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tPA and plasminogen proteins are synthesized locally in the mouse hippocampus. Immunodetection of tPA and plasminogen proteins in coronal sections through the hippocampus 12 hr after kainate injection of a wild-type mouse unilaterally into the hippocampus. High- magnification photomicrographs of the CA1 field reacting with ( a ) IgG, ( b ) normal sheep serum, ( c ) anti-tPA antibody at the contralateral side, ( d ) anti-plasminogen antibody at the contralateral side, ( e ) anti-tPA antibody at the ipsilateral side, and ( f ) anti- plasminogen antibody at the ipsilateral side. Scale bar, 50 m.
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Mice lacking the serine protease tissue plasminogen activator (tPA) are resistant to excitotoxin-mediated hippocampal neuronal degeneration. We have used genetic and cellular analyses to study the role of tPA in neuronal cell death. Mice deficient for the zymogen plasminogen, a known substrate for tPA, are also resistant to excitotoxins, implicatin...
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Context 1
... hippocampus could be derived from the blood-borne protein via leakage or transport, or from local synthesis. The latter was suggested by Sappino et al. (1993), who detected low levels of plasminogen mRNA in the hippocampus by RNase-protection assay. In light of the resistance of the plg Ϫ / Ϫ mice to excitotoxins, determining whether plasminogen is synthesized in the brain would help advance the understanding of the pathway of degeneration. (2) Previous work has indicated that cells that synthesize both a plasminogen activator and plasminogen suffer deleterious consequences, possibly attributable to the generation of intracellular proteolysis (Sandgren et al., 1991). It was therefore of interest to determine whether the production of tPA and plasminogen was segregated by cell type. To determine the sites of synthesis of tPA and plasminogen, antisense mouse tPA and plasminogen digoxygenin-labeled RNA probes were hybridized to control or kainate-injected brain sections (Fig. 2). tPA mRNA was detected along the neuronal cell layers in the hippocampus (Fig. 2 c ), as described previously (Qian et al., 1993; Sappino et al., 1993). At higher magnification, the pattern of tPA mRNA along the neuronal cell layer had a complex appearance (Fig. 2 e ), suggesting that synthesis might be occurring in more than one cell type. In addition, after destruction of neurons by kainate injection, the staining in the larger cells (pyramidal cells) disappeared but remained in the darker and smaller cells (Fig. 2 g ). Several lines of evidence indicate that these smaller cells are microglia. (1) Transgenic mice that express the bacterial lacZ gene under the control of mouse tPA 5 Ј regulatory sequences (tPA / lacZ ; Carroll et al., 1994) express  -gal along the neuronal pyramidal cell layer in cells ϳ 10 m in diameter, smaller than pyramidal neurons, but of average size for satellite microglial cells. (2) In the tPA / lacZ transgenic mice, adjacent coronal brain sections were doubly stained for  -gal and with antibodies to detect markers specific for the three non-neuronal cell types in the hippocampus (data not shown).  -gal-expressing cells colocalized with perineuronal microglia (F4/80), but did not colocalize with oligodendrocytes (anti-myelin basic protein antibody) or astro- cytes (anti-glial fibrillary acidic protein). These results demonstrate that tPA mRNA is produced both in neurons (the fraction of the staining that disappears after injection of kainate) and in the satellite microglia that overlie the neuronal cell layer (the fraction that persists after kainate injection). The distribution of tPA mRNA in both cell types is in agreement with the studies on the localization of tPA in the rat cerebellum and brain stem (Ware et al., 1995). Plasminogen mRNA was also detected in the hippocampus along the neuronal cell layers, indicating local transcription of this gene (Fig. 2 d ). Plasminogen mRNA, however, was detected only in the larger pyramidal neurons (Fig. 2 f ), and the mRNA staining was completely abolished by kainate destruction of neurons (Fig. 2 h ). An intriguing finding was that plasminogen mRNA was detected in dendrites emanating from the neuronal cell bodies ( arrows in Fig. 2 f and data not shown). This cellular distribution of plasminogen mRNA suggests transport of the message into the dendrites, which could direct local protein production on neuronal activity (Miyashiro et al., 1994; Link et al., 1995; Steward, 1995). Taken together, these data demonstrate that plasminogen mRNA is synthesized in the brain exclusively by neurons, whereas tPA mRNA is produced in both neurons and perineuronal microglia. Because post-transcriptional control can often regulate protein expression, it was of interest to determine whether tPA and plasminogen proteins were also present in the hippocampus. The presence of tPA protein has been examined previously by Sappino et al. (1993), who determined tPA activity by zymography on tissue sections. Even though tPA mRNA is observed in the CA1– CA3 regions of the hippocampus and in the dentate gyrus (Sappino et al., 1993) (Fig. 2 c ), tPA enzyme activity is confined to CA2–CA3 and the dentate and is not detected in the CA1 region. This distribution of tPA activity was corroborated by subsequent, independent experiments (Tsirka et al., 1995; Gualandris et al., 1996). Sappino et al. (1993) also showed that extracts of the CA1 region contained tPA activity; because preparation of the extracts would dissociate enzyme-inhibitor complexes, they concluded that inhibitors in the CA1 were masking the tPA activity in that region. These elegant studies suggest that tPA activity in the hippocampus is under regulation by inhibitors. Antibodies against tPA revealed the presence of tPA protein over the CA2–CA3 pyramidal subfields and dentate gyrus in neuronal as well as microglial cells (Fig. 3 c,e ). Injection of kainate resulted in stronger tPA microglial expression at the ipsilateral side (Fig. 3 e ), thus confirm- ing that tPA activity is upregulated in activated microglia (Tsirka et al., 1995). We have investigated the presence of plasminogen protein by immunohistochemistry. Plasminogen was detected in neurons in the pyramidal cell layer (Fig. 3). Although plasminogen mRNA is present in most pyramidal neurons, only a small subset of neurons stained for plasminogen protein (Fig. 3 d ). Soon after injection of kainate, the levels of plasminogen protein expression appear to be increased (Fig. 3 f ), suggesting that post-transcriptional regulation controls its expression. Such regulation may represent a mechanism for enhanced expression associated with neuronal activity, in agreement with proposed functions of tPA /plasmin in plasticity and restructuring in the brain (Krystosek and Seeds, 1981; Carroll et al., 1994; Frey et al., 1996). The injection of kainate into the hippocampus results in local activation of microglial cells (Andersson et al., 1991), made man- ifest by changes in morphology and increased expression of the surface antigen F4/80 (Lawson et al., 1990) (Fig. 4 a– d ). Mice deficient for tPA displayed activation of microglial cells after kainate administration, but the response was attenuated compared to that of wild-type animals, both in F4/80 staining intensity and in morphological changes (Tsirka et al., 1995). These results suggested that tPA participates in the activation pathway of microglial cells, which in turn could be affecting the degeneration process. To examine the effect of plasminogen on microglial activation, F4/80 immunostaining was performed on brain sections from kainate-injected plg Ϫ / Ϫ mice. Both the staining intensity and morphology of microglial cells from plg Ϫ / Ϫ mice were comparable to those of wild-type cells (Fig. 4 c–f ). These results show that resistance to neuronal degeneration can occur in spite of normal microglial activation, and that a critical proteolytic event is down- stream of activation. They also suggest that the role of tPA in microglial activation is independent of its activation of plasminogen. The above results identify a genetic and cellular pathway, involving tPA and plasminogen, that can result in neuronal cell death after excitotoxic injury. To evaluate directly whether plasmin, the product generated by the action of tPA on plasminogen, is mediating neuronal death, an inhibitor of the activity of plasmin ( ␣ 2 -antiplasmin) was delivered to the brain. Because tPA is required acutely after excitotoxic insult to mediate neuronal degeneration (Tsirka et al., 1996), inhibition of tPA /plasmin proteolytic activity might retard neuronal cell death. Wild-type mice were infused with ␣ 2 -antiplasmin, injected 2 d later with kainate, and then evaluated for their neuronal status in the hippocampus. Control mice infused with buffer were sensitive to neuronal degeneration in the hippocampus (Fig. 5, top ). In contrast, ␣ 2 antiplasmin retarded neuronal degeneration induced by kainate (Fig. 5, bottom ). The resistance of the protease inhibitor-infused mice was comparable to that observed with untreated tPA Ϫ / Ϫ mice (Table 1). Therefore, plasmin is a product of the proteolytic cascade that promotes hippocampal excitotoxic neuronal death, and inhibition of plasmin activity in the adult animal can protect against degeneration. The results presented here demonstrate that extracellular proteolysis plays a central role in excitotoxin-mediated hippocampal neuronal degeneration. A critical observation in defining this pathway is the protection against degeneration in mice that lack plasminogen. The comparable resistant phenotype conferred by both tPA and plasminogen deficiency is consistent with the function of these enzymes in other contexts: namely, they operate sequentially within a cascade whose final proteolytic product is plasmin. This view is strengthened by the results of local delivery of ␣ 2 -antiplasmin, because this inhibitor confers resistance to wild-type mice. Previous work has implicated proteases in neuronal destruction. (1) The degeneration of ganglion neuronal cells after transection of the optic nerve can be retarded by the injection of protease inhibitors into the vitreous body (Thanos, 1991; Thanos et al., 1993). (2) Protease nexin-1, an inhibitor of plasminogen activators and plasmin, can protect cultured hippocampal neurons against hypoglycemic damage, suggesting that proteases can modulate vulnerability to neurotoxicity (Smith-Swintosky et al., 1995). (3) In the cerebellum of the mouse mutant weaver , increased neuronal cell death is evident, coinciding with 10-fold higher than normal tPA activity. This cell death is also observed in cultured cerebellar weaver neurons and can be prevented by inclusion of aprotinin, a serine protease inhibitor, in the culture medium (Murtom ̈ki et al., 1995). The weaver phenotype results from a mutation in the potassium channel gene Girk2 (Patil et al., 1995), which alters the ...
Context 2
... of the resistance of the plg Ϫ / Ϫ mice to excitotoxins, determining whether plasminogen is synthesized in the brain would help advance the understanding of the pathway of degeneration. (2) Previous work has indicated that cells that synthesize both a plasminogen activator and plasminogen suffer deleterious consequences, possibly attributable to the generation of intracellular proteolysis (Sandgren et al., 1991). It was therefore of interest to determine whether the production of tPA and plasminogen was segregated by cell type. To determine the sites of synthesis of tPA and plasminogen, antisense mouse tPA and plasminogen digoxygenin-labeled RNA probes were hybridized to control or kainate-injected brain sections (Fig. 2). tPA mRNA was detected along the neuronal cell layers in the hippocampus (Fig. 2 c ), as described previously (Qian et al., 1993; Sappino et al., 1993). At higher magnification, the pattern of tPA mRNA along the neuronal cell layer had a complex appearance (Fig. 2 e ), suggesting that synthesis might be occurring in more than one cell type. In addition, after destruction of neurons by kainate injection, the staining in the larger cells (pyramidal cells) disappeared but remained in the darker and smaller cells (Fig. 2 g ). Several lines of evidence indicate that these smaller cells are microglia. (1) Transgenic mice that express the bacterial lacZ gene under the control of mouse tPA 5 Ј regulatory sequences (tPA / lacZ ; Carroll et al., 1994) express  -gal along the neuronal pyramidal cell layer in cells ϳ 10 m in diameter, smaller than pyramidal neurons, but of average size for satellite microglial cells. (2) In the tPA / lacZ transgenic mice, adjacent coronal brain sections were doubly stained for  -gal and with antibodies to detect markers specific for the three non-neuronal cell types in the hippocampus (data not shown).  -gal-expressing cells colocalized with perineuronal microglia (F4/80), but did not colocalize with oligodendrocytes (anti-myelin basic protein antibody) or astro- cytes (anti-glial fibrillary acidic protein). These results demonstrate that tPA mRNA is produced both in neurons (the fraction of the staining that disappears after injection of kainate) and in the satellite microglia that overlie the neuronal cell layer (the fraction that persists after kainate injection). The distribution of tPA mRNA in both cell types is in agreement with the studies on the localization of tPA in the rat cerebellum and brain stem (Ware et al., 1995). Plasminogen mRNA was also detected in the hippocampus along the neuronal cell layers, indicating local transcription of this gene (Fig. 2 d ). Plasminogen mRNA, however, was detected only in the larger pyramidal neurons (Fig. 2 f ), and the mRNA staining was completely abolished by kainate destruction of neurons (Fig. 2 h ). An intriguing finding was that plasminogen mRNA was detected in dendrites emanating from the neuronal cell bodies ( arrows in Fig. 2 f and data not shown). This cellular distribution of plasminogen mRNA suggests transport of the message into the dendrites, which could direct local protein production on neuronal activity (Miyashiro et al., 1994; Link et al., 1995; Steward, 1995). Taken together, these data demonstrate that plasminogen mRNA is synthesized in the brain exclusively by neurons, whereas tPA mRNA is produced in both neurons and perineuronal microglia. Because post-transcriptional control can often regulate protein expression, it was of interest to determine whether tPA and plasminogen proteins were also present in the hippocampus. The presence of tPA protein has been examined previously by Sappino et al. (1993), who determined tPA activity by zymography on tissue sections. Even though tPA mRNA is observed in the CA1– CA3 regions of the hippocampus and in the dentate gyrus (Sappino et al., 1993) (Fig. 2 c ), tPA enzyme activity is confined to CA2–CA3 and the dentate and is not detected in the CA1 region. This distribution of tPA activity was corroborated by subsequent, independent experiments (Tsirka et al., 1995; Gualandris et al., 1996). Sappino et al. (1993) also showed that extracts of the CA1 region contained tPA activity; because preparation of the extracts would dissociate enzyme-inhibitor complexes, they concluded that inhibitors in the CA1 were masking the tPA activity in that region. These elegant studies suggest that tPA activity in the hippocampus is under regulation by inhibitors. Antibodies against tPA revealed the presence of tPA protein over the CA2–CA3 pyramidal subfields and dentate gyrus in neuronal as well as microglial cells (Fig. 3 c,e ). Injection of kainate resulted in stronger tPA microglial expression at the ipsilateral side (Fig. 3 e ), thus confirm- ing that tPA activity is upregulated in activated microglia (Tsirka et al., 1995). We have investigated the presence of plasminogen protein by immunohistochemistry. Plasminogen was detected in neurons in the pyramidal cell layer (Fig. 3). Although plasminogen mRNA is present in most pyramidal neurons, only a small subset of neurons stained for plasminogen protein (Fig. 3 d ). Soon after injection of kainate, the levels of plasminogen protein expression appear to be increased (Fig. 3 f ), suggesting that post-transcriptional regulation controls its expression. Such regulation may represent a mechanism for enhanced expression associated with neuronal activity, in agreement with proposed functions of tPA /plasmin in plasticity and restructuring in the brain (Krystosek and Seeds, 1981; Carroll et al., 1994; Frey et al., 1996). The injection of kainate into the hippocampus results in local activation of microglial cells (Andersson et al., 1991), made man- ifest by changes in morphology and increased expression of the surface antigen F4/80 (Lawson et al., 1990) (Fig. 4 a– d ). Mice deficient for tPA displayed activation of microglial cells after kainate administration, but the response was attenuated compared to that of wild-type animals, both in F4/80 staining intensity and in morphological changes (Tsirka et al., 1995). These results suggested that tPA participates in the activation pathway of microglial cells, which in turn could be affecting the degeneration process. To examine the effect of plasminogen on microglial activation, F4/80 immunostaining was performed on brain sections from kainate-injected plg Ϫ / Ϫ mice. Both the staining intensity and morphology of microglial cells from plg Ϫ / Ϫ mice were comparable to those of wild-type cells (Fig. 4 c–f ). These results show that resistance to neuronal degeneration can occur in spite of normal microglial activation, and that a critical proteolytic event is down- stream of activation. They also suggest that the role of tPA in microglial activation is independent of its activation of plasminogen. The above results identify a genetic and cellular pathway, involving tPA and plasminogen, that can result in neuronal cell death after excitotoxic injury. To evaluate directly whether plasmin, the product generated by the action of tPA on plasminogen, is mediating neuronal death, an inhibitor of the activity of plasmin ( ␣ 2 -antiplasmin) was delivered to the brain. Because tPA is required acutely after excitotoxic insult to mediate neuronal degeneration (Tsirka et al., 1996), inhibition of tPA /plasmin proteolytic activity might retard neuronal cell death. Wild-type mice were infused with ␣ 2 -antiplasmin, injected 2 d later with kainate, and then evaluated for their neuronal status in the hippocampus. Control mice infused with buffer were sensitive to neuronal degeneration in the hippocampus (Fig. 5, top ). In contrast, ␣ 2 antiplasmin retarded neuronal degeneration induced by kainate (Fig. 5, bottom ). The resistance of the protease inhibitor-infused mice was comparable to that observed with untreated tPA Ϫ / Ϫ mice (Table 1). Therefore, plasmin is a product of the proteolytic cascade that promotes hippocampal excitotoxic neuronal death, and inhibition of plasmin activity in the adult animal can protect against degeneration. The results presented here demonstrate that extracellular proteolysis plays a central role in excitotoxin-mediated hippocampal neuronal degeneration. A critical observation in defining this pathway is the protection against degeneration in mice that lack plasminogen. The comparable resistant phenotype conferred by both tPA and plasminogen deficiency is consistent with the function of these enzymes in other contexts: namely, they operate sequentially within a cascade whose final proteolytic product is plasmin. This view is strengthened by the results of local delivery of ␣ 2 -antiplasmin, because this inhibitor confers resistance to wild-type mice. Previous work has implicated proteases in neuronal destruction. (1) The degeneration of ganglion neuronal cells after transection of the optic nerve can be retarded by the injection of protease inhibitors into the vitreous body (Thanos, 1991; Thanos et al., 1993). (2) Protease nexin-1, an inhibitor of plasminogen activators and plasmin, can protect cultured hippocampal neurons against hypoglycemic damage, suggesting that proteases can modulate vulnerability to neurotoxicity (Smith-Swintosky et al., 1995). (3) In the cerebellum of the mouse mutant weaver , increased neuronal cell death is evident, coinciding with 10-fold higher than normal tPA activity. This cell death is also observed in cultured cerebellar weaver neurons and can be prevented by inclusion of aprotinin, a serine protease inhibitor, in the culture medium (Murtom ̈ki et al., 1995). The weaver phenotype results from a mutation in the potassium channel gene Girk2 (Patil et al., 1995), which alters the specificity of the channel (Slesinger et al., 1996) and may result in depolarization of granule neurons (Goldowitz and Smeyne, 1995). Such a depolarization could lead to increased tPA levels, because we have found that chemically induced depolarization of PC12 ...
Context 3
... the understanding of the pathway of degeneration. (2) Previous work has indicated that cells that synthesize both a plasminogen activator and plasminogen suffer deleterious consequences, possibly attributable to the generation of intracellular proteolysis (Sandgren et al., 1991). It was therefore of interest to determine whether the production of tPA and plasminogen was segregated by cell type. To determine the sites of synthesis of tPA and plasminogen, antisense mouse tPA and plasminogen digoxygenin-labeled RNA probes were hybridized to control or kainate-injected brain sections (Fig. 2). tPA mRNA was detected along the neuronal cell layers in the hippocampus (Fig. 2 c ), as described previously (Qian et al., 1993; Sappino et al., 1993). At higher magnification, the pattern of tPA mRNA along the neuronal cell layer had a complex appearance (Fig. 2 e ), suggesting that synthesis might be occurring in more than one cell type. In addition, after destruction of neurons by kainate injection, the staining in the larger cells (pyramidal cells) disappeared but remained in the darker and smaller cells (Fig. 2 g ). Several lines of evidence indicate that these smaller cells are microglia. (1) Transgenic mice that express the bacterial lacZ gene under the control of mouse tPA 5 Ј regulatory sequences (tPA / lacZ ; Carroll et al., 1994) express  -gal along the neuronal pyramidal cell layer in cells ϳ 10 m in diameter, smaller than pyramidal neurons, but of average size for satellite microglial cells. (2) In the tPA / lacZ transgenic mice, adjacent coronal brain sections were doubly stained for  -gal and with antibodies to detect markers specific for the three non-neuronal cell types in the hippocampus (data not shown).  -gal-expressing cells colocalized with perineuronal microglia (F4/80), but did not colocalize with oligodendrocytes (anti-myelin basic protein antibody) or astro- cytes (anti-glial fibrillary acidic protein). These results demonstrate that tPA mRNA is produced both in neurons (the fraction of the staining that disappears after injection of kainate) and in the satellite microglia that overlie the neuronal cell layer (the fraction that persists after kainate injection). The distribution of tPA mRNA in both cell types is in agreement with the studies on the localization of tPA in the rat cerebellum and brain stem (Ware et al., 1995). Plasminogen mRNA was also detected in the hippocampus along the neuronal cell layers, indicating local transcription of this gene (Fig. 2 d ). Plasminogen mRNA, however, was detected only in the larger pyramidal neurons (Fig. 2 f ), and the mRNA staining was completely abolished by kainate destruction of neurons (Fig. 2 h ). An intriguing finding was that plasminogen mRNA was detected in dendrites emanating from the neuronal cell bodies ( arrows in Fig. 2 f and data not shown). This cellular distribution of plasminogen mRNA suggests transport of the message into the dendrites, which could direct local protein production on neuronal activity (Miyashiro et al., 1994; Link et al., 1995; Steward, 1995). Taken together, these data demonstrate that plasminogen mRNA is synthesized in the brain exclusively by neurons, whereas tPA mRNA is produced in both neurons and perineuronal microglia. Because post-transcriptional control can often regulate protein expression, it was of interest to determine whether tPA and plasminogen proteins were also present in the hippocampus. The presence of tPA protein has been examined previously by Sappino et al. (1993), who determined tPA activity by zymography on tissue sections. Even though tPA mRNA is observed in the CA1– CA3 regions of the hippocampus and in the dentate gyrus (Sappino et al., 1993) (Fig. 2 c ), tPA enzyme activity is confined to CA2–CA3 and the dentate and is not detected in the CA1 region. This distribution of tPA activity was corroborated by subsequent, independent experiments (Tsirka et al., 1995; Gualandris et al., 1996). Sappino et al. (1993) also showed that extracts of the CA1 region contained tPA activity; because preparation of the extracts would dissociate enzyme-inhibitor complexes, they concluded that inhibitors in the CA1 were masking the tPA activity in that region. These elegant studies suggest that tPA activity in the hippocampus is under regulation by inhibitors. Antibodies against tPA revealed the presence of tPA protein over the CA2–CA3 pyramidal subfields and dentate gyrus in neuronal as well as microglial cells (Fig. 3 c,e ). Injection of kainate resulted in stronger tPA microglial expression at the ipsilateral side (Fig. 3 e ), thus confirm- ing that tPA activity is upregulated in activated microglia (Tsirka et al., 1995). We have investigated the presence of plasminogen protein by immunohistochemistry. Plasminogen was detected in neurons in the pyramidal cell layer (Fig. 3). Although plasminogen mRNA is present in most pyramidal neurons, only a small subset of neurons stained for plasminogen protein (Fig. 3 d ). Soon after injection of kainate, the levels of plasminogen protein expression appear to be increased (Fig. 3 f ), suggesting that post-transcriptional regulation controls its expression. Such regulation may represent a mechanism for enhanced expression associated with neuronal activity, in agreement with proposed functions of tPA /plasmin in plasticity and restructuring in the brain (Krystosek and Seeds, 1981; Carroll et al., 1994; Frey et al., 1996). The injection of kainate into the hippocampus results in local activation of microglial cells (Andersson et al., 1991), made man- ifest by changes in morphology and increased expression of the surface antigen F4/80 (Lawson et al., 1990) (Fig. 4 a– d ). Mice deficient for tPA displayed activation of microglial cells after kainate administration, but the response was attenuated compared to that of wild-type animals, both in F4/80 staining intensity and in morphological changes (Tsirka et al., 1995). These results suggested that tPA participates in the activation pathway of microglial cells, which in turn could be affecting the degeneration process. To examine the effect of plasminogen on microglial activation, F4/80 immunostaining was performed on brain sections from kainate-injected plg Ϫ / Ϫ mice. Both the staining intensity and morphology of microglial cells from plg Ϫ / Ϫ mice were comparable to those of wild-type cells (Fig. 4 c–f ). These results show that resistance to neuronal degeneration can occur in spite of normal microglial activation, and that a critical proteolytic event is down- stream of activation. They also suggest that the role of tPA in microglial activation is independent of its activation of plasminogen. The above results identify a genetic and cellular pathway, involving tPA and plasminogen, that can result in neuronal cell death after excitotoxic injury. To evaluate directly whether plasmin, the product generated by the action of tPA on plasminogen, is mediating neuronal death, an inhibitor of the activity of plasmin ( ␣ 2 -antiplasmin) was delivered to the brain. Because tPA is required acutely after excitotoxic insult to mediate neuronal degeneration (Tsirka et al., 1996), inhibition of tPA /plasmin proteolytic activity might retard neuronal cell death. Wild-type mice were infused with ␣ 2 -antiplasmin, injected 2 d later with kainate, and then evaluated for their neuronal status in the hippocampus. Control mice infused with buffer were sensitive to neuronal degeneration in the hippocampus (Fig. 5, top ). In contrast, ␣ 2 antiplasmin retarded neuronal degeneration induced by kainate (Fig. 5, bottom ). The resistance of the protease inhibitor-infused mice was comparable to that observed with untreated tPA Ϫ / Ϫ mice (Table 1). Therefore, plasmin is a product of the proteolytic cascade that promotes hippocampal excitotoxic neuronal death, and inhibition of plasmin activity in the adult animal can protect against degeneration. The results presented here demonstrate that extracellular proteolysis plays a central role in excitotoxin-mediated hippocampal neuronal degeneration. A critical observation in defining this pathway is the protection against degeneration in mice that lack plasminogen. The comparable resistant phenotype conferred by both tPA and plasminogen deficiency is consistent with the function of these enzymes in other contexts: namely, they operate sequentially within a cascade whose final proteolytic product is plasmin. This view is strengthened by the results of local delivery of ␣ 2 -antiplasmin, because this inhibitor confers resistance to wild-type mice. Previous work has implicated proteases in neuronal destruction. (1) The degeneration of ganglion neuronal cells after transection of the optic nerve can be retarded by the injection of protease inhibitors into the vitreous body (Thanos, 1991; Thanos et al., 1993). (2) Protease nexin-1, an inhibitor of plasminogen activators and plasmin, can protect cultured hippocampal neurons against hypoglycemic damage, suggesting that proteases can modulate vulnerability to neurotoxicity (Smith-Swintosky et al., 1995). (3) In the cerebellum of the mouse mutant weaver , increased neuronal cell death is evident, coinciding with 10-fold higher than normal tPA activity. This cell death is also observed in cultured cerebellar weaver neurons and can be prevented by inclusion of aprotinin, a serine protease inhibitor, in the culture medium (Murtom ̈ki et al., 1995). The weaver phenotype results from a mutation in the potassium channel gene Girk2 (Patil et al., 1995), which alters the specificity of the channel (Slesinger et al., 1996) and may result in depolarization of granule neurons (Goldowitz and Smeyne, 1995). Such a depolarization could lead to increased tPA levels, because we have found that chemically induced depolarization of PC12 cells leads to an enhanced rate of tPA secretion (Gualandris et al., 1996). Both neurons and microglia synthesize tPA, but it is not ...
Context 4
... the blood– brain barrier (BBB) without a specialized transport system. Therefore, plasminogen in the hippocampus could be derived from the blood-borne protein via leakage or transport, or from local synthesis. The latter was suggested by Sappino et al. (1993), who detected low levels of plasminogen mRNA in the hippocampus by RNase-protection assay. In light of the resistance of the plg Ϫ / Ϫ mice to excitotoxins, determining whether plasminogen is synthesized in the brain would help advance the understanding of the pathway of degeneration. (2) Previous work has indicated that cells that synthesize both a plasminogen activator and plasminogen suffer deleterious consequences, possibly attributable to the generation of intracellular proteolysis (Sandgren et al., 1991). It was therefore of interest to determine whether the production of tPA and plasminogen was segregated by cell type. To determine the sites of synthesis of tPA and plasminogen, antisense mouse tPA and plasminogen digoxygenin-labeled RNA probes were hybridized to control or kainate-injected brain sections (Fig. 2). tPA mRNA was detected along the neuronal cell layers in the hippocampus (Fig. 2 c ), as described previously (Qian et al., 1993; Sappino et al., 1993). At higher magnification, the pattern of tPA mRNA along the neuronal cell layer had a complex appearance (Fig. 2 e ), suggesting that synthesis might be occurring in more than one cell type. In addition, after destruction of neurons by kainate injection, the staining in the larger cells (pyramidal cells) disappeared but remained in the darker and smaller cells (Fig. 2 g ). Several lines of evidence indicate that these smaller cells are microglia. (1) Transgenic mice that express the bacterial lacZ gene under the control of mouse tPA 5 Ј regulatory sequences (tPA / lacZ ; Carroll et al., 1994) express  -gal along the neuronal pyramidal cell layer in cells ϳ 10 m in diameter, smaller than pyramidal neurons, but of average size for satellite microglial cells. (2) In the tPA / lacZ transgenic mice, adjacent coronal brain sections were doubly stained for  -gal and with antibodies to detect markers specific for the three non-neuronal cell types in the hippocampus (data not shown).  -gal-expressing cells colocalized with perineuronal microglia (F4/80), but did not colocalize with oligodendrocytes (anti-myelin basic protein antibody) or astro- cytes (anti-glial fibrillary acidic protein). These results demonstrate that tPA mRNA is produced both in neurons (the fraction of the staining that disappears after injection of kainate) and in the satellite microglia that overlie the neuronal cell layer (the fraction that persists after kainate injection). The distribution of tPA mRNA in both cell types is in agreement with the studies on the localization of tPA in the rat cerebellum and brain stem (Ware et al., 1995). Plasminogen mRNA was also detected in the hippocampus along the neuronal cell layers, indicating local transcription of this gene (Fig. 2 d ). Plasminogen mRNA, however, was detected only in the larger pyramidal neurons (Fig. 2 f ), and the mRNA staining was completely abolished by kainate destruction of neurons (Fig. 2 h ). An intriguing finding was that plasminogen mRNA was detected in dendrites emanating from the neuronal cell bodies ( arrows in Fig. 2 f and data not shown). This cellular distribution of plasminogen mRNA suggests transport of the message into the dendrites, which could direct local protein production on neuronal activity (Miyashiro et al., 1994; Link et al., 1995; Steward, 1995). Taken together, these data demonstrate that plasminogen mRNA is synthesized in the brain exclusively by neurons, whereas tPA mRNA is produced in both neurons and perineuronal microglia. Because post-transcriptional control can often regulate protein expression, it was of interest to determine whether tPA and plasminogen proteins were also present in the hippocampus. The presence of tPA protein has been examined previously by Sappino et al. (1993), who determined tPA activity by zymography on tissue sections. Even though tPA mRNA is observed in the CA1– CA3 regions of the hippocampus and in the dentate gyrus (Sappino et al., 1993) (Fig. 2 c ), tPA enzyme activity is confined to CA2–CA3 and the dentate and is not detected in the CA1 region. This distribution of tPA activity was corroborated by subsequent, independent experiments (Tsirka et al., 1995; Gualandris et al., 1996). Sappino et al. (1993) also showed that extracts of the CA1 region contained tPA activity; because preparation of the extracts would dissociate enzyme-inhibitor complexes, they concluded that inhibitors in the CA1 were masking the tPA activity in that region. These elegant studies suggest that tPA activity in the hippocampus is under regulation by inhibitors. Antibodies against tPA revealed the presence of tPA protein over the CA2–CA3 pyramidal subfields and dentate gyrus in neuronal as well as microglial cells (Fig. 3 c,e ). Injection of kainate resulted in stronger tPA microglial expression at the ipsilateral side (Fig. 3 e ), thus confirm- ing that tPA activity is upregulated in activated microglia (Tsirka et al., 1995). We have investigated the presence of plasminogen protein by immunohistochemistry. Plasminogen was detected in neurons in the pyramidal cell layer (Fig. 3). Although plasminogen mRNA is present in most pyramidal neurons, only a small subset of neurons stained for plasminogen protein (Fig. 3 d ). Soon after injection of kainate, the levels of plasminogen protein expression appear to be increased (Fig. 3 f ), suggesting that post-transcriptional regulation controls its expression. Such regulation may represent a mechanism for enhanced expression associated with neuronal activity, in agreement with proposed functions of tPA /plasmin in plasticity and restructuring in the brain (Krystosek and Seeds, 1981; Carroll et al., 1994; Frey et al., 1996). The injection of kainate into the hippocampus results in local activation of microglial cells (Andersson et al., 1991), made man- ifest by changes in morphology and increased expression of the surface antigen F4/80 (Lawson et al., 1990) (Fig. 4 a– d ). Mice deficient for tPA displayed activation of microglial cells after kainate administration, but the response was attenuated compared to that of wild-type animals, both in F4/80 staining intensity and in morphological changes (Tsirka et al., 1995). These results suggested that tPA participates in the activation pathway of microglial cells, which in turn could be affecting the degeneration process. To examine the effect of plasminogen on microglial activation, F4/80 immunostaining was performed on brain sections from kainate-injected plg Ϫ / Ϫ mice. Both the staining intensity and morphology of microglial cells from plg Ϫ / Ϫ mice were comparable to those of wild-type cells (Fig. 4 c–f ). These results show that resistance to neuronal degeneration can occur in spite of normal microglial activation, and that a critical proteolytic event is down- stream of activation. They also suggest that the role of tPA in microglial activation is independent of its activation of plasminogen. The above results identify a genetic and cellular pathway, involving tPA and plasminogen, that can result in neuronal cell death after excitotoxic injury. To evaluate directly whether plasmin, the product generated by the action of tPA on plasminogen, is mediating neuronal death, an inhibitor of the activity of plasmin ( ␣ 2 -antiplasmin) was delivered to the brain. Because tPA is required acutely after excitotoxic insult to mediate neuronal degeneration (Tsirka et al., 1996), inhibition of tPA /plasmin proteolytic activity might retard neuronal cell death. Wild-type mice were infused with ␣ 2 -antiplasmin, injected 2 d later with kainate, and then evaluated for their neuronal status in the hippocampus. Control mice infused with buffer were sensitive to neuronal degeneration in the hippocampus (Fig. 5, top ). In contrast, ␣ 2 antiplasmin retarded neuronal degeneration induced by kainate (Fig. 5, bottom ). The resistance of the protease inhibitor-infused mice was comparable to that observed with untreated tPA Ϫ / Ϫ mice (Table 1). Therefore, plasmin is a product of the proteolytic cascade that promotes hippocampal excitotoxic neuronal death, and inhibition of plasmin activity in the adult animal can protect against degeneration. The results presented here demonstrate that extracellular proteolysis plays a central role in excitotoxin-mediated hippocampal neuronal degeneration. A critical observation in defining this pathway is the protection against degeneration in mice that lack plasminogen. The comparable resistant phenotype conferred by both tPA and plasminogen deficiency is consistent with the function of these enzymes in other contexts: namely, they operate sequentially within a cascade whose final proteolytic product is plasmin. This view is strengthened by the results of local delivery of ␣ 2 -antiplasmin, because this inhibitor confers resistance to wild-type mice. Previous work has implicated proteases in neuronal destruction. (1) The degeneration of ganglion neuronal cells after transection of the optic nerve can be retarded by the injection of protease inhibitors into the vitreous body (Thanos, 1991; Thanos et al., 1993). (2) Protease nexin-1, an inhibitor of plasminogen activators and plasmin, can protect cultured hippocampal neurons against hypoglycemic damage, suggesting that proteases can modulate vulnerability to neurotoxicity (Smith-Swintosky et al., 1995). (3) In the cerebellum of the mouse mutant weaver , increased neuronal cell death is evident, coinciding with 10-fold higher than normal tPA activity. This cell death is also observed in cultured cerebellar weaver neurons and can be prevented by inclusion of aprotinin, a serine protease inhibitor, in the culture medium (Murtom ̈ki et al., 1995). The weaver ...
Context 5
... a plasminogen activator and plasminogen suffer deleterious consequences, possibly attributable to the generation of intracellular proteolysis (Sandgren et al., 1991). It was therefore of interest to determine whether the production of tPA and plasminogen was segregated by cell type. To determine the sites of synthesis of tPA and plasminogen, antisense mouse tPA and plasminogen digoxygenin-labeled RNA probes were hybridized to control or kainate-injected brain sections (Fig. 2). tPA mRNA was detected along the neuronal cell layers in the hippocampus (Fig. 2 c ), as described previously (Qian et al., 1993; Sappino et al., 1993). At higher magnification, the pattern of tPA mRNA along the neuronal cell layer had a complex appearance (Fig. 2 e ), suggesting that synthesis might be occurring in more than one cell type. In addition, after destruction of neurons by kainate injection, the staining in the larger cells (pyramidal cells) disappeared but remained in the darker and smaller cells (Fig. 2 g ). Several lines of evidence indicate that these smaller cells are microglia. (1) Transgenic mice that express the bacterial lacZ gene under the control of mouse tPA 5 Ј regulatory sequences (tPA / lacZ ; Carroll et al., 1994) express  -gal along the neuronal pyramidal cell layer in cells ϳ 10 m in diameter, smaller than pyramidal neurons, but of average size for satellite microglial cells. (2) In the tPA / lacZ transgenic mice, adjacent coronal brain sections were doubly stained for  -gal and with antibodies to detect markers specific for the three non-neuronal cell types in the hippocampus (data not shown).  -gal-expressing cells colocalized with perineuronal microglia (F4/80), but did not colocalize with oligodendrocytes (anti-myelin basic protein antibody) or astro- cytes (anti-glial fibrillary acidic protein). These results demonstrate that tPA mRNA is produced both in neurons (the fraction of the staining that disappears after injection of kainate) and in the satellite microglia that overlie the neuronal cell layer (the fraction that persists after kainate injection). The distribution of tPA mRNA in both cell types is in agreement with the studies on the localization of tPA in the rat cerebellum and brain stem (Ware et al., 1995). Plasminogen mRNA was also detected in the hippocampus along the neuronal cell layers, indicating local transcription of this gene (Fig. 2 d ). Plasminogen mRNA, however, was detected only in the larger pyramidal neurons (Fig. 2 f ), and the mRNA staining was completely abolished by kainate destruction of neurons (Fig. 2 h ). An intriguing finding was that plasminogen mRNA was detected in dendrites emanating from the neuronal cell bodies ( arrows in Fig. 2 f and data not shown). This cellular distribution of plasminogen mRNA suggests transport of the message into the dendrites, which could direct local protein production on neuronal activity (Miyashiro et al., 1994; Link et al., 1995; Steward, 1995). Taken together, these data demonstrate that plasminogen mRNA is synthesized in the brain exclusively by neurons, whereas tPA mRNA is produced in both neurons and perineuronal microglia. Because post-transcriptional control can often regulate protein expression, it was of interest to determine whether tPA and plasminogen proteins were also present in the hippocampus. The presence of tPA protein has been examined previously by Sappino et al. (1993), who determined tPA activity by zymography on tissue sections. Even though tPA mRNA is observed in the CA1– CA3 regions of the hippocampus and in the dentate gyrus (Sappino et al., 1993) (Fig. 2 c ), tPA enzyme activity is confined to CA2–CA3 and the dentate and is not detected in the CA1 region. This distribution of tPA activity was corroborated by subsequent, independent experiments (Tsirka et al., 1995; Gualandris et al., 1996). Sappino et al. (1993) also showed that extracts of the CA1 region contained tPA activity; because preparation of the extracts would dissociate enzyme-inhibitor complexes, they concluded that inhibitors in the CA1 were masking the tPA activity in that region. These elegant studies suggest that tPA activity in the hippocampus is under regulation by inhibitors. Antibodies against tPA revealed the presence of tPA protein over the CA2–CA3 pyramidal subfields and dentate gyrus in neuronal as well as microglial cells (Fig. 3 c,e ). Injection of kainate resulted in stronger tPA microglial expression at the ipsilateral side (Fig. 3 e ), thus confirm- ing that tPA activity is upregulated in activated microglia (Tsirka et al., 1995). We have investigated the presence of plasminogen protein by immunohistochemistry. Plasminogen was detected in neurons in the pyramidal cell layer (Fig. 3). Although plasminogen mRNA is present in most pyramidal neurons, only a small subset of neurons stained for plasminogen protein (Fig. 3 d ). Soon after injection of kainate, the levels of plasminogen protein expression appear to be increased (Fig. 3 f ), suggesting that post-transcriptional regulation controls its expression. Such regulation may represent a mechanism for enhanced expression associated with neuronal activity, in agreement with proposed functions of tPA /plasmin in plasticity and restructuring in the brain (Krystosek and Seeds, 1981; Carroll et al., 1994; Frey et al., 1996). The injection of kainate into the hippocampus results in local activation of microglial cells (Andersson et al., 1991), made man- ifest by changes in morphology and increased expression of the surface antigen F4/80 (Lawson et al., 1990) (Fig. 4 a– d ). Mice deficient for tPA displayed activation of microglial cells after kainate administration, but the response was attenuated compared to that of wild-type animals, both in F4/80 staining intensity and in morphological changes (Tsirka et al., 1995). These results suggested that tPA participates in the activation pathway of microglial cells, which in turn could be affecting the degeneration process. To examine the effect of plasminogen on microglial activation, F4/80 immunostaining was performed on brain sections from kainate-injected plg Ϫ / Ϫ mice. Both the staining intensity and morphology of microglial cells from plg Ϫ / Ϫ mice were comparable to those of wild-type cells (Fig. 4 c–f ). These results show that resistance to neuronal degeneration can occur in spite of normal microglial activation, and that a critical proteolytic event is down- stream of activation. They also suggest that the role of tPA in microglial activation is independent of its activation of plasminogen. The above results identify a genetic and cellular pathway, involving tPA and plasminogen, that can result in neuronal cell death after excitotoxic injury. To evaluate directly whether plasmin, the product generated by the action of tPA on plasminogen, is mediating neuronal death, an inhibitor of the activity of plasmin ( ␣ 2 -antiplasmin) was delivered to the brain. Because tPA is required acutely after excitotoxic insult to mediate neuronal degeneration (Tsirka et al., 1996), inhibition of tPA /plasmin proteolytic activity might retard neuronal cell death. Wild-type mice were infused with ␣ 2 -antiplasmin, injected 2 d later with kainate, and then evaluated for their neuronal status in the hippocampus. Control mice infused with buffer were sensitive to neuronal degeneration in the hippocampus (Fig. 5, top ). In contrast, ␣ 2 antiplasmin retarded neuronal degeneration induced by kainate (Fig. 5, bottom ). The resistance of the protease inhibitor-infused mice was comparable to that observed with untreated tPA Ϫ / Ϫ mice (Table 1). Therefore, plasmin is a product of the proteolytic cascade that promotes hippocampal excitotoxic neuronal death, and inhibition of plasmin activity in the adult animal can protect against degeneration. The results presented here demonstrate that extracellular proteolysis plays a central role in excitotoxin-mediated hippocampal neuronal degeneration. A critical observation in defining this pathway is the protection against degeneration in mice that lack plasminogen. The comparable resistant phenotype conferred by both tPA and plasminogen deficiency is consistent with the function of these enzymes in other contexts: namely, they operate sequentially within a cascade whose final proteolytic product is plasmin. This view is strengthened by the results of local delivery of ␣ 2 -antiplasmin, because this inhibitor confers resistance to wild-type mice. Previous work has implicated proteases in neuronal destruction. (1) The degeneration of ganglion neuronal cells after transection of the optic nerve can be retarded by the injection of protease inhibitors into the vitreous body (Thanos, 1991; Thanos et al., 1993). (2) Protease nexin-1, an inhibitor of plasminogen activators and plasmin, can protect cultured hippocampal neurons against hypoglycemic damage, suggesting that proteases can modulate vulnerability to neurotoxicity (Smith-Swintosky et al., 1995). (3) In the cerebellum of the mouse mutant weaver , increased neuronal cell death is evident, coinciding with 10-fold higher than normal tPA activity. This cell death is also observed in cultured cerebellar weaver neurons and can be prevented by inclusion of aprotinin, a serine protease inhibitor, in the culture medium (Murtom ̈ki et al., 1995). The weaver phenotype results from a mutation in the potassium channel gene Girk2 (Patil et al., 1995), which alters the specificity of the channel (Slesinger et al., 1996) and may result in depolarization of granule neurons (Goldowitz and Smeyne, 1995). Such a depolarization could lead to increased tPA levels, because we have found that chemically induced depolarization of PC12 cells leads to an enhanced rate of tPA secretion (Gualandris et al., 1996). Both neurons and microglia synthesize tPA, but it is not known whether the function of the enzyme from these two sources is the same or distinct. There are previous reports ...
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Mice lacking the serine protease tissue plasminogen activator (tPA) are resistant to excitotoxin-mediated hippocampal neuronal degeneration. We have used genetic and cellular analyses to study the role of tPA in neuronal cell death. Mice deficient for the zymogen plasminogen, a known substrate for tPA, are also resistant to excitotoxins, implicatin...
Citations
... In addition, t-PA treatment carries life-threatening risks, mainly for development of symptomatic intracerebral hemorrhage (sICH), which has been associated with t-PA-mediated disruption of the blood brain-barrier (BBB) (4). Together with additional evidence linking the thrombolytic to neurotoxicity (5,6), t-PA complications are major reasons for concern when the decision to treat is made. These issues are particularly relevant for patients who fail to reanalyze in response to the treatment, hence failing to gain any therapeutic benefit of the drug. ...
Introduction: Acute ischemic stroke (AIS) is a potent trigger of immunosuppression, resulting in increased infection risk. While thrombolytic therapy with tissue-type plasminogen activator (t-PA) is still the only pharmacological treatment for AIS, plasmin, the effector protease, has been reported to suppress dendritic cells (DCs), known for their potent antigen-presenting capacity. Accordingly, in the major group of thrombolyzed AIS patients who fail to reanalyze (>60%), t-PA might trigger unintended and potentially harmful immunosuppressive consequences instead of beneficial reperfusion. To test this hypothesis, we performed an exploratory study to investigate the immunomodulatory properties of t-PA treatment in a mouse model of ischemic stroke.
Methods: C57Bl/6J wild-type mice and plasminogen-deficient (plg−/−) mice were subjected to middle cerebral artery occlusion (MCAo) for 60 min followed by mouse t-PA treatment (0.9 mg/kg) at reperfusion. Behavioral testing was performed 23 h after occlusion, pursued by determination of blood counts and plasma cytokines at 24 h. Spleens and cervical lymph nodes (cLN) were also harvested and characterized by flow cytometry.
Results: MCAo resulted in profound attenuation of immune activation, as anticipated. t-PA treatment not only worsened neurological deficit, but further reduced lymphocyte and monocyte counts in blood, enhanced plasma levels of both IL-10 and TNFα and decreased various conventional DC subsets in the spleen and cLN, consistent with enhanced immunosuppression and systemic inflammation after stroke. Many of these effects were abolished in plg−/− mice, suggesting plasmin as a key mediator of t-PA-induced immunosuppression.
Conclusion: t-PA, via plasmin generation, may weaken the immune response post-stroke, potentially enhancing infection risk and impairing neurological recovery. Due to the large number of comparisons performed in this study, additional pre-clinical work is required to confirm these significant possibilities. Future studies will also need to ascertain the functional implications of t-PA-mediated immunosuppression for thrombolyzed AIS patients, particularly for those with failed recanalization.
... Direct injections of P(g) into the brain parenchyma enhance neuronal apoptosis, brain tissue damage and inflammatory cell infiltration [3]. Within the brain parenchyma, P(g) has been shown to contribute to excitotoxic neuronal cell death, in a non-fibrin-dependent manner [4], in part by acting as a chemokine activator of MCP-1 [5]. Plasmin has been linked to microglial activation [6] and laminin degradation [7] that alter the survival of neurons and astrocytes. ...
Background and objectives:
Plasminogen appears to affect brain inflammation, cell movement, fibrinolysis, neuronal excitotoxicity and cell death. However, brain tissue and circulating blood plasminogen may have different roles and, there is large individual variation in blood plasminogen levels. The aim of this study was to determine the integrated effect of blood plasminogen levels on ischemic brain injury.
Methods:
We examined thromboembolic stroke in mice with varying, experimentally-determined, blood plasminogen levels. Ischemic brain injury, blood-brain barrier breakdown, matrix metalloproteinase-9 expression and microvascular thrombosis were determined.
Results:
Within the range of normal variation, plasminogen levels were strongly associated with ischemic brain injury (p<0.0001); higher blood plasminogen levels had dose-related, protective effects (p<0.0001). Higher plasminogen levels were associated with increased dissolution of the middle cerebral artery thrombus (p<0.0001). Higher plasminogen levels decreased blood-brain barrier breakdown (p<0.05), matrix metalloproteinase-9 expression (p<0.01) and reduced microvascular thrombosis (p<0.0001) in the ischemic brain. In plasminogen-deficient mice, selective restoration of blood plasminogen levels reversed the harmful effects of plasminogen deficiency on ischemic brain injury. Specific inhibition of thrombin also reversed the effect of plasminogen deficiency on ischemic injury by diminishing microvascular thrombosis, blood-brain barrier breakdown and matrix metalloproteinase-9 expression.
Conclusions:
Variation in blood plasminogen levels, within the range seen in normal individuals, had marked effects on experimental ischemic brain injury. Higher plasminogen levels protected against ischemic brain injury, decreased blood-brain barrier breakdown, matrix metalloproteinase-9 expression and microvascular thrombosis. The protective effects of blood plasminogen appear to be mediated largely through reduction of microvascular thrombosis in the ischemic territory. This article is protected by copyright. All rights reserved.
... Plasminogen is essentially present in both blood and the brain under most pathologic brain scenarios, especially together with rt-PA during its utilization in ischemic stroke. Plasminogen is exclusively localized in neurons of the cerebral cortex, hippocampus, hypothalamus, and the cerebellum in rodents (Tsirka et al., 1997;Basham and Seeds, 2001;Taniguchi et al., 2011). Hence, it is not likely to be obviously assumed that brain-derived plasminogen is activated at the BBB during stroke by endogenous t-PA. ...
Cerebrovascular homeostasis is maintained by the blood-brain barrier (BBB), which forms a mechanical and functional barrier between systemic circulation and the central nervous system (CNS). In patients with ischemic stroke, the recombinant tissue-type plasminogen activator (rt-PA) is used to accelerate recanalization of the occluded vessels. However, rt-PA is associated with a risk of increasing intracranial bleeding (ICB). This effect is thought to be caused by the increase in cerebrovascular permeability though various factors such as ischemic reperfusion injury and the activation of matrix metalloproteinases (MMPs), but the detailed mechanisms are unknown. It was recently found that rt-PA treatment enhances BBB permeability not by disrupting the BBB, but by activating the vascular endothelial growth factor (VEGF) system. The VEGF regulates both the dissociation of endothelial cell (EC) junctions and endothelial endocytosis, and causes a subsequent increase in vessel permeability through the VEGF receptor-2 (VEGFR-2) activation in ECs. Here, we review the possibility that rt-PA increases the penetration of toxic molecules derived from the bloodstream including rt-PA itself, without disrupting the BBB, and contributes to these detrimental processes in the cerebral parenchyma.
... The role of tPA within the central nervous system (CNS) is controversial ( Su et al., 2009;Yepes et al., 2009;Lemarchant et al., 2012;Schielke and Lawrence, 2012). It has been proposed that tPA directly affects multiple processes, including neuronal development/plasticity/excitotoxicity ( Tsirka et al., 1996;Seeds et al., 2003;Li et al., 2013), microglial activation ( Tsirka et al., 1997;Rogove and Tsirka, 1998), as well as regulation of cerebrovascular permeability ( Yepes et al., 2003;Su et al., 2008). In a recent paper, we proposed that the neurovascular events regulated by tPA might provide a unifying pathway for many of these pleotropic effects of tPA in the CNS ( Fredriksson et al., 2015). ...
The serine protease tissue-type plasminogen activator (tPA) is used as a thrombolytic agent in the management of ischemic stroke, but concerns for hemorrhagic conversion greatly limits the number of patients that receive this treatment. It has been suggested that the bleeding complications associated with thrombolytic tPA may be due to unanticipated roles of tPA in the brain. Recent work has suggested tPA regulation of neurovascular barrier integrity, mediated via platelet derived growth factor (PDGF)-C/ PDGF receptor-α (PDGFRα) signaling, as a possible molecular mechanism affecting the outcome of stroke. To better understand the role of tPA in neurovascular regulation we conducted a detailed analysis of the cerebrovasculature in brains from adult tPA deficient (tPA-/-) mice. Our analysis demonstrates that life-long deficiency of tPA is associated with rearrangements in the cerebrovascular tree, including a reduction in the number of vascular smooth-muscle cell covered, large diameter, vessels and a decrease in vessel-associated PDGFRα expression as compared to wild-type littermate controls. In addition, we found that ablation of tPA results in an increased number of ERG (ETS related gene) positive endothelial cells and increased junctional localization of the tight junction protein ZO1. This is intriguing since ERG is an endothelial transcription factor implicated in regulation of vascular integrity. Based on these results we propose that the protection of barrier properties seen utilizing these tPA-/- mice might be due, at least in part, to these cerebrovascular rearrangements. In addition, we found that tPA-/- mice displayed mild cerebral ventricular malformations, a feature previously associated with ablation of PDGF-C, thereby providing an in vivo link between tPA and PDGF signaling in CNS development. Taken together, the data presented here will advance our understanding of the role of tPA within the CNS and in regulation of cerebrovascular permeability.
... In a mouse model of MCAO, Kaplan et al. [28] showed that MMP-9 expression and activity were elevated and BBB permeability increased as early as 2-4 h after ischemia. tPA is widely expressed in the developing and mature brain [29] and by neurons and microglia [30]; it is thought to mediate neuronal death and microglial activation after excitotoxic injury [31]. In addition to its impact on the intravascular compartment, exogenous tPA can cross the intact or injured BBB into the brain parenchyma, where it produces neurotoxic effects [32]. ...
Intracerebral hemorrhagic transformation (HT) is well recognized as a common cause of hemorrhage in patients with ischemic stroke. HT after acute ischemic stroke contributes to early mortality and adversely affects functional recovery. The risk of HT is especially high when patients receive thrombolytic reperfusion therapy with tissue plasminogen activator, the only available treatment for ischemic stroke. Although many important publications address preclinical models of ischemic stroke, there are no current recommendations regarding the conduct of research aimed at understanding the mechanisms and prediction of HT. In this review, we discuss the underlying mechanisms for HT after ischemic stroke, provide an overview of the models commonly used for the study of HT, and discuss biomarkers that might be used for the early detection of this challenging clinical problem.
... Although the ECASS III trial showed that the therapeutic time frame of intravenous thrombolysis with tPA for acute ischemic stroke was extended from 3 hr to 4.5 hr after the onset of symptoms, the trial simultaneously showed that tPA was more frequently associated with symptomatic intracranial hemorrhage (Hacke et al., 2008). Endogenous tPA is a major parenchymal serine protease in the brain that regulates physiological tissue remodeling and plasticity (Tsirka et al., 1997; Gravanis and Tsirka, 2005). However, a high dose of exogenous tPA could also cause hemorrhagic transformation by disturbing the neurovascular unit (NVU; Yamashita et al., 2009) and by direct neurotoxicity (Lukic-Panin et al., 2010), presenting a threat to the safe use for thrombolytic therapy. ...
... In the present study, administration of tPA decreased both pericyte coverage and PDGFRb protein expression at 4 days after tMCAO (Fig. 3). A small amount of endogenous tPA is normally produced in neurons , astrocytes, microglia, and endothelial cells of the rodent brain (Tsirka et al., 1997; Schreiber et al., 1998; Kim et al., 2006; Xin et al., 2010). This tPA is secreted Fig. 5 ...
Pericytes play a pivotal role in contraction, mediating inflammation and regulation of blood flow in the brain. In this study, changes of pericytes in the neurovascular unit (NVU) were examined in relation to the effects of exogenous tissue plasminogen activator (tPA) and a free radical scavenger, edaravone. Immunohistochemistry and Western blot analyses showed that the overlap between platelet-derived growth factor receptor β-positive pericytes and N-acetylglucosamine oligomers (NAGO)-positive endothelial cells increased significantly at 4 days after 90 min of transient middle cerebral artery occlusion (tMCAO). The number of pericytes and the overlap with NAGO decreased with tPA but recovered with edaravone 4 days after tMCAO with proliferation. Thus, tPA treatment damaged pericytes, resulting in the detachment from astrocytes and a decrease in glial cell line-derived neurotrophic factor secretion. However, treatment with edaravone greatly improved tPA-induced damage to pericytes. The present study demonstrates that exogenous tPA strongly damages pericytes and destroys the integrity of the NVU, but edaravone treatment can greatly ameliorate such damage after acute cerebral ischemia in rats. © 2014 The Authors. Journal of Neuroscience Research Published by Wiley Periodicals, Inc.
... 15, 16 Yet, within the neuronal and vascular compartments, a2AP and serpins that block TPA-initiated proteolytic pathways, such as the activation of matrix metalloproteinase-9 (MMP-9), may protect the brain by reducing cell death or neurotoxicity and may prevent bleeding complications. [17][18][19][20] In this report we investigated how circulating and thrombus-bound a2AP affect endogenous fibrinolysis, microvascular thrombosis, hemorrhage, brain injury and other outcomes in an experimental thromboembolic model with translational relevance to human ischemic stroke. We find that thrombus-bound a2AP modulates dissolution of the culprit thromboembolus, while circulating a2AP activity also has dynamic, deleterious effects on the development of microvascular thrombosis, MMP-9 expression, brain injury, hemorrhage, disability and death following cerebral thromboembolism. ...
Objective:
Ischemic stroke is primarily attributable to thrombotic vascular occlusion. Elevated α2-antiplasmin (a2AP) levels correlate with increased stroke risk, but whether a2AP contributes to the pathogenesis of stroke is unknown. We examined how a2AP affects thrombosis, ischemic brain injury, and survival after experimental cerebral thromboembolism.
Approach and results:
We evaluated the effects of a2AP on stroke outcomes in mice with increased, normal, or no circulating a2AP, as well as in mice given an a2AP-inactivating antibody. Higher a2AP levels were correlated with greater ischemic brain injury (rs=0.88, P<0.001), brain swelling (rs=0.82, P<0.001), and reduced middle cerebral artery thrombus dissolution (rs=-0.93, P<0.001). In contrast, a2AP deficiency enhanced thrombus dissolution, increased cerebral blood flow, reduced brain infarction, and decreased brain swelling. By comparison to tissue plasminogen activator (TPA), a2AP inactivation hours after thromboembolism still reduced brain infarction (P<0.001) and hemorrhage (P<0.05). Microvascular thrombosis, a process that enhances brain ischemia, was markedly reduced in a2AP-deficient or a2AP-inactivated mice compared with TPA-treated mice or mice with increased a2AP levels (all P<0.001). Matrix metalloproteinase-9 expression, which contributes to acute brain injury, was profoundly decreased in a2AP-deficient or a2AP-inactivated mice versus TPA-treated mice or mice with increased a2AP levels (all P<0.001). a2AP inactivation markedly reduced stroke mortality versus TPA (P<0.0001).
Conclusions:
a2AP has profound, dose-related effects on ischemic brain injury, swelling, hemorrhage, and survival after cerebral thromboembolism. By comparison to TPA, the protective effects of a2AP deficiency or inactivation seem to be mediated through reductions in microvascular thrombosis and matrix metalloproteinase-9 expression.
... Moreover, administration of a2AP has been shown to decrease experimental bleeding after TPA therapy (Weitz, et al., 1993). Other studies indicate that a2AP may be neuroprotective during TPA therapy because in the brain tissue TPA enhances excitotoxicity and a2AP injection reduces excitotoxicity (Tsirka, et al., 1997). However, these findings are at odds with clinical studies linking high a2AP levels to increased stroke risk and the failure of TPA to reperfuse thrombotically-occluded vessels during stroke therapy (Marti-Fabregas, et al., 2005, Suri, et al., 2010 Therefore, in these studies we sought to determine whether a2AP modulates the therapeutic effects of TPA given after different intervals of ischemia in a thromboembolic model of stroke. ...
High blood levels of α2-antiplasmin have been associated with failed tissue plasminogen activator (TPA) therapy for ischemic stroke. Yet, other data suggests that α2-antiplasmin may be protective in stroke, because it defends against bleeding and excitotoxicity. To address this paradox, we examined the effects of high α2-antiplasmin levels and α2-antiplasmin inactivation in mice treated with TPA 0.5-2.5 hour after middle cerebral artery (MCA) thromboembolism. Brain infarction, swelling, hemorrhage, blood brain barrier breakdown and neuronal apoptosis were measured by a blinded observer. Thrombus dissolution was determined by gamma counting. During TPA treatment, high α2-antiplasmin blood levels increased brain infarction (2.2-fold) and swelling (3.7-fold), but decreased MCA thrombus dissolution. Conversely, α2-antiplasmin inactivation during TPA treatment reduced brain infarction, hemorrhage and swelling, but increased MCA thrombus dissolution. Inactivation of α2-antiplasmin during TPA treatment reduced neuronal apoptosis and blood brain barrier breakdown. Inactivation of α2-antiplasmin also reduced short-term mortality. Taken together these data show that α2-antiplasmin opposes the effects of TPA therapy and contributes to enhanced brain injury after experimental thromboembolic stroke. Conversely, α2-antiplasmin inactivation during TPA treatment improves thrombus dissolution and reduces brain infarction, swelling and hemorrhage. Consistent with clinical observations, these data suggest that α2-antiplasmin exerts deleterious effects that reduce the efficacy and safety of TPA therapy for ischemic stroke.
... In the brain, PAI-1 is produced predominantly by astrocytes [21], whereas its main function is to inhibit t-PA [22]. After injury, an excessive amount of t-PA is released into the extracellular space of the brain, which can trigger both neuronal degeneration [23] and disruption of the BBB [24]. Thus, PAI-1 is needed to reduce the deleterious effects of excessive t-PA activity. ...
Background:
Previous studies have shown impaired fibrinolysis in multiple sclerosis (MS) and implicated extracellular proteolytic enzymes as important factors in demyelinating neuroinflammatory disorders. Tissue-type plasminogen activator (t-PA) and its inhibitor (PAI-1) are key molecules in both fibrinolysis and extracellular proteolysis. In the present study, an association of the TPA Alu I/D and PAI-1 4G/5G polymorphisms with MS was analyzed within the Genomic Network for Multiple Sclerosis (GENoMS).
Methods:
The GENoMS includes four populations (Croatian, Slovenian, Serbian, and Bosnian and Herzegovinian) sharing the same geographic location and a similar ethnic background. A total of 885 patients and 656 ethnically matched healthy blood donors with no history of MS in their families were genotyped using PCR-RFLP.
Results:
TPA DD homozygosity was protective (OR = 0.79, 95% CI 0.63-0.99, P = 0.037) and PAI 5G5G was a risk factor for MS (OR = 1.30, 95% CI 1.01-1.66, P = 0.038). A significant effect of the genotype/carrier combination was detected in 5G5G/I carriers (OR = 1.39 95% CI 1.06-1.82, P = 0.017).
Conclusions:
We found a significantly harmful effect of the combination of the PAI-1 5G/5G genotype and TPA I allele on MS susceptibility, which indicates the importance of gene-gene interactions in complex diseases such as MS.
... In primary cultured embryonic cortical neurons, we found that Plg deficiency significantly reduced neuritogenesis, and neurite sprouting and outgrowth. tPA is primarily produced by neurons and microglia, whereas plasminogen is exclusively expressed by neurons [42]. Previous studies suggested that non-proteolytic effects of tPA derived from microglia may indirectly affect hippocampal mossy fiber pathfinding and outgrowth [43], while the neuron derived tPA/plasmin proteolytic system facilitates continued neurite extension via degradation of the extracellular matrix proteoglycans and cell surface components [44]. ...
Tissue plasminogen activator (tPA) has been implicated in neurite outgrowth and neurological recovery post stroke. tPA converts the zymogen plasminogen (Plg) into plasmin. In this study, using plasminogen knockout (Plg-/-) mice and their Plg-native littermates (Plg+/+), we investigated the role of Plg in axonal remodeling and neurological recovery after stroke. Plg+/+ and Plg-/- mice (n = 10/group) were subjected to permanent intraluminal monofilament middle cerebral artery occlusion (MCAo). A foot-fault test and a single pellet reaching test were performed prior to and on day 3 after stroke, and weekly thereafter to monitor functional deficit and recovery. Biotinylated dextran amine (BDA) was injected into the left motor cortex to anterogradely label the corticospinal tract (CST). Animals were euthanized 4 weeks after stroke. Neurite outgrowth was also measured in primary cultured cortical neurons harvested from Plg+/+ and Plg-/- embryos. In Plg+/+ mice, the motor functional deficiency after stroke progressively recovered with time. In contrast, recovery in Plg-/- mice was significantly impaired compared to Plg+/+ mice (p<0.01). BDA-positive axonal density of the CST originating from the contralesional cortex in the denervated side of the cervical gray matter was significantly reduced in Plg-/- mice compared with Plg+/+ mice (p<0.05). The behavioral outcome was highly correlated with the midline-crossing CST axonal density (R2>0.82, p<0.01). Plg-/- neurons exhibited significantly reduced neurite outgrowth. Our data suggest that plasminogen-dependent proteolysis has a beneficial effect during neurological recovery after stroke, at least in part, by promoting axonal remodeling in the denervated spinal cord.
