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Interferon-β administration confers a beneficial outcome in a rabbit model of thromboembolic cerebral ischemia
Neuroscience Letters 327 (2002) 146–148 www.elsevier.com/locate/neulet
Interferon-b administration confers a beneficial outcome in a rabbit model of thromboembolic cerebral ischemia Haiping Liu a, Lixia Xin a, Bernard P.L. Chan b, Robert Teoh a, Bor Luen Tang a,c,*, Y.H. Tan a a
Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore 117609, Singapore Department of Medicine, National University Hospital, 5 Lower Kent Ridge Road, Singapore 119260, Singapore c Department of Biochemistry, National University of Singapore, 10 Kent Ridge Crescent, Singapore, Singapore
b
Received 4 March 2002; received in revised form 27 March 2002; accepted 2 April 2002
Abstract We demonstrate that treatment with the cytokine human interferon-b (IFN-b) resulted in a beneficial outcome in a rabbit model of thromboembolic cerebral ischemia. Rabbits administered subcutaneously with IFN-b prior to (pretreatment) or after (post-treatment) the introduction of an autologous blood clot into the middle cerebral artery have consistently smaller subcortical infarct volumes compared to control (untreated) counterparts. The average subcortical infarct volume of pre-treatment rabbits is 46.3 ^ 9.3 mm 3 (n ¼ 4), and that of post-treatment rabbit is 40.0 ^ 23.1 mm 3 (n ¼ 4). Both are significantly lower (P ¼ 0:003 and P ¼ 0:004, respectively) than the average subcortical infarct volume of control rabbits (121.6 ^ 32.9 mm 3, n ¼ 5). Although the precise mechanism responsible for the beneficial effect of IFNb on stroke is not yet clear, our results are in line with the known anti-inflammatory potential and anti-apoptotic function of IFN-b. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Animal model; Interferon; Ischemia; Stroke
Stroke is a major health cost burden in terms of exclusion of its victims from the work force and in terms of rehabilitation needs. Despite the obvious clinical importance, there is virtually no effective therapeutic intervention except for thrombolysis by the tissue plasminogen activator (tPA). To be beneficial, however, tPA must be introduced within 3 h upon stroke onset [8], an extremely narrow therapeutic window that eliminates more than 95% of stroke victims. Many neuroprotective agents and other drugs that have been promising in animal studies of ischemic brain injury have subsequently been proven to be either ineffective or toxic in clinical trials [1]. There is, therefore, an acute need for a safe pharmacological agent that is functional within a wider therapeutic window. One of the major mechanisms leading to neuronal cell death during cerebral ischemia is the onset of detrimental inflammatory processes [3,5,6,13]. Proinflammatory cytokines such as tumor necrosis factor a (TNF-a) and interleukin 1b (IL-1b) that are expressed and released by * Corresponding author. Tel.: 165-6874-3732; fax: 65-67791117. E-mail address: [email protected] (B.L. Tang).
neurons, microglia and endothelial cells during the initial episode of ischemia promotes the expression of adhesion molecules such as selectins, intercellular adhesion molecule-1 and integrins. These adhesion molecules enhance leukocyte adhesion to endothelial cells, resulting in microvascular plugging and secondary ischemia. They may also enhance infiltration of leukocytes and macrophages across the blood brain barrier, thereby exerting inflammatory cytotoxicity by releasing free radicals and other agents. Interferon-b (IFN-b) is a broad spectrum antiviral drug. It also has a proven beneficial influence on the course of patients with the demyelinating disease multiple sclerosis (MS), and has become a standard treatment for relapsingremitting MS [2,8,9,12]. Similar therapeutic effects were observed after treating mice with IFN-b at the onset of clinical signs of experimental autoimmune encephalomyelitis, a demyelinating disease animal model widely used in MS studies [7,12]. The mechanism behind the beneficial effects of IFN-b in MS is not known with certainty. However, it is clear that there are two major potential ways of beneficial influence: on cytokine expression and on the entry of leukocytes into the central nervous system. We surmise that, in correlative view of these aspects, IFN-b
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S0 30 4- 39 40 ( 02) 0 03 71- 3
H. Liu et al. / Neuroscience Letters 327 (2002) 146–148
treatment may also help to reduce the extent of cerebral ischemic injury resulting from inflammation. We report here that human IFN-b confers a beneficial outcome in an animal model of stroke. To our knowledge, the use of interferon for the treatment of stroke has not been previously reported. We used human IFN-b produced in a mammalian cell expression system, Glycoferon w (US Patent no. 6,207,146) in our studies. In deciding on a model animal to test our hypothesis, we had taken into consideration that Glycoferon w has a distinct antiviral effect in rabbit kidney RK13 cells (Tan YH, unpublished results), an indication that the rabbit type I interferon receptor could recognize the human ligand, and to elicit downstream responses. Rodent models were ruled out based on the functional basis that Glycoferon w did not elicit a significant antiviral response in rodent cell lines. New Zealand White rabbits weighing 2.5–3.2 kg were employed in this study. Cerebral ischemia was induced with a protocol adapted from published procedures [4], with some modifications. Briefly, rabbits are tranquilized with 0.2 ml/kg of Hypnorm (Janssen Phamaceutica, Belgium). One ml of autologous blood sample was drawn from the ear vein for clot preparation. It was mixed well with 50 mg tin powder (Sigma, USA) and slowly injected into a polyethylene (PE-90) tubing previously flushed with thrombin (1000 U/ml, Sigma, USA). The subsequent clot was cut into a 1.5 cm segment for embolization. Anesthesia was maintained by intravenous injection with a mixture containing equal parts of ketamine (100 mg/ml) and diazepam (10 mg/2 ml, F. Hoffmann-La Roche Ltd, Basel). Once adequate anesthesia was established, a midline neck incision was made and the right common (CCA), internal (ICA) and external carotid arteries (ECA) were isolated. Temporary aneurysm clips were placed on the CCA, and proximal ECA and ICA. An arteriotomy was then made in the CCA distal to the CCA aneurysm clip, and the artery cannulated with the PE tubing containing a 1.5 cm-long segment of the autologous clot. The clot-containing tubing was then advanced to the origin of the ICA. The ICA clip was then momentarily opened, and the clot embolus was injected into the middle cerebral artery via the ICA under microscopic visualization. Glycoferon w was administered in treated animals according to the following regime. ‘Pre-treatment’ rabbits received 1 £ 10 7 IU Glycoferon w subcutaneously 4 h before clot placement and 0.5 £ 10 7 IU Glycoferon w within 30 min after. ‘Post-treatment’ rabbits received 1 £ 10 7 IU Glycoferon w subcutaneously immediately after clot placement and 0.5 £ 10 7 IU Glycoferon w 4 h later. Control rabbits were similarly operated with clot placement but received nothing. The subcutaneous mode of administration was used as this allowed serum Glycoferon w levels to be better sustained (administration via the intravenous route resulted in more rapid metabolic clearance, data not shown). Rabbits that survived were sacrificed after 24 h and their brains harvested. The position of the clot was noted. Brains were
147
cut into 2 mm slices and incubated with 1.5% triphenyltetrazolium chloride (TTC) in saline to delineate the infarct region. Photographs of the slices were taken using a digital camera (Olympus Optics Co.) and the infarct volume calculated with appropriate compensations using the Micro Image Lite 4.0 image analysis software (Fig. 1). Induction of thromboembolic stroke by the introduction of an autologous blood clot into the middle cerebral artery provided a moderately reproducible model. Animals that satisfied all four criteria below are included in the final analysis. These are: (1) clear neurological defects after surgery; (2) survival after 24 h; (3) clot discernable in middle cerebral artery; and (4) a clear subcortical infarct as revealed by TTC staining. Glycoferon w treated rabbits have consistently lower infarct volume compared to untreated rabbits. The average infarct volume of pre-treatment rabbits is 46.3 ^ 9.3 mm 3 (n ¼ 4), and that of posttreatment rabbit is 40.0 ^ 23.1 mm 3 (n ¼ 4). Both are significantly lower (P ¼ 0:003 and P ¼ 0:004, respectively) than the average infarct volume of control rabbits (121.6 ^ 32.9 mm 3, n ¼ 5) as assessed using the Student’s t-test. The results are summarized in Table 1. Human IFN-b has a 10-fold lower efficacy in terms of antiviral effect in rabbit cells as compared to human cells.
Fig. 1. (A) Representative picture showing the presence of a tintagged clot in the middle cerebral artery (MCA). Shown here is the optic chiasm, the MCA and the position of the clot (arrows). Note that the artery itself is translucent and not particular visible, and is located above the underlying red colored vessels that are veins. (B) A representative brain slice stained by the vital dye TTC. Living tissues are stained red while the infarct region (arrow) is unstained.
H. Liu et al. / Neuroscience Letters 327 (2002) 146–148
148
Table 1 A summary of the infarct volumes (in mm 3) of control, pre-treatment and post-treatment rabbits that satisfied all primary assessment criteria and are included in the final analysis a
animal stroke model suggest that it may be therapeutically useful to humans. Preliminary results reported on the neuroprotective effect of IFN-b in a rat model of ischemic stroke [10] appears to be in full agreement with our findings.
Infarct volume (mm 3)
Mean ^ SD
Control
Pre-treatment
Post-treatment
132 113 124 165 74
43 43 39 60
43 71 25 20
121.6 ^ 32.9
46.3 ^ 9.3
40.0 ^ 23.1
a
Statistical outliers were excluded based on Chauvenet’s criteria.
There is also a lag time period required to achieve a maximum serum Glycoferon w concentration upon subcutaneous administration (unpublished results). In both the pre- and post-treatment regime, Glycoferon w administration confers a beneficial outcome to stroke, manifested in this case by the reduction of infarct volume. The mere fact that the beneficial effect of Glycoferon w is already obvious by direct measurement of infarct volume is encouraging. We expect the beneficial effect of Glycoferon w to become even more significant in more refined assessments, including alternative models of transient, global ischemia and detail neurological examination of animals recovering from stroke. Furthermore, the effect of Glycoferon w would be expected to be even greater when used on human subjects, if only because human type I interferon receptors have a much higher affinity for its ligand. IFN-b has been shown to be a relatively safe and non-toxic therapeutic agent in anti-viral treatment and MS trials. As such, it has tremendous potential to be part of an anti-stroke therapeutic regime. Although sustained high serum level of IFN-b can be achieved via subcutaneous injections, it is difficult to determine its concentration at the site of infarct. The drug is unlikely to penetrate the blood–brain barrier but may reach brain tissues when the integrity of the barrier is compromised during primary infarct. We have not yet determined the extent at which this may be occurring in our treated animals. Further investigations would be also needed to fully delineate the cellular mechanisms underlying IFNb’s beneficial influence in stroke. Two possibilities are currently being considered. Firstly, IFN-b may suppress the activities of the pro-inflammatory cytokines such as IL-1 and TNFa that are expressed acutely during stroke and that contribute greatly to neuronal injury and damage via leukocyte adhesion and vascular plugging [13]. Secondly, IFN-b may induce the expression of anti-apoptotic genes and proteins, such as NF-kB [11]. Whatever the mechanism may be, the beneficial effects of IFN-b in the
Bernie Murugesu-Oei provided logistic help and coordination. We are grateful to Huub Schellekens for helpful advice and Masafumi Inoue for the supply of Glycoferon. BLT and YHT were supported by research grants from the Agency for Science, Technology and Research of Singapore to the Institute of Molecular and Cell Biology. [1] del Zoppo, G.J., Clinical trials in acute stroke: why have they not been successful? Neurology, 51 (1998) S59–S61. [2] European Study Group on Interferon b-1b in Secondary Progressive Multiple Sclerosis, Placebo-controlled multicentre randomized trial of interferon b-1b in treatment of secondary progressive multiple sclerosis, Lancet, 352 (1998) 1491–1497. [3] Lee, J.-M., Grabb, M.C., Zipfel, G.J. and Choi, D.W., Brain tissue responses to ischemia, J. Clin. Invest., 106 (2000) 723–731. [4] Lew, S.M., Gross, C.E., Bednar, M.M., Russel, S.J., Fuller, S.P., Ellenberger, C.L. and Howard, D., Complement depletion does not reduce brain injury in a rabbit model of thromboembolic stroke, Brain Res. Bull., 48 (1999) 325–331. [5] Mattson, M.P., Culmsee, C. and Yu, Z.F., Apoptotic and antiapoptotic mechanisms in stroke, Cell Tissue Res., 301 (2000) 173–187. [6] Neumar, R.W., Molecular mechanisms of ischemic neuronal injury, Ann. Emerg. Med., 36 (2000) 483–506. [7] Ruuls, S.R., de Labie, M.C.D.C., Weber, K.S., Botman, C.A.D., Groenestein, R.J., Dijkstra, C.D., Olsson, T. and Van der Meide, P.H., The length of treatment determines whether IFN-b prevents or aggravates experimental autoimmune encephalomyelitis in Lewis rats, J. Immunol., 157 (1996) 5721–5731. [8] The Interferon b Multiple Sclerosis Study Group, Interferon beta-1b is effective in relapsing-remitting multiple sclerosis. 1. Clinical results of a multicenter, randomized, double blind, placebo-controlled trial, Neurology, 43 (1993) 655– 661. [9] The National Institute of Neurological Disorders and Stroke rt-PA Stroke Study Group, Tissue plasminogen activator for acute ischemic stroke, N. Engl. J. Med., 333 (1995) 1581– 1587. [10] Veldhuis, W.B., van der Meide, P.H., Bar, D. and Nicolay, K., Delayed treatment with interferon-b protects against ischemic stroke, Stroke, 33 (2002) 346. [11] Yang, C.H., Murti, A., Pfeffer, S.R., Kim, J.G., Donner, D.B. and Pfeffer, L.M., Interferon alpha/beta promotes cell survival by activating nuclear factor kappa B through phosphatidylinositol 3-kinase and Akt, J. Biol. Chem., 276 (2001) 13756–13761. [12] Yu, M., Nishiyama, A., Trapp, B.D. and Tuohy, V.K., Interferon-b inhibits progression of relapsing-remitting experimental autoimmune encephalomyelitis, J. Neuroimmunol., 64 (1996) 91–100. [13] Zhao, B. and Schwartz, J.P., Involvement of cytokines in normal CNS development and neurological diseases: recent progress and perspectives, J. Neurosci. Res., 52 (1998) 7–16.
Interferon-b administration confers a beneficial outcome in a rabbit model of thromboembolic cerebral ischemia Haiping Liu a, Lixia Xin a, Bernard P.L. Chan b, Robert Teoh a, Bor Luen Tang a,c,*, Y.H. Tan a a
Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore 117609, Singapore Department of Medicine, National University Hospital, 5 Lower Kent Ridge Road, Singapore 119260, Singapore c Department of Biochemistry, National University of Singapore, 10 Kent Ridge Crescent, Singapore, Singapore
b
Received 4 March 2002; received in revised form 27 March 2002; accepted 2 April 2002
Abstract We demonstrate that treatment with the cytokine human interferon-b (IFN-b) resulted in a beneficial outcome in a rabbit model of thromboembolic cerebral ischemia. Rabbits administered subcutaneously with IFN-b prior to (pretreatment) or after (post-treatment) the introduction of an autologous blood clot into the middle cerebral artery have consistently smaller subcortical infarct volumes compared to control (untreated) counterparts. The average subcortical infarct volume of pre-treatment rabbits is 46.3 ^ 9.3 mm 3 (n ¼ 4), and that of post-treatment rabbit is 40.0 ^ 23.1 mm 3 (n ¼ 4). Both are significantly lower (P ¼ 0:003 and P ¼ 0:004, respectively) than the average subcortical infarct volume of control rabbits (121.6 ^ 32.9 mm 3, n ¼ 5). Although the precise mechanism responsible for the beneficial effect of IFNb on stroke is not yet clear, our results are in line with the known anti-inflammatory potential and anti-apoptotic function of IFN-b. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Animal model; Interferon; Ischemia; Stroke
Stroke is a major health cost burden in terms of exclusion of its victims from the work force and in terms of rehabilitation needs. Despite the obvious clinical importance, there is virtually no effective therapeutic intervention except for thrombolysis by the tissue plasminogen activator (tPA). To be beneficial, however, tPA must be introduced within 3 h upon stroke onset [8], an extremely narrow therapeutic window that eliminates more than 95% of stroke victims. Many neuroprotective agents and other drugs that have been promising in animal studies of ischemic brain injury have subsequently been proven to be either ineffective or toxic in clinical trials [1]. There is, therefore, an acute need for a safe pharmacological agent that is functional within a wider therapeutic window. One of the major mechanisms leading to neuronal cell death during cerebral ischemia is the onset of detrimental inflammatory processes [3,5,6,13]. Proinflammatory cytokines such as tumor necrosis factor a (TNF-a) and interleukin 1b (IL-1b) that are expressed and released by * Corresponding author. Tel.: 165-6874-3732; fax: 65-67791117. E-mail address: [email protected] (B.L. Tang).
neurons, microglia and endothelial cells during the initial episode of ischemia promotes the expression of adhesion molecules such as selectins, intercellular adhesion molecule-1 and integrins. These adhesion molecules enhance leukocyte adhesion to endothelial cells, resulting in microvascular plugging and secondary ischemia. They may also enhance infiltration of leukocytes and macrophages across the blood brain barrier, thereby exerting inflammatory cytotoxicity by releasing free radicals and other agents. Interferon-b (IFN-b) is a broad spectrum antiviral drug. It also has a proven beneficial influence on the course of patients with the demyelinating disease multiple sclerosis (MS), and has become a standard treatment for relapsingremitting MS [2,8,9,12]. Similar therapeutic effects were observed after treating mice with IFN-b at the onset of clinical signs of experimental autoimmune encephalomyelitis, a demyelinating disease animal model widely used in MS studies [7,12]. The mechanism behind the beneficial effects of IFN-b in MS is not known with certainty. However, it is clear that there are two major potential ways of beneficial influence: on cytokine expression and on the entry of leukocytes into the central nervous system. We surmise that, in correlative view of these aspects, IFN-b
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S0 30 4- 39 40 ( 02) 0 03 71- 3
H. Liu et al. / Neuroscience Letters 327 (2002) 146–148
treatment may also help to reduce the extent of cerebral ischemic injury resulting from inflammation. We report here that human IFN-b confers a beneficial outcome in an animal model of stroke. To our knowledge, the use of interferon for the treatment of stroke has not been previously reported. We used human IFN-b produced in a mammalian cell expression system, Glycoferon w (US Patent no. 6,207,146) in our studies. In deciding on a model animal to test our hypothesis, we had taken into consideration that Glycoferon w has a distinct antiviral effect in rabbit kidney RK13 cells (Tan YH, unpublished results), an indication that the rabbit type I interferon receptor could recognize the human ligand, and to elicit downstream responses. Rodent models were ruled out based on the functional basis that Glycoferon w did not elicit a significant antiviral response in rodent cell lines. New Zealand White rabbits weighing 2.5–3.2 kg were employed in this study. Cerebral ischemia was induced with a protocol adapted from published procedures [4], with some modifications. Briefly, rabbits are tranquilized with 0.2 ml/kg of Hypnorm (Janssen Phamaceutica, Belgium). One ml of autologous blood sample was drawn from the ear vein for clot preparation. It was mixed well with 50 mg tin powder (Sigma, USA) and slowly injected into a polyethylene (PE-90) tubing previously flushed with thrombin (1000 U/ml, Sigma, USA). The subsequent clot was cut into a 1.5 cm segment for embolization. Anesthesia was maintained by intravenous injection with a mixture containing equal parts of ketamine (100 mg/ml) and diazepam (10 mg/2 ml, F. Hoffmann-La Roche Ltd, Basel). Once adequate anesthesia was established, a midline neck incision was made and the right common (CCA), internal (ICA) and external carotid arteries (ECA) were isolated. Temporary aneurysm clips were placed on the CCA, and proximal ECA and ICA. An arteriotomy was then made in the CCA distal to the CCA aneurysm clip, and the artery cannulated with the PE tubing containing a 1.5 cm-long segment of the autologous clot. The clot-containing tubing was then advanced to the origin of the ICA. The ICA clip was then momentarily opened, and the clot embolus was injected into the middle cerebral artery via the ICA under microscopic visualization. Glycoferon w was administered in treated animals according to the following regime. ‘Pre-treatment’ rabbits received 1 £ 10 7 IU Glycoferon w subcutaneously 4 h before clot placement and 0.5 £ 10 7 IU Glycoferon w within 30 min after. ‘Post-treatment’ rabbits received 1 £ 10 7 IU Glycoferon w subcutaneously immediately after clot placement and 0.5 £ 10 7 IU Glycoferon w 4 h later. Control rabbits were similarly operated with clot placement but received nothing. The subcutaneous mode of administration was used as this allowed serum Glycoferon w levels to be better sustained (administration via the intravenous route resulted in more rapid metabolic clearance, data not shown). Rabbits that survived were sacrificed after 24 h and their brains harvested. The position of the clot was noted. Brains were
147
cut into 2 mm slices and incubated with 1.5% triphenyltetrazolium chloride (TTC) in saline to delineate the infarct region. Photographs of the slices were taken using a digital camera (Olympus Optics Co.) and the infarct volume calculated with appropriate compensations using the Micro Image Lite 4.0 image analysis software (Fig. 1). Induction of thromboembolic stroke by the introduction of an autologous blood clot into the middle cerebral artery provided a moderately reproducible model. Animals that satisfied all four criteria below are included in the final analysis. These are: (1) clear neurological defects after surgery; (2) survival after 24 h; (3) clot discernable in middle cerebral artery; and (4) a clear subcortical infarct as revealed by TTC staining. Glycoferon w treated rabbits have consistently lower infarct volume compared to untreated rabbits. The average infarct volume of pre-treatment rabbits is 46.3 ^ 9.3 mm 3 (n ¼ 4), and that of posttreatment rabbit is 40.0 ^ 23.1 mm 3 (n ¼ 4). Both are significantly lower (P ¼ 0:003 and P ¼ 0:004, respectively) than the average infarct volume of control rabbits (121.6 ^ 32.9 mm 3, n ¼ 5) as assessed using the Student’s t-test. The results are summarized in Table 1. Human IFN-b has a 10-fold lower efficacy in terms of antiviral effect in rabbit cells as compared to human cells.
Fig. 1. (A) Representative picture showing the presence of a tintagged clot in the middle cerebral artery (MCA). Shown here is the optic chiasm, the MCA and the position of the clot (arrows). Note that the artery itself is translucent and not particular visible, and is located above the underlying red colored vessels that are veins. (B) A representative brain slice stained by the vital dye TTC. Living tissues are stained red while the infarct region (arrow) is unstained.
H. Liu et al. / Neuroscience Letters 327 (2002) 146–148
148
Table 1 A summary of the infarct volumes (in mm 3) of control, pre-treatment and post-treatment rabbits that satisfied all primary assessment criteria and are included in the final analysis a
animal stroke model suggest that it may be therapeutically useful to humans. Preliminary results reported on the neuroprotective effect of IFN-b in a rat model of ischemic stroke [10] appears to be in full agreement with our findings.
Infarct volume (mm 3)
Mean ^ SD
Control
Pre-treatment
Post-treatment
132 113 124 165 74
43 43 39 60
43 71 25 20
121.6 ^ 32.9
46.3 ^ 9.3
40.0 ^ 23.1
a
Statistical outliers were excluded based on Chauvenet’s criteria.
There is also a lag time period required to achieve a maximum serum Glycoferon w concentration upon subcutaneous administration (unpublished results). In both the pre- and post-treatment regime, Glycoferon w administration confers a beneficial outcome to stroke, manifested in this case by the reduction of infarct volume. The mere fact that the beneficial effect of Glycoferon w is already obvious by direct measurement of infarct volume is encouraging. We expect the beneficial effect of Glycoferon w to become even more significant in more refined assessments, including alternative models of transient, global ischemia and detail neurological examination of animals recovering from stroke. Furthermore, the effect of Glycoferon w would be expected to be even greater when used on human subjects, if only because human type I interferon receptors have a much higher affinity for its ligand. IFN-b has been shown to be a relatively safe and non-toxic therapeutic agent in anti-viral treatment and MS trials. As such, it has tremendous potential to be part of an anti-stroke therapeutic regime. Although sustained high serum level of IFN-b can be achieved via subcutaneous injections, it is difficult to determine its concentration at the site of infarct. The drug is unlikely to penetrate the blood–brain barrier but may reach brain tissues when the integrity of the barrier is compromised during primary infarct. We have not yet determined the extent at which this may be occurring in our treated animals. Further investigations would be also needed to fully delineate the cellular mechanisms underlying IFNb’s beneficial influence in stroke. Two possibilities are currently being considered. Firstly, IFN-b may suppress the activities of the pro-inflammatory cytokines such as IL-1 and TNFa that are expressed acutely during stroke and that contribute greatly to neuronal injury and damage via leukocyte adhesion and vascular plugging [13]. Secondly, IFN-b may induce the expression of anti-apoptotic genes and proteins, such as NF-kB [11]. Whatever the mechanism may be, the beneficial effects of IFN-b in the
Bernie Murugesu-Oei provided logistic help and coordination. We are grateful to Huub Schellekens for helpful advice and Masafumi Inoue for the supply of Glycoferon. BLT and YHT were supported by research grants from the Agency for Science, Technology and Research of Singapore to the Institute of Molecular and Cell Biology. [1] del Zoppo, G.J., Clinical trials in acute stroke: why have they not been successful? Neurology, 51 (1998) S59–S61. [2] European Study Group on Interferon b-1b in Secondary Progressive Multiple Sclerosis, Placebo-controlled multicentre randomized trial of interferon b-1b in treatment of secondary progressive multiple sclerosis, Lancet, 352 (1998) 1491–1497. [3] Lee, J.-M., Grabb, M.C., Zipfel, G.J. and Choi, D.W., Brain tissue responses to ischemia, J. Clin. Invest., 106 (2000) 723–731. [4] Lew, S.M., Gross, C.E., Bednar, M.M., Russel, S.J., Fuller, S.P., Ellenberger, C.L. and Howard, D., Complement depletion does not reduce brain injury in a rabbit model of thromboembolic stroke, Brain Res. Bull., 48 (1999) 325–331. [5] Mattson, M.P., Culmsee, C. and Yu, Z.F., Apoptotic and antiapoptotic mechanisms in stroke, Cell Tissue Res., 301 (2000) 173–187. [6] Neumar, R.W., Molecular mechanisms of ischemic neuronal injury, Ann. Emerg. Med., 36 (2000) 483–506. [7] Ruuls, S.R., de Labie, M.C.D.C., Weber, K.S., Botman, C.A.D., Groenestein, R.J., Dijkstra, C.D., Olsson, T. and Van der Meide, P.H., The length of treatment determines whether IFN-b prevents or aggravates experimental autoimmune encephalomyelitis in Lewis rats, J. Immunol., 157 (1996) 5721–5731. [8] The Interferon b Multiple Sclerosis Study Group, Interferon beta-1b is effective in relapsing-remitting multiple sclerosis. 1. Clinical results of a multicenter, randomized, double blind, placebo-controlled trial, Neurology, 43 (1993) 655– 661. [9] The National Institute of Neurological Disorders and Stroke rt-PA Stroke Study Group, Tissue plasminogen activator for acute ischemic stroke, N. Engl. J. Med., 333 (1995) 1581– 1587. [10] Veldhuis, W.B., van der Meide, P.H., Bar, D. and Nicolay, K., Delayed treatment with interferon-b protects against ischemic stroke, Stroke, 33 (2002) 346. [11] Yang, C.H., Murti, A., Pfeffer, S.R., Kim, J.G., Donner, D.B. and Pfeffer, L.M., Interferon alpha/beta promotes cell survival by activating nuclear factor kappa B through phosphatidylinositol 3-kinase and Akt, J. Biol. Chem., 276 (2001) 13756–13761. [12] Yu, M., Nishiyama, A., Trapp, B.D. and Tuohy, V.K., Interferon-b inhibits progression of relapsing-remitting experimental autoimmune encephalomyelitis, J. Neuroimmunol., 64 (1996) 91–100. [13] Zhao, B. and Schwartz, J.P., Involvement of cytokines in normal CNS development and neurological diseases: recent progress and perspectives, J. Neurosci. Res., 52 (1998) 7–16.