Abstract
Possible strategies for treating stroke include: 1) thrombolytic therapy with tissue plasminogen activator (tPA): restoring cerebral blood flow in the acute phase of ischemic stroke but sometimes causing hemorrhagic transformation (HT); 2) stem cell therapy: the repair of disrupted neuronal networks with newly born neurons in the chronic phase of ischemic stroke. Firstly, we estimated the vascular protective effect of a free radical scavenger, edaravone, in the tPA-treated rat model of middle cerebral artery occlusion. Edaravone prevented dramatically decreased the hemorrhagic transformation and improved the neurologic score and survival rate of tPA-treated rats. Secondly, we attempted to restore brain tissue using a novel biomaterial, polydimethysiloxane-tetraethoxysilane (PDMS-TEOS) hybrid with or without vascular endothelial growth factor (VEGF), and we could show that implantation of a PDMS-TEOS scaffold with VEGF might be effective for treating old brain infarction or trauma. In the future, we will combine these strategies to develop more effective therapies for treatment of strokes.
Introduction
Because strokes are a major cause of death and result in a severe reduction in the quality of life, new strategies for patients suffering from stroke are needed. We currently focus on two possible strategies for treating ischemic strokes. 1) Thrombolytic therapy with tissue plasminogen activator (tPA) can ameliorate ischemic brain damage in the acute phase of a stroke. However, in a clinical setting, thrombolytic therapy is strictly limited because delayed reperfusion with tPA can cause hemorrhagic transformation (HT). 2) Stem cell therapy, which allows for the repair of disrupted neuronal networks of newly born neurons in the chronic phase of a stroke (1). In this article, we focus on vascular protection preventing ischemic brain treated with tPA from HT, and on a bioaffinitive scaffold supporting neurovascular supplementation for stem cell therapy.
Vascular Protection Inhibiting HT
After a stroke, the thrombolytic effect of tPA is beneficial, if it is used within a short time. However, delayed reperfusion with tPA sometimes causes catastrophic HT, in which case its bedside use is strictly limited. Both an understanding of the mechanism underlying HT and new therapeutic methods are needed. We used a spontaneously hypertensive rat model of the middle cerebral artery occlusion (MCAO). Rats were treated with vehicle or tPA alone, or with edaravone (a free radical scavenger) plus tPA. Edaravone was injected IV at 3 mg/kg every 1.5 h during 4.5 h of MCAO, followed by tPA treatment (10 mg/kg, IV) at reperfusion. At 24 h after MCAO, the rats that survived were sacrificed. Administration of tPA alone significantly worsened the survival rate compared with those rats treated with vehicle. However, treatment with edaravone plus tPA significantly increased the survival rate, improved motor function, and dramatically decreased HT. Immunostaining revealed that edaravone suppressed tPA-induced lipid peroxidation and matrix metalloproteinase-9 (MMP-9) activation at and around cerebral microvessels. Moreover, we found that the basement membrane, which underlies endothelial cells, disintegrated and became detached from the astrocyte endfeet in the rats treated with tPA alone, whereas treatment with edaravone plus tPA prevent the microvessels from dissociation. These results suggested that edaravone can protect cerebral microvascular integrity, seemingly because it safeguards the basement membrane from excess free radicals and MMP-9, leading to a subsequent decrease in HT, improvement in the survival rate, and neurological outcome (14) (Fig. 1).
Dissociation of the neurovascular unit after ischemia and reperfusion with tPA. Not the inner lumen of cerebral endothelial cells, but the outer, perivascular side of the basement membrane was severely degraded in the rat ischemic brain treated with tPA. It was suggest that the basement membrane/extracellular matrix linking the endothelial cells might be essential for maintaining the integrity of the neurovascular unit, which is disrupted by tPA treatment.
We reported earlier that edaravone reduced edema in the ischemic brain (2), and reduced the infarct size after 1.5 h of MCAO in rat brain reperfused with tPA (15). In a clinical trial, edaravone attenuated the resulting disability in humans 90 days after acute ischemic stroke without serious adverse events (12), and it has been used clinically in Japan as a neuroprotective agent for acute stroke patients since 2001. In a large clinical trial, another free radical scavenger, NXY-059, initially appeared to reduce disability after stroke (the first Stroke-Acute-Ischemic-NXY-Treatment trial, SAINT I) (8), but this effect could not be reproduced (SAINT II) (11). However, one alternate characteristic of NXY-059 was the potential to inhibit symptomatic HT after tPA treatment (8). NXY-059 is water soluble (octanol/water partition coefficients; cLog P = −2.09), and a study using a rat model showed that it cannot easily pass through the blood—brain barrier (BBB) even after MCAO. In contrast, edaravone has a biphasic, water-soluble, and lipidsoluble nature (cLog P = 1.33), and when intravenously administered, can easily pass through the BBB to enter the brain parenchyma and cerebral fluid. As the site most vulnerable to free radical damage after reperfusion with tPA is on the outer side of the vascular endothelium (e.g., basal membrane), an intravenously administered compound should have a biphasic soluble nature to reach the most vulnerable site of the brain. Thus, this unique chemical property of edaravone might be an advantage for its delivery to the basement membrane. Therefore, we propose that a combination therapy with edaravone and tPA could provide important therapeutic benefits for acute stroke patients, not only in reducing the infarct size but also in minimizing the catastrophic HT.
Stem Cell Therapy
To supply new neurons into the infarcted brain, two tactics are proposed. One is the activation of intrinsic neural stem cells. The other is the transplantation of extrinsic stem cells (7,13). Recent studies reported that the scaffold is an important factor for intrinsic/extrinsic stem cells to survive in necrotic brain tissue forming a cavity in the injured brain (5). We have recently investigated brain tissue regeneration after implantation of gelatin and a 3-(glycidoxypropyl) trimethoxysilane scaffold (3,9). Addition of basic fibroblast growth factor and epidermal growth factor increased the number of infiltrated cells, but we felt that we needed a better biomaterial. In this context we recently used a novel biomaterial from polydimethylsiloxane (PDMS) and tetraethoxysilane (TEOS) as a new scaffold. This novel biomaterial has an advantage over GPSM in that it has an Si-OH moiety and should therefore be more hydrophilic, which would be favorable for the cells to adhere and grow (6). Furthermore, we added the most potent angiogenic factor vascular endothelial growth factor (VEGF) (4,10) and observed the difference in the regenerative process in the PDMS-TEOS scaffold. When the PDMS-TEOS scaffold was implanted into the artificial brain defect, it remained at the implanted site and kept the integrity of the brain shape. At 30 days after implantation, the marginal territory of the PDMS-TEOS scaffold became occupied by newly formed tissue. Immunohistochemical analysis revealed that the new tissue was constituted by astrocytes and endothelial cells. Addition of VEGF increased the newly produced tissue volume, and immunohistochemical analysis showed that the numbers of astrocytes and endothelial cells had increased. These findings suggested that implantation of a PDMS-TEOS scaffold with VEGF might be effective for treating old brain infarction or trauma.
In this article, we briefly highlighted recent progress in the development of these distinct new strategies for the treatment of damaged brains following a stroke. To realize more effective therapies for patients suffering from stroke, it is important to combine these strategies in the acute or chronic phase following a stroke.