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Effects of minocycline alone and in combination with mild hypothermia in embolic stroke
Brain Research 963 (2003) 327–329 www.elsevier.com / locate / brainres
Short communication
Effects of minocycline alone and in combination with mild hypothermia in embolic stroke Chen Xu Wang 1 , Tao Yang, Ashfaq Shuaib* Stroke Research Laboratory, 533 HMRC, University of Alberta, Edmonton, Canada T6 G 2 S2 Accepted 13 November 2002
Abstract Inflammatory reactions occurring in the brain after ischemia may contribute to secondary damage. In the present study effects of minocycline, an anti-inflammatory agent, alone or in combination with mild hypothermia, on focal embolic brain ischemia have been examined. Focal ischemic injury was induced by embolizing a preformed clot into the middle cerebral artery (MCA). Infarct volume was measured at 48 h after the injury. Administration of minocycline alone or minocycline plus mild hypothermia reduced infarct volume significantly. However, mild hypothermia in combination with minocycline did not show any additive effect. These results suggest that minocycline is beneficial in focal ischemic brain injury, and the lack of the enhanced neuroprotection may be due to the brief exposure to hypothermia. 2002 Published by Elsevier Science B.V. Theme: Disorders of the nervous system Topic: Ischemia Keywords: Cerebral; Infarction; Inflammatory; Ischemia; Occlusion; Rat
Inflammatory reactions occur within the brain after ischemic injury and these may contribute to secondary cerebral damage. Because inflammation reactions occur rapidly and persist for at least a few days after ischemic brain injury [1,3,4], these secondary responses are potential targets for human therapy with a sufficiently wide therapeutic window. Minocycline is a semisynthetic second-generation drug of tetracycline and it exerts antiinflammatory effects that are completely separate and distinct from its antimicrobial action [5]. Minocycline is used clinically for treatment of severe inflammatory diseases and rheumatoid arthritis [5]. Recent studies have shown that minocycline reduces the loss of hippocampal pyramidal neurons when used in a gerbil model of global ischemia [11]. In a rat model of brain ischemia induced by occlusion of the middle cerebral artery (MCA) with a suture, minocycline is also an effective neuroprotective *Corresponding author. Tel.: 11-780-492-7503; fax: 11-780-4921617. E-mail address: [email protected] (A. Shuaib). 1 Reprint requests: E-mail address: [email protected] (C.X. Wang).
agent [12]. Mild hypothermia induced before and after ischemic brain injury reduces the structural, metabolic and behavioral changes due to ischemia [2,6,7]. In a model of repetitive forebrain ischemia, we have demonstrated that mild hypothermia protects neurons from damage [6]. We have also shown that the protective effect of mild hypothermia can be enhanced by combination with a neuroprotective agent [7]. However, effects of minocycline and hypothermia on the embolic model of stroke have not been examined. In the present study, we studied whether: (1) minocycline is neuroprotective in a focal embolic model of brain ischemia; (2) mild hypothermia after ischemic injury is neuroprotective in this model; (3) combination of minocycline and mild hypothermia is superior to either minocycline or hypothermia alone. Male Wistar rats, weighing 300–350 g, were used. Embolic focal brain ischemia was induced by embolizing pre-formed clot into the MCA [8,9]. In brief, after rat was anesthetized the right common carotid artery (CCA), right internal carotid artery (ICA) and right external carotid artery (ECA) were exposed. The distal portion of the ECA was ligated and cut. A modified PE-10 catheter, filled with
0006-8993 / 02 / $ – see front matter 2002 Published by Elsevier Science B.V. PII: S0006-8993( 02 )04045-3
328
C.X. Wang et al. / Brain Research 963 (2003) 327–329
bovine thrombin was introduced into the lumen of the right ECA via a small puncture. Ten microliters of blood were withdrawn into the catheter and retained for 15 min to allow formation of a clot. Once the clot formed, the catheter was advanced 17 mm in the ICA until its tip was 1–2 mm away from the origin of the MCA. The preformed clot in the catheter was then injected, and the catheter was removed. The wound was closed and the animal returned to its cage. Body temperature was maintained at 37 8C with a heating pad, for the duration of surgery and immediate post-operation period, until the animal recovered fully from anesthesia; except the hypothermia groups. Hypothermia was induced by spraying 100% alcohol on the body of the rat while a fan circulated room air around the animal [6,7]. The rectal temperature was kept at 34–35 8C during the hypothermia period. Hypothermia was started at 1 h after embolization and it lasted for a period of 2 h. At 48 h after embolization the brain was removed for measurement of infarct volume, as described previously [10]. The infarct volume was expressed as a percentage of the total volume from the ipsilateral hemisphere. The study consisted of four groups. In the control group (n510), saline was injected. In the second group (n59), minocycline (Sigma) was administered 1 and 4 h after embolization on the first day, 45 mg / kg body weight i.p., and 24 and 32 h on the second day, 22.5 mg / kg. In the third group (n59), minocycline was administered and the procedure for the drug administration is the same as in the second group, but hypothermia was also instituted. In the fourth group (n59), animals received hypothermia treatment alone. Minocycline was dissolved in distilled water and injection volume was 2 ml / rat. The dose of minocycline used in the study was based on previous work that showed it effectively reduces inflammation and protects against focal cerebral ischemia in the ischemic brain [11,12]. Embolizing a pre-formed clot resulted in an infarction in the territory irrigated by the MCA. In the control group that received the saline injection, the infarct volume was 3263.2% of the total volume of the ipsilateral hemisphere (mean6S.E.M.) at 48 h after embolization (Fig. 1). In the second group, administration of minocycline alone reduced the infarct volume by 42%, which was significantly smaller than in the control group (P,0.05). In the third group, administration of minocycline plus hypothermia resulted in the infarct volume being reduced by 44% which was also significantly smaller than in the control group (P,0.05). However, the infarct volume was not significantly different between the groups that received minocycline alone and minocycline plus hypothermia. Compared to the control group, infarct volume was reduced by 14% in the ischemic animals that received hypothermia alone. This was not a significant difference from the control group. The mortality rates (Table 1) were three out of 10 rats (30%, one died within 24 h and two within 36 h after
Fig. 1. Effects of minocycline alone or in combination with hypothermia on focal ischemic brain injury. Infarct volume was measured at 48 h after embolization. Number of rats in each group is indicated in the parenthesis above each bar. Bar represents mean6S.E.M. and * denotes significant difference from the saline group (P,0.05). The difference of infarct volume was analyzed with one-way ANOVA followed by Tukey test. Mino, minocycline; hypo, hypothermia.
embolization) in the control group; one out of nine rats (11%, within 36 h) in the second group (minocycline alone). There was no significant difference in mortality rates between those two groups. In the third group (minocycline and hypothermia) and the fourth group (hypothermia alone), all rats reached the end of the experiments. However, statistical analyses showed mortality rates in the third or fourth group were not significantly different from the control group, which may be due to small sample numbers. The present study demonstrates that minocycline, injected after MCA occlusion, was neuroprotective. Data showed that infarct volume was decreased significantly following treatment with minocycline. The infarct volume was also reduced significantly in animals that received treatment of minocycline in combination with hypothermia. The reduction in infarct volume was however the same in these two groups, i.e. minocycline alone and minocycline plus hypothermia. Mild hypothermia instituted 1 h after ischemia with a period of 2 h did not reduce infarct volume significantly. Interestingly, no rat died prematurely in the groups treated with either hypothermia Table 1 Mortality in different groups a Group
Saline (n510)
Mino (n59)
Mino1hypo (n59)
Hypo (n59)
24 h 36 h Total
1 2 3 (20%)
0 1 1 (11%)
0 0 0 (0%)
0 0 0 (0%)
a
Mortality was recorded at 24 and 36 h after embolization. No death occurred later than 36 h after embolization. Mino, minocycline; Hypo, hypothermia.
C.X. Wang et al. / Brain Research 963 (2003) 327–329
alone or in combination with minocycline. These data suggest that hypothermia tended to reduce mortality. The beneficial actions of minocycline treatment in the focal brain ischemia may be due to several mechanisms. Firstly, minocycline may reduce the infarction by inhibiting inflammatory reactions. Minocycline has been used as an anti-inflammatory agent in many diseases and, recently, it has been shown that minocycline prevents microglia activation in the injured brain [5]. Minocycline also inhibits production of other inflammation mediators, for example, prostaglandin (PG) E 2 [12]. Secondly, the protective actions of minocycline may also arise through other mechanisms such as, inhibition of caspase, inducible nitric oxide synthase (iNOS) and p38 mitogen-activated protein kinase (MAPK) [12]. Studies have shown that mild or moderate hypothermia is protective in models of transient or permanent MCA occlusion [2,6,7]. Further, mild hypothermia in combination with drug therapy is a more effective treatment than hypothermia alone in transient cerebral ischemia [7]. Previously, our studies showed that mild hypothermia is neuroprotective in the global model of ischemic brain injury when hypothermia was introduced during the induction of ischemia [6]. In the present study, mild hypothermia was instituted 1 h after ischemic injury, since this will be more clinically relevant. This is the first time we have used the new protocol of hypothermia for treatment of ischemic brain injury. Data showed that mild hypothermia started 1 h after ischemia, with a duration of 2 h, did not significantly reduce infarction size. Hypothermia, in combination with minocycline, reduced infarction volume but it did not further improve stroke outcome than using minocycline alone. These data may indicate that treatment with hypothermia should start earlier and a prolonged hypothermia may be required to achieve neuroprotective effects in this model. The present study has demonstrated that treatment with minocycline alone or in combination with hypothermia reduces infarction volume in focal embolic brain injury. Minocycline has been used clinically for treatment of
329
patients with inflammatory diseases and rheumatoid arthritis [5]. It has minimal side-effects and is well tolerated in the patients. Minocycline, therefore, could potentially be a useful drug for treatment of stroke patients.
References [1] C. Baker, S.T. Onesti, R.A. Solomon, Reduction by delayed hypothermia of cerebral infarction following middle cerebral artery occlusion in the rat, J. Neurosurg. 77 (1992) 438–444. [2] F. Colbourne, G. Sutherland, D. Corbett, Postischemic hypothermia. A critical appraisal with implications for clinical treatment, Mol. Neurobiol. 14 (1997) 171–201. [3] G.Z. Feuerstein, X. Wang, F.C. Barone, The role of cytokines in the neuropathology of stroke and neurotrauma, Neuroimmunomodulation 5 (1998) 143–159. [4] K.Y. Ng, J. Nimmannitya, Massive cerebral infarction with severe brain swelling: a clinicopathological study, Stroke 1 (1970) 158– 163. [5] J.R. O’Dell, Is there a role for antibiotics in the treatment of patients with rheumatoid arthritis?, Drugs 3 (1999) 279–282. [6] A. Shuaib, S. Ijaz, J. Kalra, W. Code, During repetitive forebrain ischemia, post-ischemic hypothermia protects neurons from damage, Can. J. Neurol. Sci. 19 (1992) 428–432. [7] A. Shuaib, S. Ijaz, R. Mazagri, A. Senthilsevlvan, CGS-19755 is neuroprotective during repetitive ischemia: this effect is significantly enhanced when combined with hypothermia, Neuroscience 56 (1993) 915–920. [8] C.X. Wang, Y. Yang, A. Shuaib, A focal embolic model of cerebral ischemia in rats, Brain Res. Brain Res. Protoc. 7 (2001) 115–120. [9] C.X. Wang, K.G. Todd, Y. Yang, T. Gordon, A. Shuaib, Patency of cerebral microvessels after focal embolic stroke in the rat, J. Cereb. Blood Flow Metab. 21 (2001) 413–421. [10] Y. Yang, A. Shuaib, Q. Li, Quantification of infarct size on focal cerebral ischemia model of rats using a simple and economical method, J. Neurosci. Methods 84 (1998) 9–16. [11] J. Yrjanheikki, R. Keinanen, M. Pellikka, T. Hokfelt, J. Koistinaho, Tetracyclines inhibit microglial activation and are neuroprotective in global brain ischemia, Proc. Natl. Acad. Sci. USA 95 (1998) 15769–15774. [12] J. Yrjanheikki, T. Tikka, R. Keinanen, G. Goldsteins, P.H. Chan, J. Koistinaho, A tetracycline derivative, minocycline, reduces inflammation and protects against focal cerebral ischemia with a wide therapeutic window, Proc. Natl. Acad. Sci. USA 96 (1999) 13496– 13500.
Short communication
Effects of minocycline alone and in combination with mild hypothermia in embolic stroke Chen Xu Wang 1 , Tao Yang, Ashfaq Shuaib* Stroke Research Laboratory, 533 HMRC, University of Alberta, Edmonton, Canada T6 G 2 S2 Accepted 13 November 2002
Abstract Inflammatory reactions occurring in the brain after ischemia may contribute to secondary damage. In the present study effects of minocycline, an anti-inflammatory agent, alone or in combination with mild hypothermia, on focal embolic brain ischemia have been examined. Focal ischemic injury was induced by embolizing a preformed clot into the middle cerebral artery (MCA). Infarct volume was measured at 48 h after the injury. Administration of minocycline alone or minocycline plus mild hypothermia reduced infarct volume significantly. However, mild hypothermia in combination with minocycline did not show any additive effect. These results suggest that minocycline is beneficial in focal ischemic brain injury, and the lack of the enhanced neuroprotection may be due to the brief exposure to hypothermia. 2002 Published by Elsevier Science B.V. Theme: Disorders of the nervous system Topic: Ischemia Keywords: Cerebral; Infarction; Inflammatory; Ischemia; Occlusion; Rat
Inflammatory reactions occur within the brain after ischemic injury and these may contribute to secondary cerebral damage. Because inflammation reactions occur rapidly and persist for at least a few days after ischemic brain injury [1,3,4], these secondary responses are potential targets for human therapy with a sufficiently wide therapeutic window. Minocycline is a semisynthetic second-generation drug of tetracycline and it exerts antiinflammatory effects that are completely separate and distinct from its antimicrobial action [5]. Minocycline is used clinically for treatment of severe inflammatory diseases and rheumatoid arthritis [5]. Recent studies have shown that minocycline reduces the loss of hippocampal pyramidal neurons when used in a gerbil model of global ischemia [11]. In a rat model of brain ischemia induced by occlusion of the middle cerebral artery (MCA) with a suture, minocycline is also an effective neuroprotective *Corresponding author. Tel.: 11-780-492-7503; fax: 11-780-4921617. E-mail address: [email protected] (A. Shuaib). 1 Reprint requests: E-mail address: [email protected] (C.X. Wang).
agent [12]. Mild hypothermia induced before and after ischemic brain injury reduces the structural, metabolic and behavioral changes due to ischemia [2,6,7]. In a model of repetitive forebrain ischemia, we have demonstrated that mild hypothermia protects neurons from damage [6]. We have also shown that the protective effect of mild hypothermia can be enhanced by combination with a neuroprotective agent [7]. However, effects of minocycline and hypothermia on the embolic model of stroke have not been examined. In the present study, we studied whether: (1) minocycline is neuroprotective in a focal embolic model of brain ischemia; (2) mild hypothermia after ischemic injury is neuroprotective in this model; (3) combination of minocycline and mild hypothermia is superior to either minocycline or hypothermia alone. Male Wistar rats, weighing 300–350 g, were used. Embolic focal brain ischemia was induced by embolizing pre-formed clot into the MCA [8,9]. In brief, after rat was anesthetized the right common carotid artery (CCA), right internal carotid artery (ICA) and right external carotid artery (ECA) were exposed. The distal portion of the ECA was ligated and cut. A modified PE-10 catheter, filled with
0006-8993 / 02 / $ – see front matter 2002 Published by Elsevier Science B.V. PII: S0006-8993( 02 )04045-3
328
C.X. Wang et al. / Brain Research 963 (2003) 327–329
bovine thrombin was introduced into the lumen of the right ECA via a small puncture. Ten microliters of blood were withdrawn into the catheter and retained for 15 min to allow formation of a clot. Once the clot formed, the catheter was advanced 17 mm in the ICA until its tip was 1–2 mm away from the origin of the MCA. The preformed clot in the catheter was then injected, and the catheter was removed. The wound was closed and the animal returned to its cage. Body temperature was maintained at 37 8C with a heating pad, for the duration of surgery and immediate post-operation period, until the animal recovered fully from anesthesia; except the hypothermia groups. Hypothermia was induced by spraying 100% alcohol on the body of the rat while a fan circulated room air around the animal [6,7]. The rectal temperature was kept at 34–35 8C during the hypothermia period. Hypothermia was started at 1 h after embolization and it lasted for a period of 2 h. At 48 h after embolization the brain was removed for measurement of infarct volume, as described previously [10]. The infarct volume was expressed as a percentage of the total volume from the ipsilateral hemisphere. The study consisted of four groups. In the control group (n510), saline was injected. In the second group (n59), minocycline (Sigma) was administered 1 and 4 h after embolization on the first day, 45 mg / kg body weight i.p., and 24 and 32 h on the second day, 22.5 mg / kg. In the third group (n59), minocycline was administered and the procedure for the drug administration is the same as in the second group, but hypothermia was also instituted. In the fourth group (n59), animals received hypothermia treatment alone. Minocycline was dissolved in distilled water and injection volume was 2 ml / rat. The dose of minocycline used in the study was based on previous work that showed it effectively reduces inflammation and protects against focal cerebral ischemia in the ischemic brain [11,12]. Embolizing a pre-formed clot resulted in an infarction in the territory irrigated by the MCA. In the control group that received the saline injection, the infarct volume was 3263.2% of the total volume of the ipsilateral hemisphere (mean6S.E.M.) at 48 h after embolization (Fig. 1). In the second group, administration of minocycline alone reduced the infarct volume by 42%, which was significantly smaller than in the control group (P,0.05). In the third group, administration of minocycline plus hypothermia resulted in the infarct volume being reduced by 44% which was also significantly smaller than in the control group (P,0.05). However, the infarct volume was not significantly different between the groups that received minocycline alone and minocycline plus hypothermia. Compared to the control group, infarct volume was reduced by 14% in the ischemic animals that received hypothermia alone. This was not a significant difference from the control group. The mortality rates (Table 1) were three out of 10 rats (30%, one died within 24 h and two within 36 h after
Fig. 1. Effects of minocycline alone or in combination with hypothermia on focal ischemic brain injury. Infarct volume was measured at 48 h after embolization. Number of rats in each group is indicated in the parenthesis above each bar. Bar represents mean6S.E.M. and * denotes significant difference from the saline group (P,0.05). The difference of infarct volume was analyzed with one-way ANOVA followed by Tukey test. Mino, minocycline; hypo, hypothermia.
embolization) in the control group; one out of nine rats (11%, within 36 h) in the second group (minocycline alone). There was no significant difference in mortality rates between those two groups. In the third group (minocycline and hypothermia) and the fourth group (hypothermia alone), all rats reached the end of the experiments. However, statistical analyses showed mortality rates in the third or fourth group were not significantly different from the control group, which may be due to small sample numbers. The present study demonstrates that minocycline, injected after MCA occlusion, was neuroprotective. Data showed that infarct volume was decreased significantly following treatment with minocycline. The infarct volume was also reduced significantly in animals that received treatment of minocycline in combination with hypothermia. The reduction in infarct volume was however the same in these two groups, i.e. minocycline alone and minocycline plus hypothermia. Mild hypothermia instituted 1 h after ischemia with a period of 2 h did not reduce infarct volume significantly. Interestingly, no rat died prematurely in the groups treated with either hypothermia Table 1 Mortality in different groups a Group
Saline (n510)
Mino (n59)
Mino1hypo (n59)
Hypo (n59)
24 h 36 h Total
1 2 3 (20%)
0 1 1 (11%)
0 0 0 (0%)
0 0 0 (0%)
a
Mortality was recorded at 24 and 36 h after embolization. No death occurred later than 36 h after embolization. Mino, minocycline; Hypo, hypothermia.
C.X. Wang et al. / Brain Research 963 (2003) 327–329
alone or in combination with minocycline. These data suggest that hypothermia tended to reduce mortality. The beneficial actions of minocycline treatment in the focal brain ischemia may be due to several mechanisms. Firstly, minocycline may reduce the infarction by inhibiting inflammatory reactions. Minocycline has been used as an anti-inflammatory agent in many diseases and, recently, it has been shown that minocycline prevents microglia activation in the injured brain [5]. Minocycline also inhibits production of other inflammation mediators, for example, prostaglandin (PG) E 2 [12]. Secondly, the protective actions of minocycline may also arise through other mechanisms such as, inhibition of caspase, inducible nitric oxide synthase (iNOS) and p38 mitogen-activated protein kinase (MAPK) [12]. Studies have shown that mild or moderate hypothermia is protective in models of transient or permanent MCA occlusion [2,6,7]. Further, mild hypothermia in combination with drug therapy is a more effective treatment than hypothermia alone in transient cerebral ischemia [7]. Previously, our studies showed that mild hypothermia is neuroprotective in the global model of ischemic brain injury when hypothermia was introduced during the induction of ischemia [6]. In the present study, mild hypothermia was instituted 1 h after ischemic injury, since this will be more clinically relevant. This is the first time we have used the new protocol of hypothermia for treatment of ischemic brain injury. Data showed that mild hypothermia started 1 h after ischemia, with a duration of 2 h, did not significantly reduce infarction size. Hypothermia, in combination with minocycline, reduced infarction volume but it did not further improve stroke outcome than using minocycline alone. These data may indicate that treatment with hypothermia should start earlier and a prolonged hypothermia may be required to achieve neuroprotective effects in this model. The present study has demonstrated that treatment with minocycline alone or in combination with hypothermia reduces infarction volume in focal embolic brain injury. Minocycline has been used clinically for treatment of
329
patients with inflammatory diseases and rheumatoid arthritis [5]. It has minimal side-effects and is well tolerated in the patients. Minocycline, therefore, could potentially be a useful drug for treatment of stroke patients.
References [1] C. Baker, S.T. Onesti, R.A. Solomon, Reduction by delayed hypothermia of cerebral infarction following middle cerebral artery occlusion in the rat, J. Neurosurg. 77 (1992) 438–444. [2] F. Colbourne, G. Sutherland, D. Corbett, Postischemic hypothermia. A critical appraisal with implications for clinical treatment, Mol. Neurobiol. 14 (1997) 171–201. [3] G.Z. Feuerstein, X. Wang, F.C. Barone, The role of cytokines in the neuropathology of stroke and neurotrauma, Neuroimmunomodulation 5 (1998) 143–159. [4] K.Y. Ng, J. Nimmannitya, Massive cerebral infarction with severe brain swelling: a clinicopathological study, Stroke 1 (1970) 158– 163. [5] J.R. O’Dell, Is there a role for antibiotics in the treatment of patients with rheumatoid arthritis?, Drugs 3 (1999) 279–282. [6] A. Shuaib, S. Ijaz, J. Kalra, W. Code, During repetitive forebrain ischemia, post-ischemic hypothermia protects neurons from damage, Can. J. Neurol. Sci. 19 (1992) 428–432. [7] A. Shuaib, S. Ijaz, R. Mazagri, A. Senthilsevlvan, CGS-19755 is neuroprotective during repetitive ischemia: this effect is significantly enhanced when combined with hypothermia, Neuroscience 56 (1993) 915–920. [8] C.X. Wang, Y. Yang, A. Shuaib, A focal embolic model of cerebral ischemia in rats, Brain Res. Brain Res. Protoc. 7 (2001) 115–120. [9] C.X. Wang, K.G. Todd, Y. Yang, T. Gordon, A. Shuaib, Patency of cerebral microvessels after focal embolic stroke in the rat, J. Cereb. Blood Flow Metab. 21 (2001) 413–421. [10] Y. Yang, A. Shuaib, Q. Li, Quantification of infarct size on focal cerebral ischemia model of rats using a simple and economical method, J. Neurosci. Methods 84 (1998) 9–16. [11] J. Yrjanheikki, R. Keinanen, M. Pellikka, T. Hokfelt, J. Koistinaho, Tetracyclines inhibit microglial activation and are neuroprotective in global brain ischemia, Proc. Natl. Acad. Sci. USA 95 (1998) 15769–15774. [12] J. Yrjanheikki, T. Tikka, R. Keinanen, G. Goldsteins, P.H. Chan, J. Koistinaho, A tetracycline derivative, minocycline, reduces inflammation and protects against focal cerebral ischemia with a wide therapeutic window, Proc. Natl. Acad. Sci. USA 96 (1999) 13496– 13500.