Cerebral Ischemia Induces Neuronal Vulnerability and Astrocytic Dysfunction in Stroke-Prone Spontaneously Hypertensive Rats Cerebral Ischemia Induces Neuronal Vulnerability and Astrocytic Dysfunction in Stroke-Prone Spontaneously Hypertensive Rats

Stroke-prone spontaneously hypertensive (SHRSP) rats develop severe hypertension, and more than 95% of them die of cerebral stroke. Cerebral ischemia or hypoxia and/or subsequent oxygen reperfusion strongly induces neuronal damage in SHRSP rats. The biochemical features of brain cells such as neuronal cells and astrocytes of SHRSP rats might contribute to the strong tendency of SHRSP rats to suffer strokes. In SHRSP rats, the production of hydroxyl radicals was strongly elevated after reperfusion. Neuronal expression of thioredoxin ( Txn1 ) and Bcl2 genes was significantly reduced in SHRSP rats compared with Wistar Kyoto (WKY) rats. In SHRSP rats, the susceptibility of neuronal cells to death is partly due to an insufficiency of mitochondrial redox regulation and a deficiency of the apoptosis-inhibitory protein Bcl-2. Antioxidant vitamin E may regulate the expression of redox and apoptosis-related proteins in neuronal damage. In astrocytes isolated from SHRSP rats, the cells’ proliferative ability and expression of vascular cell adhesion molecule-1 (VCAM-1) and high-mobility group box 1 (HMGB1) are strongly increased compared with those in the WKY rat strain. Astrocytic lactate production, an energy source for neuronal cells, was reduced in SHRSP rats in comparison with the WKY rat strain. SHRSP astrocytes reduced their production of glial cell line–derived neurotrophic factor (GDNF) and l-serine compared to WKY astrocytes during hypoxia and reoxygenation (H/R). Furthermore, sphingosine-1-phosphate (S1P) reduced the expression of GDNF in primary SHRSP rat astrocytes. On the other hand, production of l-serine and the expression of alanine/serine/cysteine/threonine transporter (ASCT1) were lower in SHRSP than in WKY rat astrocytes after exposure to arginine vasopressin (AVP). In this chapter, we describe the neuronal vulnerabilities and astrocytic dysfunctions of SHRSP rats induced by cerebral ischemia.


Introduction
Stroke involves cerebral infarction and hemorrhaging and is associated with very high mortality. Stroke causes a loss of brain function due to an insufficient blood supply to the brain. The stroke-prone spontaneously hypertensive (SHRSP) rat is an experimental model of human malignant hypertension (>200 mm Hg), and this rat strain has a high incidence of cerebrovascular disease [1,2]. Namely, SHRSP rats develop severe hypertension of more than 200 mm Hg, and more than 95% die of stroke [1,2]. In SHRSP rats, the increase of sodium intake accelerates the rise of blood pressure (BP), and cerebral ischemia induces the appearance of cerebral vasogenic edema [3]. Therefore, SHRSP rats are widely used as a model of human stroke [2].
Studies of SHRSP rats may provide considerable useful information regarding human strokes and should indicate genetic susceptibility of particular types of cerebrovascular diseases [4]. Indeed, this strain shares features with human lacunar stroke [5]. Twenty minutes of cerebral ischemia in SHRSP rats induced a large efflux of glutamate, causing strong delayed neuronal death in region CA1 of the hippocampus, whereas the parental strain of SHRSP rats, Wistar Kyoto (WKY) rats, lacked these characteristics under the same conditions [6]. The hippocampal neurons of SHRSP rats were innately vulnerable to ischemic stimulation, and the Ca 2+ channel blockers prevented neuronal cell death in SHRSP rats [7]. The production of hydroxyl radicals by neurons was strongly elevated after reperfusion of SHRSP rats. In neuronal cells, expression of the thioredoxin gene (Txn1) and the Bcl2 gene was significantly reduced in SHRSP rats compared with WKY rats [8]. We showed that SHRSP rat neurons were more vulnerable than WKY rat neurons during cerebral ischemia-hypoxia [9,10]. In these findings, we noticed that unknown factors other than a hereditary weakness in the neurons themselves played additional roles in accelerating cell death in SHRSP rats during cerebral ischemia [9,10]. On the other hand, lactate production from astrocytes (an energy source for neuronal cells) was reduced in SHRSP rat cells in comparison with the WKY rat strain [11]. Moreover, astrocytes from SHRSP rats reduced lactate production, glial cell line-derived neurotrophic factor (GDNF), and l-serine in comparison with WKY rat astrocytes during hypoxia and reoxygenation (H/R) [12]. In addition, the release of l-serine and the expression of lactate transporter were lower in SHRSP rats than in WKY rat astrocytes after exposure to arginine vasopressin (AVP) [13].
Cerebral ischemia promotes blood-brain barrier (BBB) destruction, increases edema, and increases nervous system cell death. In particular, the reperfusion after cerebral ischemia rapidly generates a large quantity of reactive oxygen species (ROS), but the pathogenic mechanism of SHRSP rats in stroke is not well understood. In SHRSP rats, endothelial injury is induced at multiple sites following BBB leakage. Ultimately, this results in vessel rupture [14,15]. Thus, we asked whether there were significant differences between the functions of WKY and SHRSP rat neurons and astrocytes during cerebral ischemia. Here, we present an overview of the cellular characteristic of SHRSP rat and WKY rat neurons and astrocytes during cerebral ischemia.

Cell death of neuronal cells isolated from SHRSP rats during ischemia
In SHRSP rats, cerebral ischemia of 20 min duration enhances the production of large amounts of glutamate, causing delayed neuronal cell death in the CA1 region of the hippocampus [6]. Briefly, the neurons of SHRSP rats are more susceptible to H/R states than those of WKY rats [7,9,10]. We examined cultured neuronal cells isolated from the brains of SHRSP rats and WKY rats. The cells were cultured for 6-24 h under hypoxic conditions (1% O 2 ) and subsequently for 1.5-5 h in a reoxygenated state to assess cell viability [9]. None of the neuronal cells were stainable by trypan blue, indicating the absence of cell death in both strains. The majority (65-85%) of neuronal cells survived even after 36 h of hypoxic culture. Following hypoxia, after 1.5 h of reoxygenation, only 10-30% of neurons survived. The percentages of neuronal cell deaths in WKY rats and SHRSP rats were 41% (necrosis, 12%; apoptosis, 29%) and 78% (necrosis, 15%; apoptosis, 63%), respectively. Following hypoxia, 3 h of reoxygenation led to 68% cell death in WKY rats, whereas 99% of the neuronal cells from SHRSP rats were dead. Using the TdT-mediated dUTP nick end labeling (TUNEL) method, we found little or no DNA fragmentation in SHRSP rat neuronal cells after culture in 20% oxygen. In contrast, following 36 h of hypoxia and 3 h of reoxygenation, we noted a markedly increased fragmentation of DNA that was generally localized to areas containing many lipid droplets [9]. We classified the levels of apoptosis in H/R status by a morphological analysis of neuronal cell death [9,10]. Briefly, we showed the characteristics of neuronal apoptosis in SHRSP rats in Figure 1. In the initial stage of apoptosis, neuronal cell axons and dendrites were lost, and many lipid droplets appeared in the neuronal cell body (A). In the next stage of apoptosis, cell shrinkage was observed (B) and, next, continued development of apoptotic morphology (C). Finally, the neuronal cell membrane was lost and the nucleus disappeared (D). These processes eventually led to neuronal cell death. In SHRSP rats, a report demonstrated that the angiotensin II type 1 receptor-activated caspase-3 in the rostral ventrolateral medulla and was involved in sympathoexcitation [16]. These features may be associated with stroke pathogenic mechanisms of SHRSP rats.

Oxidative stress-induced neuronal cell death and redox changes in SHRSP rats
In the brain, ischemia leads to the rapid generation of a large amount of ROS and induces neuronal cell injury through self-perpetuating reactions. Cerebral ischemia increases the intracellular level of calcium ions and activates calcium-dependent proteases. These reactions activate xanthine dehydrogenase (XDH) and generate xanthine oxidase (XOD). The superoxide anion radicals generated via this pathway cause neuronal cell death in the brain [17]. Free radicals are generated by reoxygenation after cerebral ischemia, and they enhance the injury of brain neuronal cells [18]. These findings suggest that reducing the ROS (such as hydroxyl radicals produced during H/R) would be beneficial for preventing neuronal injury [19]. This might be achieved by increasing the level of antioxidant substances such as vitamin E.
Ischemic stimulation is considered to be the process that most strongly enhances cell death in cerebral ischemia in the SHRSP rat stroke model. In SHRSP rats, ischemic stimulation, i.e., the reoxygenation that occurs after hypoxia, generates a large amount of ROS that lead directly to neuronal death [20]. The expression of the thioredoxin gene (Txn1) was significantly reduced in neurons isolated from SHRSP rats compared with WKY rats [8]. Txn protein acts against ROS via its SH group. Furthermore, Txn proteins have many functions that are involved in intracellular signal transduction. For that reason, reduced expression of Txn1 is associated with an attenuation of the defense system during oxidative stress in SHRSP rats. This in turn causes H/R-induced neuronal cell death. These findings indicated that redox regulatory functions in SHRSP rat neurons were markedly reduced by oxygen stimulation after hypoxia, and such changes may be involved in neuronal vulnerability. From these results, we suggested that the susceptibility of neurons to apoptosis in SHRSP rats is partly due to an insufficiency of mitochondrial thioredoxin and apoptosis-inhibitory proteins.

Protective effects of antioxidant vitamin E in neuronal cell death of SHRSP rats
Vitamin E, which is present in biological membranes, contains a hydroxyl group that reacts with unpaired electrons and can be reduced to form peroxyl radicals. The main antioxidant effect of vitamin E is to rapidly add alkoxy radicals (RO•) and hydrogen to peroxy radicals (ROO•). This is the mechanism of a chain-breaking antioxidant that blocks reactive oxygen metabolic cascades [21,22].
We demonstrated the preventive effects of vitamin E against neuronal cell death associated with cerebral ischemia-reperfusion, particularly apoptosis, in WKY and SHRSP rat strains [9,19]. Hypoxic stimuli followed by oxygen reperfusion induced strong neuronal damage in both WKY and SHRSP rats [9,10]. The rate of neuronal cell death (mainly apoptosis) occurring during H/R was markedly higher in the neurons of SHRSP rats than in those of WKY rats. Vitamin E is mostly enriched in microsomal cell membranes of mitochondria of the liver and heart. Transport of vitamin E to mitochondria or the microsomes is achieved by vitamin E binding to proteins [23]. Reports demonstrated that the α-tocopherol-binding protein, afamin, transported α-tocopherol across an in vitro model of the BBB. The model consisted of porcine brain capillary endothelial cells and cultured astrocytoma cells [24]. Thus, afamin might function to maintain α-tocopherol homeostasis at the BBB in vivo. Furthermore, scavenger receptor class B type I (SR-BI) facilitates selective uptake of HDL-related α-tocopherol at the BBB [25]. Damage related to oxidative stress alters traffic to neuronal cells and changes the levels of vitamin E in mitochondria. The addition of vitamin E almost completely inhibited neuronal death in SHRSP and WKY rat lines. Vitamin E decreased neuronal cell death in a dose-dependent manner over the range of 10-50 μg/mL. With vitamin E at 50-300 μg/mL, neuronal death was almost completely prevented [9]. In SHRSP rat neurons, we used HPLC to investigate the vitamin E levels in neuronal mitochondria after vitamin E supplementation (20-300 μg/mL) [9]. The accumulation of vitamin E in mitochondria of neuronal cells after 36 h of hypoxia was confirmed. Vitamin E accumulated most effectively into the mitochondria of neuronal cells at 50 μg/mL. When neuronal cell death was induced by reoxygenation for 3 h after 36 h of hypoxia, vitamin E prevented neuronal cell death at 50 μg/mL. Vitamin E prevented stroke and loss of both memory and cognition functions [26]. Vitamin E might have a marked inhibitory effect against neuronal damage after being incorporated into biological membranes, particularly mitochondrial membranes, and capturing the reactive oxygen and free radicals formed.

Regulation of the Bcl-2 family proteins in neuronal cell death
We pointed out that apoptosis is the likely mechanism responsible for ischemic neuronal death in SHRSP rats [9,27]. The expression of apoptosis-inducing molecules such as Bax was  enhanced in injured neuronal cells [8]. On the other hand, in surviving neurons, expression of apoptosis-inhibiting molecules such as Bcl-2 and Bcl-XL was elevated in rat brains following global ischemia [28]. The expression of Bcl2 mRNA after H/R was investigated by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) using cultured neuronal cells isolated from SHRSP and WKY rats [8]. An analysis of Bcl2 mRNA expression in SHRSP and WKY rats showed that the most remarkable difference occurred after 30 min of reoxygenation. The expression of Bcl2 mRNA was significantly decreased in SHRSP compared to WKY rats. The vulnerability of neuronal cells to ischemic stress promotes neuronal cell death during stroke in SHRSP rats (Figure 2) [8].

Gliosis and the proliferation rate of astrocytes in SHRSP rats
After brain injury, the number of reactive astrocytes increases [29]. These reactive astrocytes have prominent characteristics of growing cells [30]. The growth state of astrocytes in vitro reflects the physiological abnormalities occurring in brain damage. In astrocytes cultured from SHRSP rat brains in 10% fetal bovine serum (FBS), the cell growth rate was faster than astrocytes from WKY rats. For example, the doubling time of cultured astrocytes isolated from SHRSP rats was 21 h, whereas that of WKY rat astrocytes was 30 h [31]. These results indicated the elevated growth capacity of SHRSP rat astrocytes. The greater increase in the numbers of reactive astrocytes in SHRSP rats may have pathological consequences. In addition, reports have indicated enhanced astrocytic reactivity to epidermal growth factor (EGF). SHRSP rat astrocytes responded more strongly to EGF than WKY rat astrocytes, with larger increases in cell number. In the ischemic brain, proliferative astrocytes were found around the infarcted tissue [32]. Likewise, these proliferating astrocytes were immunoreactive for the EGF receptor (EGFR). The increased growth activity of SHRSP rat astrocytes suggests that cerebral vascular lesions in cerebral ischemia may be due to dysfunctional responses to EGF [32].

Production of neurotrophic factor by SHRSP astrocytes
Astrocytes modulate several functions such as the uptake of glutamate [33] and the induction of the blood-brain barrier (BBB) [34] and induce production of cytokines [35] and neurotrophic factors [36]. In SHRSP rat strain, astrocytic properties relate to the development of brain disorders [37][38][39]. For example, neuron regeneration is controlled through the production of neurotrophic factors from astrocytes after brain injury [32].
Neuronal vulnerability of SHRSP rats under ischemic conditions was correlated with reduced GDNF production by SHRSP rat astrocytes [13]. The study focused on the production of GDNF under normal conditions and H/R in cultured astrocytes from WKY and SHRSP rat strains. SHRSP rat astrocytes released higher levels of GDNF than did WKY rats under normal oxygen concentrations [13] (Figure 3). On the other hand, after hypoxia and 1.5 or 6 h reoxygenation, the expression of GDNF was significantly lower in astrocytes from the SHRSP rat strain than the WKY rat strain [13]. Furthermore, sphingosine-1-phosphate (S1P) [40] and adenosine [41] reduced the expression of GDNF in primary SHRSP rat astrocytes. S1P is a lysophospholipid released by activated platelets [42,43], and it enhances the apoptosis of neuronal cells in the central nervous system (CNS) [44]. A report demonstrated that GDNF is a potent factor for the survival of neuronal cells [45]. Thus, in SHRSP rats, upregulation of S1P attenuates GDNF production by astrocytes (Figure 4). This reduces neuronal protection mediated by the neurotrophic factor, GDNF. In traumatic injury of the CNS, the attenuated release of GDNF by astrocytes may be involved in neuronal vulnerability in the SHRSP rat strain. Adenosine enhances the levels of several growth factors in ischemic brain tissues, likely as part of a preventive response. In the CNS, adenosine in the extracellular space acts as an intercellular signaling molecule, and at high levels, it induces apoptosis [46]. Briefly, adenosine has both immediate effects such as neurotransmission and neurotrophic effects that enhance changes in cell metabolism and structure and has neuroprotective function [47,48]. The expression of GDNF was regulated differently in cultured astrocytes from SHRSP compared to WKY rats. The amount of GDNF produced was lower in astrocytes of adenosine-treated SHRSP rats compared with WKY rats. These results indicate that GDNF production is regulated dynamically during reoxygenation and ischemic conditions by S1P and adenosine. Under postischemic reoxygenation conditions, the production of GDNF, neurturin (NTN), and its receptor increases in the brain tissue [49]. The pathogenesis of GDNF released by SHRSP rats is unknown. However, the specific metabolic properties of SHRSP rats may be associated with increased expression of GDNF [13].

Altered regulation of l-serine in SHRSP astrocytes
In the CNS, l-serine is generated by astrocytes, and it accelerates neurite outgrowth from ganglion neurons and enhances neuronal survival [50]. l-Serine is synthesized by 3-phosphoglycerate dehydrogenase (3-PGDH) in glial cells but not in neurons [51]. l-Serine released from astrocytes is transported to the extracellular space by neural amino acid transporter alanine/ serine/cysteine/threonine transporter (ASCT1) proteins and becomes available to neuronal cells [52]. Induces outgrowth of ganglion neurons and neuronal survival by L-serine from astrocytes. Hence, L-serine synthesis and the expression of ASCT1 protein were essential for neuronal survival and differentiation [53]. On the other hand, the expression of 3-PGDH and ASCT1 proteins is enhanced by excitotoxic damage in the mouse brain hippocampus [54]. In astrocytes isolated from SHRSP rats, glutamate-induced stimulation of l-serine production was reduced [55]. The production of l-serine was regulated by astrocytes in response to molecules such as glutamate, kainic acid (KA), and free radicals and others that induced neurodegenerative disorders [54,56,57].
Arginine vasopressin (AVP) induced the effects of inflammatory molecules in traumatic neuronal injury [58]. Furthermore, ischemic conditions such as hypoxia and AVP affect cerebral cell volume [59,60], ion uptake by cerebral cells via Na/H exchange (NHE) [61], and Na-K-Cl cotransporter (NKCC) activities [62,63]. AVP and hypoxia contribute to ischemiainduced astrocyte swelling [64]. During cerebral ischemia, astrocyte swelling leads to ischemic neuronal cell death. AVP might contribute to astrocyte swelling induced by hypoxia and reperfusion in SHRSP rats. On the other hand, l-serine generation might be regulated by astrocytes in response to a variety of molecules such as AVP that enhance brain edema in SHRSP rats [12].

Inflammatory regulation and expression of HMGB1 and adhesion molecules in SHRSP astrocytes
High-mobility group box 1 (HMGB1) regulates nucleosomal structure stabilization, modulates inflammation, and is involved in recovery after stroke [65][66][67]. Hypoxia induces HMGB1 expression in neurons and astrocytes [68]. Furthermore, after hypoxia, HMGB1 enhances breakdown of the blood-brain barrier (BBB) during ischemic injury [69]. These results indicate that HMGB1 is involved in inflammatory responses associated with stroke after ischemia [70]. One report demonstrated that HMGB1 is produced by neuronal cells and glial cells and aggravates ischemic neurodegeneration [68]. In reactive astrocytes, production of HMGB1 accelerates endothelial progenitor cell-mediated neurovascular remodeling during stroke recovery [71]. On the other hand, in oxygen-glucose deprivation or reperfusion, HMGB1 produced from astrocytes induces endogenous neural stem or progenitor cell proliferation [67,72]. One study examined the expression of AVP-induced HMGB1 in cultured primary astrocytes isolated from WKY, SHR, SHRSP, and SHRpch1_18 rats [12]. AVP induced HMGB1 expression at 50 and 100 nM, and it was significantly higher in SHR, SHRSP, and SHRpch1_18 rat astrocytes than in WKY rat astrocytes. HMGB1 may relate to early stages of the inflammatory response [69]. Reports have indicated that myelin loss is associated with neuroinflamma-tion [73] and that it induces inflammation after MCA occlusion [74] in the SHRSP rat strain. This characteristic of SHRSP, SHR, and SHRpch1_18 rats is likely an important contributor to enhanced inflammation in astrocytes and could explain how AVP augments the inflammatory reaction and induces neuronal cell death [12].
Following exposure to tumor necrosis factor-alpha (TNF-α), the expression of vascular cell adhesion molecule-1 (VCAM-1) by SHRSP rat astrocytes was increased compared with those from WKY rats. Expression of TNF-α and adhesion molecules are related to the presence of early neurological exacerbation and infarct volume in stroke [75,76]. TNF-α is generated by microglial cells and infiltrating macrophages following ischemic stroke [77].
In H/R treatment of SHRSP rat astrocytes, the expression of monocyte chemotactic protein-1 (MCP-1) was increased compared with that under normal oxygen. Inhibition or genetic lack of these adhesion molecules decreased infarct volume, edema, and/or mortality in different animal models of ischemic stroke [78]. These enhanced levels of adhesion molecules in H/R and TNF-α treatment may be induced by stroke in SHRSP rats. In SHRSP rats, alteration and attenuation of astrocyte functions promote neuronal cell death during stroke [79].

Conclusions
The level of neuronal cell death in SHRSP rats is significantly higher than in the WKY rat strain [9,10]. In cerebral ischemia, the properties of SHRSP rat neuronal cells, unlike those of WKY rats, might be a factor in the elevated frequency of stroke. Vitamin E reduces neuronal cell damage caused by ROS generated in cerebral ischemia. Thus, antioxidants such as vitamin E could be used as a treatment for oxidative stress-mediated diseases. These antioxidants may regulate redox potential and apoptosis-related proteins. Furthermore, in SHRSP rats, astrocyte properties contribute to the development of brain disorders. In SHRSP rat astrocytes, attenuation and loss of several functions such as GDNF and l-serine were demonstrated under cerebral ischemic stroke conditions (Figure 4). In addition, after ischemic reperfusion, generation of MCP-1 is strongly enhanced in SHRSP rat astrocytes [80]. The expression of VCAM-1 and MCP-1 [81] is markedly elevated in SHRSP astrocytes compared with WKY rat astrocytes. Taken together, neuronal vulnerability and altered regulation of the neuronal supportive functions of astrocytes increase the risk of stroke in SHRSP rats.
Supply of oxygen is critical to neuronal cells viability. On the other hand, oxygen reperfusion induces cellular dysfunction, apoptosis, and necrosis. From several animal experiment data, clinical therapy by single-drug treatment may have little effect [82].