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Time window of intracisternal osteogenic protein-1 in enhancing functional recovery after stroke
Neuropharmacology 39 (2000) 860–865 www.elsevier.com/locate/neuropharm
Time window of intracisternal osteogenic protein-1 in enhancing functional recovery after stroke JingMei Ren a
a,*
, Paul L. Kaplan b, Marc F. Charette b, Heather Speller a, Seth P. Finklestein a
CNS Growth Factor Research Laboratory, Department of Neurology, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA b Creative BioMolecules Inc., Hopkinton, MA 01748, USA Accepted 14 December 1999
Abstract Osteogenic protein-1 (OP-1, BMP-7) is a member of the bone morphogenetic protein subfamily of the TGF-ß superfamily that selectively stimulates dendritic neuronal outgrowth. In previous studies, we found that the intracisternal injection of OP-1, starting at one day after stroke, enhanced sensorimotor recovery of the contralateral limbs following unilateral cerebral infarction in rats. In the current study, we further explored the time window during which intracisternal OP-1 enhances sensorimotor recovery, as assessed by limb placing tests. We found that intracisternal OP-1 (10 µg) given 1 and 3 days, or 3 and 5 days, but not 7 and 9 days after stroke, significantly enhanced recovery of forelimb and hindlimb placing. There was no difference in infarct volume between vehicle- and OP-1-treated animals. The mechanism of OP-1 action might be stimulation of new dendritic sprouting in the remaining uninjured brain. 2000 Published by Elsevier Science Ltd. All rights reserved. Keywords: Stroke; Recovery; Rat; Ischemia; Growth factor; Osteogenic protein-1
1. Introduction Cerebral infarction (stroke) is a major public health problem, affecting as many as 700,000 Americans annually (Furie et al., 1998). Stroke is generally due to the blockage of a cerebral artery, causing lack of blood flow (ischemia) and eventual death (infarction) of the region of brain supplied by the artery. Depending on the size and location of cerebral infarction, a number of neurological deficits can ensue, including focal weakness, sensory loss, visual loss, and cognitive impairment (Furie et al., 1998). Although damaged brain does not regenerate, partial neurological recovery commonly ensues in stroke patients, most likely due, in part, to neural reorganization (“rewiring”) in the remaining undamaged parts of the brain. Such reorganization is likely to include new axonal and dendritic sprouting and new synapse formation from remaining intact neurons. Indeed, studies in both animals and humans show such neural reorganiza-
* Corresponding author.
tion both in tissue surrounding focal brain infarcts and in homologous regions of the intact contralateral hemisphere (Cramer and Finklestein, 1998; Jones and Schallert, 1994; Nudo et al., 1996; Stroemer et al., 1995). Osteogenic protein-1 (OP-1), or bone morphogenetic protein-7 (BMP-7) is a member of the bone morphogenetic protein subfamily of the TGF-β superfamily. This 35 kiloDalton (kDa) homodimeric glycoprotein was initially identified by its ability to promote bone formation in a bone environment, but is also expressed in the developing and mature brain (Ozkaynak et al., 1990; Sampath et al., 1992). OP-1 binds to Type I and II highaffinity serine/threonine kinase receptors, which are also found in the developing, mature, and injured brain (Lewen et al., 1997; Soderstrom et al., 1996). In culture, OP-1 selectively promotes the outgrowth of dendritic processes from both peripheral and central neurons (Lein et al., 1995; Withers et al., 1997). This property of promoting dendritic growth distinguishes OP-1 from most other identified neural growth factors, which largely support axonal outgrowth. Because recovery likely depends on new neural out-
0028-3908/00/$ - see front matter 2000 Published by Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 8 - 3 9 0 8 ( 9 9 ) 0 0 2 6 1 - 0
J. Ren et al. / Neuropharmacology 39 (2000) 860–865
growth and synapse formation in uninjured brain regions, and because OP-1 stimulates dendritic outgrowth, we hypothesized that the exogenous administration of OP-1 might enhance functional recovery after stroke. Indeed, in previous studies (Kawamata et al., 1998), we showed that the direct administration of OP1 into the cisterna magna, at one and four days after unilateral cerebral infarction, enhanced sensorimotor recovery of the contralateral limbs. OP-1 was administered intracisternally in these studies to gain access to the intact as well as damaged cerebral cortex. We further found that the recovery-promoting effects of OP-1 were dose dependent, with a greater effect at 10 µg compared to 1 µg per injection. When OP-1 was given in this manner, there was no difference in infarct volume between OP-1- and vehicle-treated animals, although enhanced recovery was seen in OP-1-treated animals (Kawamata et al., 1998). Most “neuroprotective” treatments that reduce infarct size have an effective therapeutic time window of only a few hours in rat models of focal cerebral ischemia (Ren and Finklestein, 1997). In our previous studies using OP1, we showed that the effective time window for initiating treatment to enhance neurological recovery was at least one day following the onset of ischemia (Kawamata et al., 1998). In the current study, we further explored the time window of the recovery-promoting effects of OP-1.
2. Materials and methods Focal cerebral infarcts were made by permanent occlusion of the proximal right middle cerebral artery (MCA) using a modification of the method of Tamura et al. (Kawamata et al., 1998; Tamura et al., 1981) under institutional guidelines. Briefly, male Sprague–Dawley rats (280–330 g, Charles River) were anesthetized with 2% halothane in 70% N2O/30% O2, and anesthesia was maintained with 1–1.5% halothane. The proximal MCA was exposed through a subtemporal craniectomy without removing the zygomatic arch and orbital contents and without transecting the facial nerve. The artery was then occluded by microbipolar coagulation from just proximal to the olfactory tract to the inferior cerebral vein, and was then transected. Body temperature was maintained at 37±0.5°C during the anesthesia using a heating blanket connected to a temperature controller (Model 73A, Yellow Springs Instrument, Yellow Springs, OH, USA). Mature human recombinant OP-1 was supplied by Creative BioMolecules Inc. at a concentration of 0.2 mg/ml in 20 mM acetate buffer, pH 4.5 and 5% mannitol. The vehicle solution that was used as a control contained 0.2 mg/ml bovine serum albumin (BSA; Boehringer Mannheim, Cat. No. 711454) and all other
861
components at the same final concentrations and pH as the OP-1 solution. Solutions were stored refrigerated before use. OP-1 (10 µg in 50 µl) was given by direct percutaneous injection into the cisterna magna following halothane anesthesia (Kawamata et al., 1998) on days 1 and 3, 3 and 5, or 7 and 9 after the onset of the cerebral ischemia. Recovery of sensorimotor function of the contralateral limbs was evaluated by limb placing tests, as described previously (Kawamata et al. 1997, 1998). Rats were handled for 10 min each day for seven days before stroke surgery. Briefly, for the forelimb placing test, the examiner held the rat close to a table and scored the rat’s ability to place the forelimb on the table top in response to whisker, visual, tactile, or proprioceptive stimulation. Similarly, for the hindlimb placing test, the examiner assessed the rat’s ability to place the hindlimb on the table top in response to tactile and proprioceptive stimulation. Separate subscores were obtained for each mode of sensory input and added to give total scores (for the forelimb placing test: 0=normal, 12=maximally impaired; for the hindlimb placing test: 0=normal; 6=maximally impaired). Animals were tested just before stroke surgery, on the first day following surgery, and then every other day thereafter until 31 days after surgery (i.e., on days 0, 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29 and 31 after stroke). On day 31 following MCA occlusion, animals were anesthetized deeply with chloral hydrate and perfused transcardially with heparinized saline followed by 4% paraformaldehyde. Brains were removed and postfixed in 4% paraformaldehyde for 2 days and coronal sections (50 µm) were cut and stained with Hematoxylin and Eosin (H & E). The area of cerebral infarcts was determined on seven slices (+4.7, +2.7, +0.7, ⫺1.3, ⫺3.3, ⫺5.3, and ⫺7.3 compared to bregma, respectively), using a computer-interfaced imaging system (Bioquant, R & M Biometrics, Nashville, TN, USA). Infarct area on each slice was calculated using the “indirect method” as (the area of the intact contralateral hemisphere—the remaining uninfarcted area of the ipsilateral hemisphere) to correct for brain shrinkage during processing (Swanson et al., 1990). Infarct areas were then summed among slices and multiplied by slice thickness to give the total infarct volume, which was expressed as a percentage of the intact contralateral hemispheric volume. Infarct volumes were also determined separately for the cerebral cortex and striatum using similar methods. H & E-stained brain sections were also examined for gross histological abnormalities, including tumor formation, hydrocephalus, etc. Animals were weighed on the days of behavioral assessment. Behavioral scores and body weight were analyzed by two-way repeated measures analysis of variance (ANOVA; treatment×time). Infarct volume was
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J. Ren et al. / Neuropharmacology 39 (2000) 860–865
analyzed by one-way ANOVA. Correlations were done using simple regression analysis.
3. Results There were no differences in body weight between vehicle-treated animals, and those receiving OP-1 at days 1 and 3, 3 and 5, or 7 and 9 following stroke (Fig. 1). All groups lost weight transiently after surgery, and then steadily gained weight during the first month after stroke (Fig. 1). Histological examination of brains showed focal infarction in the territory of the right MCA, involving the dorsolateral cerebral cortex and underlying striatum (Fig. 2). Specifically, infarcts involved cortical regions controlling sensorimotor function of the contralateral (left) limbs, including forelimb (FL) and hindlimb (HL) regions of cortex (Kawamata et al. 1997, 1998). There were no differences in total, cortical, or striatal infarct volume between vehicle-treated animals and those receiving OP-1 at days 1 and 3, 3 and 5, or 7 and 9 after stroke (Table 1). There were no significant correlations between infarct volume and forelimb or hindlimb placing overall, or in the vehicle- or OP-1-treated groups individually. There was no evidence of tumor formation or hydrocephalus in any brain examined. Before stroke, all animals showed intact placing behavior of all four limbs. After stroke, there were marked deficits in placing of the contralateral (left) forelimb and hindlimb that recovered somewhat during the first month after stroke (Fig. 3). Animals treated with OP-1 at days 1 and 3 or days 3 and 5 showed significant enhancement of recovery of forelimb and hindlimb placing, beginning on the day of the first OP-1 injection (Fig. 3). By contrast, animals treated with OP-1 at 7 and 9 days after stroke showed no significant enhancement of recovery of limb placing behavior (Fig. 3).
4. Discussion In summary, we found that intracisternal OP-1, delivered at 1 and 3, or 3 and 5, but not at 7 and 9 days after stroke significantly enhanced recovery of placing behavior of the contralateral limbs. There was no reduction in infarct size in OP-1 treated animals. Moreover, intracisternal OP-1 treatment was not associated with weight loss or other apparent adverse systemic or neurological consequences. These data corroborate and extend our previous findings that intracisternal OP-1 administration enhances recovery of placing behavior of the contralateral limbs following unilateral infarction due to MCA occlusion in rats (Kawamata et al., 1998). The placing tests that we used reflect sensorimotor function of the forelimbs and
Fig. 1. Weight gain after stroke surgery. Graphs show transient weight loss of both vehicle- and OP-1-treated animals for the first day, followed by a steady weight gain for the next month after surgery. Data are expressed as mean±S.E.M. (A) Treatment given 1 and 3 days after surgery, (B) treatment given 3 and 5 days after surgery, (C) treatment given 7 and 9 days after surgery. There were no differences in post-operative weights between vehicle and OP-1-treated animals in any group by two-way repeated measures ANOVA.
J. Ren et al. / Neuropharmacology 39 (2000) 860–865
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Table 1 Volume of infarction in entire ipsilateral (right) hemisphere, cortex, and striatum, expressed as a percentage of the corresponding intact contralateral structure (mean±S.E.M.)a Infarct Vehicle volume (%) n=23
OP-1 (1, 3 days) n=7
OP-1 (3, 5 days) n=8
OP-1 (7, 9 days) n=8
Total 32.6±1.5 (hemisphere) Cortex 40.8±2.1 Striatum 72.7±2.4
27.1±3.9
29.7±1.9
28.8±1.7
34.4±5.8 75.7±9.5
35.9±1.8 85.8±2.8
31.4±3.8 78.7±1.7
a
Fig. 2. Location of cerebral infarcts. H & E-stained sections at four coronal levels through infarcts. (A) +2.7, (B) +0.7, (C) ⫺1.3, and (D) ⫺3.3 mm compared to bregma, respectively. Infarcts involve large regions of the dorsolateral cortex and underlying white matter and striatum, as described in text.
There were no differences among groups by one-way ANOVA.
hindlimbs, which is impaired following damage to the forelimb and hindlimb sensorimotor regions of the dorsolateral cortex, respectively (DeRyck et al., 1992). To some extent, impairments on these tests may also reflect damage to the striatum, which is also affected by the cerebral infarcts we produced. The mechanism by which OP-1 enhances functional recovery in our rat model of stroke is unknown, and requires further study. One possible mechanism is enhancement of dendritic sprouting in remaining uninjured parts of the brain. Previous studies in rats have shown abundant axonal and dendritic sprouting from remaining neurons in regions surrounding focal infarcts and in homologous regions of the contralateral hemisphere (Jones and Schallert, 1994; Stroemer et al., 1995). Other evidence in both rodents and primates suggests that new neuronal sprouting and synapse formation likely contribute to functional recovery following stroke (Jones and Schallert, 1994; Nudo et al., 1996). As noted above, OP-1 selectively enhances dendritic outgrowth from cultured peripheral and central neurons in vitro (Lein et al., 1995; Withers et al., 1997). Stimulation of dendritic sprouting in uninjured brain may be one mechanism by which OP-1 enhances functional recovery in vivo. Our current data using OP-1 are analogous to our previous results using the axonal growth factor bFGF (basic fibroblast growth factor). We found that intracisternal bFGF enhanced functional recovery and stimulated axonal sprouting (as evidenced by upregulation of the axonal protein GAP-43) in both the ipsilateral and contralateral hemispheres following focal infarction (Kawamata et al. 1997, 1999). By analogy, it is possible that OP-1 enhances functional recovery by stimulation of dendritic sprouting in the intact remaining brain. The major intent of the current studies was to explore the time window of enhancement of recovery by OP-1. Indeed, we found that the first injection of intracisternal OP-1 could be given as long as 3 days, but not as long as 7 days after the onset of focal ischemia. This time window of opportunity is considerably longer than that seen for “neuroprotective” agents (NMDA antagonists, free radical scavengers, growth factors, etc.) in animal
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Fig. 3. Paw placing tests following OP-1 treatment. Left panels show the results of forelimb placing tests; right panels show the results of hindlimb placing tests. Data are expressed as mean±S.E.M. and are analyzed by two-way repeated measures ANOVA. (A) Treatment given 1 and 3 days after stroke. Forelimb placing: F=31.5, P⬍0.0001; hindlimb placing: F=86.6, P⬍0.0001; (B) Treatment given 3 and 5 days after stroke. Forelimb placing: F=22.4, P⬍0.0003; hindlimb placing: F=58.7, P⬍0.0001; (C) Treatment given 7 and 9 days after stroke. Forelimb placing: F=1.5, P=n.s.; hindlimb placing: F=0.2, P=n.s.
J. Ren et al. / Neuropharmacology 39 (2000) 860–865
models of acute stroke. Typically, the time window during which such agents can reduce infarct volume is only a few hours after the onset of ischemia (Ren and Finklestein, 1997). The current data emphasize the point that the time window for enhancing stroke recovery is potentially much longer than that for reducing infarct volume. Moreover, enhancement of stroke recovery does not necessarily depend on reduction of infarct volume, but rather, perhaps, on reorganization of the remaining intact brain. Indeed, we found that OP-1 enhanced functional recovery, but did not reduce infarct volume in the current studies. These data have broad implications for the development of recovery-promoting drugs for stroke, which may have effective time windows of several days or even weeks after the onset of ischemia.
Acknowledgements Supported by NS10828 and Creative BioMolecules, Inc.
References Cramer, S.C., Finklestein, S.P., 1998. Reparative approaches: growth factors and other pharmacological treatments. In: Miller, L.P. (Ed.) Stroke Therapy: Basic, Preclinical, and Clinical Directions. John Wiley and Sons, New York, pp. 321–336. DeRyck, M., Reempts, J.V., Duytschaever, H., Deuren, B.V., Clincke, G., 1992. Neocortical localization of tactile/proprioceptive limb placing reactions in the rat. Brain Research 573, 44–60. Furie, K.L., Oglivy, C.S., Smrcka, M., Suwanwela, N., Can, U., Ay, H., Cramer, S.C., Greenberg, S.M., Rordorf, G., Finklestein, S.P., Foster, G.P., Koroshetz, W.J., Suwanwela, N., Kistler, J.P., 1998. Cerebrovascular disease. In: Rosenberg, R.N. (Ed.) The Atlas of Clinical Neurology. Current Medicine, Philadelphia. Jones, T.A., Schallert, T., 1994. Use-dependent growth of pyramidal neurons after neocortical damage. Journal of Neuroscience 14, 2140–2152. Kawamata, T., Dietrich, W.D., Schallert, T., Gotts, J.E., Cocke, R.R., Benowitz, L.I., Finklestein, S.P., 1997. Intracisternal basic fibroblast growth factor (bFGF) enhances functional recovery and upregulates the expression of a molecular marker of neuronal sprouting following focal cerebral infarction. Proceedings of the National Academy of Sciences, USA 94, 8179–8184.
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Kawamata, T., Ren, J., Chan, T.C.K., Charette, M., Finklestein, S.P., 1998. Intracisternal osteogenic protein-1 enhances functional recovery following focal stroke. NeuroReport 9, 1441–1445. Kawamata, T., Ren, J.M., Cha, C.H., Finklestein, S.P., 1999. Intracisternal antisense oligonucleotide to growth associated protein-43 (GAP-43) blocks the recovery-promoting effects of basic fibroblast growth factor (bFGF) after focal stroke. Experimental Neurology 158, 89–96. Lein, P., Johnson, M., Guo, X., Rueger, D., Higgins, D., 1995. Osteogenic protein-1 induces dendritic growth in rat sympathetic neurons. Neuron 15, 597–605. Lewen, A., Soderstrom, S., Hillered, L., Ebendal, T., 1997. Expression of serine/threonine kinase receptors in traumatic brain injury. NeuroReport 8, 475–479. Nudo, R.J., Wise, B.M., SiFuentes, F., Milliken, G.W., 1996. Neural substrates for the effects of rehabilitative training on motor recovery after ischemic infarct. Science 272, 1791–1794. Ozkaynak, E., Rueger, D.C., Drier, E.A., Corbett, C., Ridge, R.J., Sampath, T.K., Oppermann, H., 1990. Op-1 cDNA encodes an osteogenic protein in the TGF-β family. European Molecular Biology Organisation Journal 9, 2085–2093. Ren, J., Finklestein, S.P., 1997. Time window of infarct reduction by intravenous basic fibroblast growth factor in focal cerebral ischemia. European Journal of Pharmacology 327, 11–16. Sampath, T.K., Maliakal, J.C., Hauschka, P.V., Jones, W.K., Sasak, H., Tucker, R.K., White, K.H., Coughlin, J.E., Tucker, M.M., Pang, R.H.L., Corbett, C., Ozkaynak, E., Oppermann, H., Rueger, D.C., 1992. Recombinant human osteogenic protein-1 (hOP-1) induces new bone formation in vivo with a specific activity comparable with mature bovine osteogenic protein and stimulates osteoblast proliferation and differentiation in vitro. Journal of Biological Chemistry 267, 20352–20362. Soderstrom, S., Bengtsson, H., Ebendal, T., 1996. Expression of serine/threonine kinase receptors including the bone morphogenetic factor type II receptor in the developing and adult rat brain. Cell and Tissue Research 286, 269–279. Stroemer, R.P., Kent, T.A., Hulsebosch, C.E., 1995. Neocortical neural sprouting, synaptogenesis, and behavioral recovery after neocortical infarction in rats. Stroke 26, 2135–2144. Swanson, R.A., Morton, M.T., Tsao-Wu, G., Savalos, R.A., Davidson, C., Sharp, F.R., 1990. A semiautomated method for measuring brain infarct volume. Journal of Cerebral Blood Flow and Metabolism 10, 290–293. Tamura, A., Graham, D.I., McCulloch, J., Teasdale, G.M., 1981. Focal cerebral ischemia in the rat: 1. Description of technique and early neuropathological consequences following middle cerebral artery occlusion. Journal of Cerebral Blood Flow and Metabolism 1, 53–60. Withers, G., Higgins, D., Rueger, D., Banker, G., 1997. Treatment with osteogenic protein-1 increases synaptogenesis in cultured hippocampal neurons. Society of Neuroscience Abstracts 23, 1433.
Time window of intracisternal osteogenic protein-1 in enhancing functional recovery after stroke JingMei Ren a
a,*
, Paul L. Kaplan b, Marc F. Charette b, Heather Speller a, Seth P. Finklestein a
CNS Growth Factor Research Laboratory, Department of Neurology, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA b Creative BioMolecules Inc., Hopkinton, MA 01748, USA Accepted 14 December 1999
Abstract Osteogenic protein-1 (OP-1, BMP-7) is a member of the bone morphogenetic protein subfamily of the TGF-ß superfamily that selectively stimulates dendritic neuronal outgrowth. In previous studies, we found that the intracisternal injection of OP-1, starting at one day after stroke, enhanced sensorimotor recovery of the contralateral limbs following unilateral cerebral infarction in rats. In the current study, we further explored the time window during which intracisternal OP-1 enhances sensorimotor recovery, as assessed by limb placing tests. We found that intracisternal OP-1 (10 µg) given 1 and 3 days, or 3 and 5 days, but not 7 and 9 days after stroke, significantly enhanced recovery of forelimb and hindlimb placing. There was no difference in infarct volume between vehicle- and OP-1-treated animals. The mechanism of OP-1 action might be stimulation of new dendritic sprouting in the remaining uninjured brain. 2000 Published by Elsevier Science Ltd. All rights reserved. Keywords: Stroke; Recovery; Rat; Ischemia; Growth factor; Osteogenic protein-1
1. Introduction Cerebral infarction (stroke) is a major public health problem, affecting as many as 700,000 Americans annually (Furie et al., 1998). Stroke is generally due to the blockage of a cerebral artery, causing lack of blood flow (ischemia) and eventual death (infarction) of the region of brain supplied by the artery. Depending on the size and location of cerebral infarction, a number of neurological deficits can ensue, including focal weakness, sensory loss, visual loss, and cognitive impairment (Furie et al., 1998). Although damaged brain does not regenerate, partial neurological recovery commonly ensues in stroke patients, most likely due, in part, to neural reorganization (“rewiring”) in the remaining undamaged parts of the brain. Such reorganization is likely to include new axonal and dendritic sprouting and new synapse formation from remaining intact neurons. Indeed, studies in both animals and humans show such neural reorganiza-
* Corresponding author.
tion both in tissue surrounding focal brain infarcts and in homologous regions of the intact contralateral hemisphere (Cramer and Finklestein, 1998; Jones and Schallert, 1994; Nudo et al., 1996; Stroemer et al., 1995). Osteogenic protein-1 (OP-1), or bone morphogenetic protein-7 (BMP-7) is a member of the bone morphogenetic protein subfamily of the TGF-β superfamily. This 35 kiloDalton (kDa) homodimeric glycoprotein was initially identified by its ability to promote bone formation in a bone environment, but is also expressed in the developing and mature brain (Ozkaynak et al., 1990; Sampath et al., 1992). OP-1 binds to Type I and II highaffinity serine/threonine kinase receptors, which are also found in the developing, mature, and injured brain (Lewen et al., 1997; Soderstrom et al., 1996). In culture, OP-1 selectively promotes the outgrowth of dendritic processes from both peripheral and central neurons (Lein et al., 1995; Withers et al., 1997). This property of promoting dendritic growth distinguishes OP-1 from most other identified neural growth factors, which largely support axonal outgrowth. Because recovery likely depends on new neural out-
0028-3908/00/$ - see front matter 2000 Published by Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 8 - 3 9 0 8 ( 9 9 ) 0 0 2 6 1 - 0
J. Ren et al. / Neuropharmacology 39 (2000) 860–865
growth and synapse formation in uninjured brain regions, and because OP-1 stimulates dendritic outgrowth, we hypothesized that the exogenous administration of OP-1 might enhance functional recovery after stroke. Indeed, in previous studies (Kawamata et al., 1998), we showed that the direct administration of OP1 into the cisterna magna, at one and four days after unilateral cerebral infarction, enhanced sensorimotor recovery of the contralateral limbs. OP-1 was administered intracisternally in these studies to gain access to the intact as well as damaged cerebral cortex. We further found that the recovery-promoting effects of OP-1 were dose dependent, with a greater effect at 10 µg compared to 1 µg per injection. When OP-1 was given in this manner, there was no difference in infarct volume between OP-1- and vehicle-treated animals, although enhanced recovery was seen in OP-1-treated animals (Kawamata et al., 1998). Most “neuroprotective” treatments that reduce infarct size have an effective therapeutic time window of only a few hours in rat models of focal cerebral ischemia (Ren and Finklestein, 1997). In our previous studies using OP1, we showed that the effective time window for initiating treatment to enhance neurological recovery was at least one day following the onset of ischemia (Kawamata et al., 1998). In the current study, we further explored the time window of the recovery-promoting effects of OP-1.
2. Materials and methods Focal cerebral infarcts were made by permanent occlusion of the proximal right middle cerebral artery (MCA) using a modification of the method of Tamura et al. (Kawamata et al., 1998; Tamura et al., 1981) under institutional guidelines. Briefly, male Sprague–Dawley rats (280–330 g, Charles River) were anesthetized with 2% halothane in 70% N2O/30% O2, and anesthesia was maintained with 1–1.5% halothane. The proximal MCA was exposed through a subtemporal craniectomy without removing the zygomatic arch and orbital contents and without transecting the facial nerve. The artery was then occluded by microbipolar coagulation from just proximal to the olfactory tract to the inferior cerebral vein, and was then transected. Body temperature was maintained at 37±0.5°C during the anesthesia using a heating blanket connected to a temperature controller (Model 73A, Yellow Springs Instrument, Yellow Springs, OH, USA). Mature human recombinant OP-1 was supplied by Creative BioMolecules Inc. at a concentration of 0.2 mg/ml in 20 mM acetate buffer, pH 4.5 and 5% mannitol. The vehicle solution that was used as a control contained 0.2 mg/ml bovine serum albumin (BSA; Boehringer Mannheim, Cat. No. 711454) and all other
861
components at the same final concentrations and pH as the OP-1 solution. Solutions were stored refrigerated before use. OP-1 (10 µg in 50 µl) was given by direct percutaneous injection into the cisterna magna following halothane anesthesia (Kawamata et al., 1998) on days 1 and 3, 3 and 5, or 7 and 9 after the onset of the cerebral ischemia. Recovery of sensorimotor function of the contralateral limbs was evaluated by limb placing tests, as described previously (Kawamata et al. 1997, 1998). Rats were handled for 10 min each day for seven days before stroke surgery. Briefly, for the forelimb placing test, the examiner held the rat close to a table and scored the rat’s ability to place the forelimb on the table top in response to whisker, visual, tactile, or proprioceptive stimulation. Similarly, for the hindlimb placing test, the examiner assessed the rat’s ability to place the hindlimb on the table top in response to tactile and proprioceptive stimulation. Separate subscores were obtained for each mode of sensory input and added to give total scores (for the forelimb placing test: 0=normal, 12=maximally impaired; for the hindlimb placing test: 0=normal; 6=maximally impaired). Animals were tested just before stroke surgery, on the first day following surgery, and then every other day thereafter until 31 days after surgery (i.e., on days 0, 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29 and 31 after stroke). On day 31 following MCA occlusion, animals were anesthetized deeply with chloral hydrate and perfused transcardially with heparinized saline followed by 4% paraformaldehyde. Brains were removed and postfixed in 4% paraformaldehyde for 2 days and coronal sections (50 µm) were cut and stained with Hematoxylin and Eosin (H & E). The area of cerebral infarcts was determined on seven slices (+4.7, +2.7, +0.7, ⫺1.3, ⫺3.3, ⫺5.3, and ⫺7.3 compared to bregma, respectively), using a computer-interfaced imaging system (Bioquant, R & M Biometrics, Nashville, TN, USA). Infarct area on each slice was calculated using the “indirect method” as (the area of the intact contralateral hemisphere—the remaining uninfarcted area of the ipsilateral hemisphere) to correct for brain shrinkage during processing (Swanson et al., 1990). Infarct areas were then summed among slices and multiplied by slice thickness to give the total infarct volume, which was expressed as a percentage of the intact contralateral hemispheric volume. Infarct volumes were also determined separately for the cerebral cortex and striatum using similar methods. H & E-stained brain sections were also examined for gross histological abnormalities, including tumor formation, hydrocephalus, etc. Animals were weighed on the days of behavioral assessment. Behavioral scores and body weight were analyzed by two-way repeated measures analysis of variance (ANOVA; treatment×time). Infarct volume was
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J. Ren et al. / Neuropharmacology 39 (2000) 860–865
analyzed by one-way ANOVA. Correlations were done using simple regression analysis.
3. Results There were no differences in body weight between vehicle-treated animals, and those receiving OP-1 at days 1 and 3, 3 and 5, or 7 and 9 following stroke (Fig. 1). All groups lost weight transiently after surgery, and then steadily gained weight during the first month after stroke (Fig. 1). Histological examination of brains showed focal infarction in the territory of the right MCA, involving the dorsolateral cerebral cortex and underlying striatum (Fig. 2). Specifically, infarcts involved cortical regions controlling sensorimotor function of the contralateral (left) limbs, including forelimb (FL) and hindlimb (HL) regions of cortex (Kawamata et al. 1997, 1998). There were no differences in total, cortical, or striatal infarct volume between vehicle-treated animals and those receiving OP-1 at days 1 and 3, 3 and 5, or 7 and 9 after stroke (Table 1). There were no significant correlations between infarct volume and forelimb or hindlimb placing overall, or in the vehicle- or OP-1-treated groups individually. There was no evidence of tumor formation or hydrocephalus in any brain examined. Before stroke, all animals showed intact placing behavior of all four limbs. After stroke, there were marked deficits in placing of the contralateral (left) forelimb and hindlimb that recovered somewhat during the first month after stroke (Fig. 3). Animals treated with OP-1 at days 1 and 3 or days 3 and 5 showed significant enhancement of recovery of forelimb and hindlimb placing, beginning on the day of the first OP-1 injection (Fig. 3). By contrast, animals treated with OP-1 at 7 and 9 days after stroke showed no significant enhancement of recovery of limb placing behavior (Fig. 3).
4. Discussion In summary, we found that intracisternal OP-1, delivered at 1 and 3, or 3 and 5, but not at 7 and 9 days after stroke significantly enhanced recovery of placing behavior of the contralateral limbs. There was no reduction in infarct size in OP-1 treated animals. Moreover, intracisternal OP-1 treatment was not associated with weight loss or other apparent adverse systemic or neurological consequences. These data corroborate and extend our previous findings that intracisternal OP-1 administration enhances recovery of placing behavior of the contralateral limbs following unilateral infarction due to MCA occlusion in rats (Kawamata et al., 1998). The placing tests that we used reflect sensorimotor function of the forelimbs and
Fig. 1. Weight gain after stroke surgery. Graphs show transient weight loss of both vehicle- and OP-1-treated animals for the first day, followed by a steady weight gain for the next month after surgery. Data are expressed as mean±S.E.M. (A) Treatment given 1 and 3 days after surgery, (B) treatment given 3 and 5 days after surgery, (C) treatment given 7 and 9 days after surgery. There were no differences in post-operative weights between vehicle and OP-1-treated animals in any group by two-way repeated measures ANOVA.
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Table 1 Volume of infarction in entire ipsilateral (right) hemisphere, cortex, and striatum, expressed as a percentage of the corresponding intact contralateral structure (mean±S.E.M.)a Infarct Vehicle volume (%) n=23
OP-1 (1, 3 days) n=7
OP-1 (3, 5 days) n=8
OP-1 (7, 9 days) n=8
Total 32.6±1.5 (hemisphere) Cortex 40.8±2.1 Striatum 72.7±2.4
27.1±3.9
29.7±1.9
28.8±1.7
34.4±5.8 75.7±9.5
35.9±1.8 85.8±2.8
31.4±3.8 78.7±1.7
a
Fig. 2. Location of cerebral infarcts. H & E-stained sections at four coronal levels through infarcts. (A) +2.7, (B) +0.7, (C) ⫺1.3, and (D) ⫺3.3 mm compared to bregma, respectively. Infarcts involve large regions of the dorsolateral cortex and underlying white matter and striatum, as described in text.
There were no differences among groups by one-way ANOVA.
hindlimbs, which is impaired following damage to the forelimb and hindlimb sensorimotor regions of the dorsolateral cortex, respectively (DeRyck et al., 1992). To some extent, impairments on these tests may also reflect damage to the striatum, which is also affected by the cerebral infarcts we produced. The mechanism by which OP-1 enhances functional recovery in our rat model of stroke is unknown, and requires further study. One possible mechanism is enhancement of dendritic sprouting in remaining uninjured parts of the brain. Previous studies in rats have shown abundant axonal and dendritic sprouting from remaining neurons in regions surrounding focal infarcts and in homologous regions of the contralateral hemisphere (Jones and Schallert, 1994; Stroemer et al., 1995). Other evidence in both rodents and primates suggests that new neuronal sprouting and synapse formation likely contribute to functional recovery following stroke (Jones and Schallert, 1994; Nudo et al., 1996). As noted above, OP-1 selectively enhances dendritic outgrowth from cultured peripheral and central neurons in vitro (Lein et al., 1995; Withers et al., 1997). Stimulation of dendritic sprouting in uninjured brain may be one mechanism by which OP-1 enhances functional recovery in vivo. Our current data using OP-1 are analogous to our previous results using the axonal growth factor bFGF (basic fibroblast growth factor). We found that intracisternal bFGF enhanced functional recovery and stimulated axonal sprouting (as evidenced by upregulation of the axonal protein GAP-43) in both the ipsilateral and contralateral hemispheres following focal infarction (Kawamata et al. 1997, 1999). By analogy, it is possible that OP-1 enhances functional recovery by stimulation of dendritic sprouting in the intact remaining brain. The major intent of the current studies was to explore the time window of enhancement of recovery by OP-1. Indeed, we found that the first injection of intracisternal OP-1 could be given as long as 3 days, but not as long as 7 days after the onset of focal ischemia. This time window of opportunity is considerably longer than that seen for “neuroprotective” agents (NMDA antagonists, free radical scavengers, growth factors, etc.) in animal
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Fig. 3. Paw placing tests following OP-1 treatment. Left panels show the results of forelimb placing tests; right panels show the results of hindlimb placing tests. Data are expressed as mean±S.E.M. and are analyzed by two-way repeated measures ANOVA. (A) Treatment given 1 and 3 days after stroke. Forelimb placing: F=31.5, P⬍0.0001; hindlimb placing: F=86.6, P⬍0.0001; (B) Treatment given 3 and 5 days after stroke. Forelimb placing: F=22.4, P⬍0.0003; hindlimb placing: F=58.7, P⬍0.0001; (C) Treatment given 7 and 9 days after stroke. Forelimb placing: F=1.5, P=n.s.; hindlimb placing: F=0.2, P=n.s.
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models of acute stroke. Typically, the time window during which such agents can reduce infarct volume is only a few hours after the onset of ischemia (Ren and Finklestein, 1997). The current data emphasize the point that the time window for enhancing stroke recovery is potentially much longer than that for reducing infarct volume. Moreover, enhancement of stroke recovery does not necessarily depend on reduction of infarct volume, but rather, perhaps, on reorganization of the remaining intact brain. Indeed, we found that OP-1 enhanced functional recovery, but did not reduce infarct volume in the current studies. These data have broad implications for the development of recovery-promoting drugs for stroke, which may have effective time windows of several days or even weeks after the onset of ischemia.
Acknowledgements Supported by NS10828 and Creative BioMolecules, Inc.
References Cramer, S.C., Finklestein, S.P., 1998. Reparative approaches: growth factors and other pharmacological treatments. In: Miller, L.P. (Ed.) Stroke Therapy: Basic, Preclinical, and Clinical Directions. John Wiley and Sons, New York, pp. 321–336. DeRyck, M., Reempts, J.V., Duytschaever, H., Deuren, B.V., Clincke, G., 1992. Neocortical localization of tactile/proprioceptive limb placing reactions in the rat. Brain Research 573, 44–60. Furie, K.L., Oglivy, C.S., Smrcka, M., Suwanwela, N., Can, U., Ay, H., Cramer, S.C., Greenberg, S.M., Rordorf, G., Finklestein, S.P., Foster, G.P., Koroshetz, W.J., Suwanwela, N., Kistler, J.P., 1998. Cerebrovascular disease. In: Rosenberg, R.N. (Ed.) The Atlas of Clinical Neurology. Current Medicine, Philadelphia. Jones, T.A., Schallert, T., 1994. Use-dependent growth of pyramidal neurons after neocortical damage. Journal of Neuroscience 14, 2140–2152. Kawamata, T., Dietrich, W.D., Schallert, T., Gotts, J.E., Cocke, R.R., Benowitz, L.I., Finklestein, S.P., 1997. Intracisternal basic fibroblast growth factor (bFGF) enhances functional recovery and upregulates the expression of a molecular marker of neuronal sprouting following focal cerebral infarction. Proceedings of the National Academy of Sciences, USA 94, 8179–8184.
865
Kawamata, T., Ren, J., Chan, T.C.K., Charette, M., Finklestein, S.P., 1998. Intracisternal osteogenic protein-1 enhances functional recovery following focal stroke. NeuroReport 9, 1441–1445. Kawamata, T., Ren, J.M., Cha, C.H., Finklestein, S.P., 1999. Intracisternal antisense oligonucleotide to growth associated protein-43 (GAP-43) blocks the recovery-promoting effects of basic fibroblast growth factor (bFGF) after focal stroke. Experimental Neurology 158, 89–96. Lein, P., Johnson, M., Guo, X., Rueger, D., Higgins, D., 1995. Osteogenic protein-1 induces dendritic growth in rat sympathetic neurons. Neuron 15, 597–605. Lewen, A., Soderstrom, S., Hillered, L., Ebendal, T., 1997. Expression of serine/threonine kinase receptors in traumatic brain injury. NeuroReport 8, 475–479. Nudo, R.J., Wise, B.M., SiFuentes, F., Milliken, G.W., 1996. Neural substrates for the effects of rehabilitative training on motor recovery after ischemic infarct. Science 272, 1791–1794. Ozkaynak, E., Rueger, D.C., Drier, E.A., Corbett, C., Ridge, R.J., Sampath, T.K., Oppermann, H., 1990. Op-1 cDNA encodes an osteogenic protein in the TGF-β family. European Molecular Biology Organisation Journal 9, 2085–2093. Ren, J., Finklestein, S.P., 1997. Time window of infarct reduction by intravenous basic fibroblast growth factor in focal cerebral ischemia. European Journal of Pharmacology 327, 11–16. Sampath, T.K., Maliakal, J.C., Hauschka, P.V., Jones, W.K., Sasak, H., Tucker, R.K., White, K.H., Coughlin, J.E., Tucker, M.M., Pang, R.H.L., Corbett, C., Ozkaynak, E., Oppermann, H., Rueger, D.C., 1992. Recombinant human osteogenic protein-1 (hOP-1) induces new bone formation in vivo with a specific activity comparable with mature bovine osteogenic protein and stimulates osteoblast proliferation and differentiation in vitro. Journal of Biological Chemistry 267, 20352–20362. Soderstrom, S., Bengtsson, H., Ebendal, T., 1996. Expression of serine/threonine kinase receptors including the bone morphogenetic factor type II receptor in the developing and adult rat brain. Cell and Tissue Research 286, 269–279. Stroemer, R.P., Kent, T.A., Hulsebosch, C.E., 1995. Neocortical neural sprouting, synaptogenesis, and behavioral recovery after neocortical infarction in rats. Stroke 26, 2135–2144. Swanson, R.A., Morton, M.T., Tsao-Wu, G., Savalos, R.A., Davidson, C., Sharp, F.R., 1990. A semiautomated method for measuring brain infarct volume. Journal of Cerebral Blood Flow and Metabolism 10, 290–293. Tamura, A., Graham, D.I., McCulloch, J., Teasdale, G.M., 1981. Focal cerebral ischemia in the rat: 1. Description of technique and early neuropathological consequences following middle cerebral artery occlusion. Journal of Cerebral Blood Flow and Metabolism 1, 53–60. Withers, G., Higgins, D., Rueger, D., Banker, G., 1997. Treatment with osteogenic protein-1 increases synaptogenesis in cultured hippocampal neurons. Society of Neuroscience Abstracts 23, 1433.