuki, H., Itomaya, Y., 1997. Rapid induction of vascular endothelial growth factor gene expression after transient middle cerebral artery occlusion in rats. Stroke 28, 2039–2044. Hayashi, T., Abe, K., Itoyama, Y., 1998. Reduction of ischemic damage by application of vascular endothelial growth factor in rat brain after transient ischemia. J. Cereb. Blood Flow Metab. 18, 887–895. Jin, K., Mao, X.O., Batteur, S.P., Mceachron, E., Leahy, A., Greenberg, D.A., 2001. Caspase-3 and the regulation of hypoxic neuronal death by vascular endothelial growth factor. Neuroscience 108, 351–358. Jin, K., Zhu, Y., Sun, Y., Mao, X.O., Xie, L., Greenberg, D.A., 2002. Vascular endothelial growth factor (vegf) stimulates neurogenesis in vitro and in vivo. Proc. Natl. Acad. Sci. U. S. A. 99, 11946–11950. Jin, K.L., Mao, X.O., Greenberg, D.A., 2000a. Vascular endothelial growth factor rescues HN33 neural cells from death induced by serum withdrawal. J. Mol. Neurosci. 14, 197–203. Jin, K.L., Mao, X.O., Greenberg, D.A., 2000b. Vascular endothelial growth factor: direct neuroprotective effect in in vitro ischemia. Proc. Natl. Acad. Sci. U. S. A. 97, 10242–10247. Kaya, D., Gursoy-Ozdemir, Y., Yemisci, M., Tuncer, N., Aktan, S., Dalkara, T., 2005. Vegf protects brain against focal ischemia without increasing blood–brain permeability when administered intracerebroventricularly. J. Cereb. Blood Flow Metab. 25, 1111–1118. Kimura, R., Nakase, H., Tamaki, R., Sakaki, T., 2005. Vascular endothelial growth factor antagonist reduces brain edema formation and venous infarction. Stroke 36, 1259–1263. Kovacs, Z., Ikezaki, K., Samoto, K., Inamura, T., Fukui, M., 1996. Vegf and Flt: expression time kinetics in rat brain infarct. Stroke 27, 1865–1873. Landis, T., Cummings, J.L., Benson, D.F., Palmer, E.P., 1986. Loss of topographic familiarity. An environmental agnosia. Arch. Neurol. 43, 132–136. Lennmyr, F., Ata, K.A., Funa, K., Olsson, Y., Terent, A., 1998. Expression of vascular endothelial growth factor (Vegf) and its receptors (Flt-1 and Flk-1) following permanent and transient occlusion of the middle cerebral artery in the rat. J. Neuropathol. Exp. Neurol. 57, 874–882. Longa, E.Z., Weinstein, P.R., Carlson, S., Cummins, R., 1989. Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke 20, 84–91. Manoonkitiwongsa, P.S., Schultz, R.L., Mccreery, D.B., Whitter, E.F., Lyden, P.D., 2004. Neuroprotection of ischemic brain by vascular endothelial growth factor is critically dependent on proper dosage and may be compromised by angiogenesis. J. Cereb. Blood Flow Metab. 24, 693–702. Marti, H.H., Risau, W., 1999. Angiogenesis in ischemic disease. Thromb. Haemost. 82 (Suppl. 1), 44–52. Marti, H.J., Bernaudin, M., Bellail, A., Schoch, H., Euler, M., Petit, E., Risau, W., 2000. Hypoxia-induced vascular endothelial growth factor expression precedes neovascularization after cerebral ischemia. Am. J. Pathol. 156, 965–976. Meerwaldt, J.D., Van Harskamp, F., 1982. Spatial disorientation in right-hemisphere infarction. J. Neurol. Neurosurg. Psychiatry 45, 586–590. Moosmang, S., Haider, N., Klugbauer, N., Adelsberger, H., Langwieser, N., Muller, J., Stiess, M., Marais, E., Schulla, V., Lacinova, L., Goebbels, S., Nave, K.A., Storm, D.R., Hofmann, F.,
BR A I N R ES E A RC H 1 1 1 5 ( 2 00 6 ) 1 8 6 –1 93
Kleppisch, T., 2005. Role of hippocampal Cav1.2 Ca2+ channels in NMDA receptor-independent synaptic plasticity and spatial memory. J. Neurosci. 25, 9883–9892. Morris, R., 1984. Development of a water-maze procedure for studying spatial learning in the rat. J. Neurosci. Methods 11, 47–60. Mould, A.W., Greco, S.A., Cahill, M.M., Tonks, I.D., Bellomo, D., Patterson, C., Zournazi, A., Nash, A., Scotney, P., Hayward, N.K., Kay, G.F., 2005. Transgenic overexpression of vascular endothelial growth factor-B isoforms by endothelial cells potentiates postnatal vessel growth in vivo and in vitro. Circ. Res. 97, E60–E70. Plate, K.H., Beck, H., Danner, S., Allegrini, P.R., Wiessner, C., 1999. Cell type specific upregulation of vascular endothelial growth factor in an MCA-occlusion model of cerebral infarct. J. Neuropathol. Exp. Neurol. 58, 654–666. Raber, J., Fan, Y., Matsumori, Y., Liu, Z., Weinstein, P.R., Fike, J.R., Liu, J., 2004. Irradiation attenuates neurogenesis and exacerbates ischemia-induced deficits. Ann. Neurol. 55, 381–389. Roof, R.L., Schielke, G.P., Ren, X., Hall, E.D., 2001. A comparison of long-term functional outcome after 2 middle cerebral artery occlusion models in rats. Stroke 32, 2648–2657. Schallert, T., Hernandez, T.D., Barth, T.M., 1986. Recovery of function after brain damage: severe and chronic disruption by diazepam. Brain Res. 379, 104–111. Shors, T.J., Miesegaes, G., Beylin, A., Zhao, M., Rydel, T., Gould, E., 2001. Neurogenesis in the adult is involved in the formation of trace memories. Nature 410, 372–376. Snyder, J.S., Hong, N.S., Mcdonald, R.J., Wojtowicz, J.M., 2005. A role for adult neurogenesis in spatial long-term memory. Neuroscience 130, 843–852. Sun, Y., Jin, K., Xie, L., Childs, J., Mao, X.O., Logvinova, A., Greenberg, D.A., 2003. VEGF-induced neuroprotection, neurogenesis, and angiogenesis after focal cerebral ischemia. J. Clin. Invest. 111, 1843–1851. Sun, Y., Jin, K., Childs, J.T., Xie, L., Mao, X.O., Greenberg, D.A., 2004. Increased severity of cerebral ischemic injury in vascular endothelial growth factor-B-deficient mice. J. Cereb. Blood Flow Metab. 24, 1146–1152. Sun, Y., Jin, K., Childs, J.T., Xie, L., Mao, X.O., Greenberg, D.A., 2006. Vascular endothelial growth factor-B (VEGFB) stimulates
193
neurogenesis: evidence from knockout mice and growth factor administration. Dev. Biol. 289, 329–335. Thurston, G., Rudge, J.S., Ioffe, E., Zhou, H., Ross, L., Croll, S.D., Glazer, N., Holash, J., Mcdonald, D.M., Yancopoulos, G.D., 2000. Angiopoietin-1 protects the adult vasculature against plasma leakage. Nat. Med. 6, 460–463. Van Bruggen, N., Thibodeaux, H., Palmer, J.T., Lee, W.P., Fu, L., Cairns, B., Tumas, D., Gerlai, R., Williams, S.P., Van Lookeren Campagne, M., Ferrara, N., 1999. VEGF antagonism reduces edema formation and tissue damage after ischemia/ reperfusion injury in the mouse brain. J. Clin. Invest. 104, 1613–1620. Vauthey, C., De Freitas, G.R., Van Melle, G., Devuyst, G., Bogousslavsky, J., 2000. Better outcome after stroke with higher serum cholesterol levels. Neurology 54, 1944–1949. Wang, Y., Kilic, E., Kilic, U., Weber, B., Bassetti, C.L., Marti, H.H., Hermann, D.M., 2005. VEGF overexpression induces post-ischaemic neuroprotection, but facilitates haemodynamic steal phenomena. Brain 128, 52–63. Winocur, G., Moscovitch, M., Fogel, S., Rosenbaum, R.S., Sekeres, M., 2005. Preserved spatial memory after hippocampal lesions: effects of extensive experience in a complex environment. Nat. Neurosci. 8, 273–275. Xi, L., Ghosh, S., Wang, X., Das, A., Anderson, F.P., Kukreja, R.C., in press. Hypercholesterolemia enhances tolerance to lethal systemic hypoxia in middle-aged mice: possible role of VEGF downregulation in brain. Mol. Cell. Biochem. doi:10.1007/ s11010-006-9194-7. Zhang, Z.G., Zhang, L., Jiang, Q., Zhang, R., Davies, K., Powers, C., Bruggen, N., Chopp, M., 2000. VEGF enhances angiogenesis and promotes blood–brain barrier leakage in the ischemic brain. J. Clin. Invest. 106, 829–838. Zhang, Z.G., Zhang, L., Tsang, W., Soltanian-Zadeh, H., Morris, D., Zhang, R., Goussev, A., Powers, C., Yeich, T., Chopp, M., 2002. Correlation of VEGF and angiopoietin expression with disruption of blood–brain barrier and angiogenesis after focal cerebral ischemia. J. Cereb. Blood Flow Metab. 22, 379–392. Zhu, W., Mao, Y., Zhao, Y., Zhou, L.F., Wang, Y., Zhu, J.H., Zhu, Y., Yang, G.Y., 2005. Transplantation of vascular endothelial growth factor-transfected neural stem cells into the rat brain provides neuroprotection after transient focal cerebral ischemia. Neurosurgery 57, 325–333 (Discussion 325–33).