Cerebral ischemia reperfusion (IR) injury may occur in many clin- ical pathophysiological processes. Considerable efforts have been devoted to reduce the extent of IR injury after ischemic episodes. In human stroke, recombinant tissue plasminogen activator (rtPA) has been proved to be beneficial [13]. However, overall patient benefit remains less than optimal partly due to the increase in the risk of symptomatic intracranial hemorrhage among the patients treated with rtPA [14]. Various processes have been investigated exten- sively and are believed to play an important role during IR injury. Factors such as the enhancement of inflammatory processes and free radical formations contribute to reperfusion injury.
Uridine 5"-triphosphate (UTP) is a P2 Y receptor agonist with anti-inflammatory and protective effect in a model of lung inflam- mation [5]. P2 purinergic receptors are very important in protecting ischemic and reperfused myocardium. UTP reduces infarct size and improves rat heart function after myocardial infarct in LAD-ligated rat hearts via the reduction of mitochondrial calcium overload [17]. In vitro studies show that the beneficial effect of UTP on cardiomy- ocytes is related to P2 Y receptors [16]. In Langendorff perfused murine hearts during 20 min of ischemia and 45 min of reperfu- sion, cardioprotection was achieved with low concentrations of UTP, which were consistent with P2 Y involvement [15]. However, the protective effects of UTP on cerebral IR have not been demon- strated. The present study was designed to test the hypothesis that single dose of UTP infusion had a protective effect in a model of rat cerebral IR injury in vivo.
The study was approved by the animal ethics committee of SMMU.
Adult male Sprague–Dawley rats weighing 250–350 g were anesthetized with 3 ml/kg i.p. 10% chloral hydrate. Rectal temperature was maintained at 37 ± 0.5 ◦ C by a heating pad and room humidity was controlled at 50 ± 5%. Different number of animals were used in different type of determination.
Ischemia was induced by intraluminal middle cerebral artery (MCA) suture of middle cerebral artery occlusion (MCAO) as previ- ously reported by Longa et al. [9]. Using microsurgical techniques, the right external cerebral artery (ECA) was exposed through a mid- line neck incision and ligated with a 6–0 silk suture and dissected distally. The right internal cerebral artery (ICA) was isolated and separated from the adjacent vagus nerve carefully. A 6–0 silk suture was used to ligate the extracranial branch of the right ICA close to its origin. A 6-cm length of 4–0 surgical monofilament nylon suture with a tip smoothly rounded by heating was introduced into the right ECA stump, advanced 20–21 mm beyond the carotid bifurcation for the occlusion of the MCA. After 120 min, the suture was withdrawn and reperfusion was obtained. Neurological deficit score (NDS) was examined in 50 rats (n = 10) and a six-grade scale was used to score the neurologic findings as previously published [1].
UTP solution was delivered through an indwelling tail venous catheter via microinfusion pump (Graseby 3500 Anaesthesia Pump) 30 min after the occlusion of MCA at a rate of 0.5 ml/100 g/min. Dif- ferent concentrations were used throughout the study and 1 ml/kg of fluid was administered to each rat. To determine the dose- dependent reduction in tissue water content and NDS, different doses (10, 30 and 90 g/kg) were used.
Brain water content was qualitatively analyzed using the stan- dard wet–dry method [12]. After completion of NDS, 40 rats (n = 8) were decapitated under deep chloral hydrate anesthesia and brains were harvested. The right/left brain hemispheres were separately dissected in a humidified chamber. A tissue paper was used to absorb the small quantity of cerebral spinal fluid on the surface of the sample. The wet weight was immediately determined with a basic precision scale to within 0.1 mg. The dry weight was measured after the tissue was dried to a constant weight at 100 ◦ C in a vacuum oven. Tissue water content was then calculated as %H2 O = (Wet Weight − Dry Weight)/Wet Weight × 100%.
The MRI experiments were performed with a 1.5-T Signa EXCITE HD system (GE Company, USA). The rat was positioned supine with the head on a 3 in. round coil. T2-weighted imaging was per- formed at 6, 30 and 54 h after withdrawal of the suture. The axial images were acquired with a 256 × 160 matrix, field of view of 3 mm × 4 mm, repetition time (TR) of 3 s, echo time (TE) of 110 ms, 6 NEX, 8 coronal slices, slice thickness of 2.5 mm, and inter-slice gap of 0 mm. On the images, the infarct area was measured with Func- Tool software package (GE Advanced Workstation Version 4.2, HP Workstation xw8000). The volume of infarct was the sum of each slice.
At 24 h after MCA occlusion, the rats were anesthetized with chloral hydrate and then decapitated, brains were harvested rapidly and frozen at −80 ◦ C and cut into 1–1.5 mm slices, which were then defrosted and incubated in sodium phosphate buffer containing 2% 2,3,5-triphenyl-tetrazolium chloride (TTC) for 20 min at 37 ◦ C. The infarct tissue was stained pale, and non-ischemia area red. To determine the infarct volume by TTC staining, 7 slices per rat were analyzed by a blinded observer using a standard computer- assisted image analysis software HPIAS-2000 (Hua Hai Medical Info-Tech Co., Ltd).
After determination of NDS in 50 rats, 6 of them (n = 2) were used to study the cellular organelles under an electron microscope, brain tissues of 1 mm3 were fixed with 4% paraformaldehyde for 4 h and rinsed with 0.1 mol/l phosphate buffer. The sections were postfixed with 1% osmium tetroxide, dehydrated by ethanol and acetone, and then embedded in Epon812. Fragments of brain tis- sues were ultramicrotome into 70-nm-thick sections which were then stained with uranil acetate and lead citrate and evaluated in a Hitachi H-600 electron microscope. The number of damaged cells and total number of cells in 10 fields were counted by a blinded observer.
Statistical analysis was performed by SPSS 13.0 software. The data were expressed as mean ± Standard Deviation (x¯ ± S). Multi- ple comparisons were tested by analysis of variance of repeated
measurements. The mean values between groups were compared by the Student–Newman–Keuls method. P < 0.05 was regarded as statistically significant. Compared with the rats given 0 g/kg UTP (normal saline group,NS group), the rats given 10, 30 and 90 g/kg UTP (G10, G30 and G90, respectively) showed better NDS (Fig. 1).
The mean difference in the right brain hemisphere water con- tent between the rats in NS group and those in G10 (1.1%) represented 37.9% of the mean difference resulting from the much larger dose of 90 g/kg (2.9%) (Fig. 2). The mean difference of inter- hemisphereic water content of the rats in NS group was 4.6%, significantly greater than that of the rats in G90 (1.6%, P < 0.01). Therefore, 90 g/kg UTP led to the greatest protective effect on cerebral IR among the 4 doses.
T2WI confirmed the successful reperfusion after withdrawal of the suture. Fig. 3 illustrated the characteristic MRI patterns observed at 6, 30 and 54 h after reperfusion in 2 rats with (Fig. 3D–F) or without (Fig. 3A–C) UTP. The signal intensity was significantly greater at both 30 and 54 h than that at 6 h. After reperfusion, the T2- weighted image of the ischemic regions was significantly greater in NS group than that in G90. The qualitative analysis of the infarct volume by MRI was shown in Fig. 3G.
As shown in Fig. 4, the infarct tissue was stained pale and non-ischemia area red. As doses of UTP went up, the infarct area decreased. The percent infarct volume by TTC was shown in Fig. 4F. Compared with NS group, the rats in G10, G30 and G90 showed a decrease in percent infarct volume.
Electron microscopy was used to investigate nerve cell death 24 h after cerebral IR. The nerve cells of the left brain appeared to be healthy with normal rough endoplasmic reticulum, mitochon- dria and synapses (data not shown). In contrast, seriously damaged structures appeared in hippocampus and cortex of the right side in NS group (Fig. 5B and E). However, the neurons in the rats given 90 g/kg UTP were less damaged compared with those in NS group (Fig. 5C and F). The qualitative results of morphological analysis were shown in the Table 1.
The treatment of cerebral IR is clinically important. The majority of fatalities that occur during the first 2 weeks after serious hemi- spheric stroke is due to tissue swelling caused by the formation of brain edema [2]. There are no optimal therapeutic interventions in ameliorating brain swelling caused by ischemia edema. Com- pounds such as mannitol are often used in intensive medical care to offer osmotic therapy. However, a nonmetabolized compound such as mannitol may worsen brain edema [8]. To search an inter- vention to ameliorate brain edema after cerebral IR is of clinical significance. In the present experiment, we administered differ- ent doses of UTP through the tail vein to investigate the protective effect of UTP on cerebral IR in a MCAO rat model. We found a dose-dependent effect and 90 g/kg UTP had the slightest edema among the 4 UTP groups.
Similar phenomena were observed in the results of NDS and infarct volume. We used a 6-point scale [1] to appraise NDS and found that the rats in UTP groups scored lower than those in NS group. Qualitative analysis of infarct volume was done via both TTC staining and MRI. The results showed that the infarct volume in the UTP groups was lower than that in NS group. Severe brain edema evolves from 6 h to 7 days [6]. We undertook imaging studies at 6, 30, and 54 h after reperfusion. And the quantitative analysis indi- cated that edema formation leading to brain swelling was slighter in G90. Morphological features were detected by electron microscopy, which showed vacuolization and extensively lysis of cytoplasmic contents in NS group. In the rats given 90 g/kg UTP, neurons were less damaged. 
We only undertook a phenomenological observation in this study. The mechanisms whereby UTP infusions protect cerebral IR cannot be explained directly from the results. However, several possibilities emerge from a brief review of the relevant literature. UTP were found to provoke an increase in coronary flow in a dose- dependent manner [11]. Yitzhaki found that UTP had a beneficial effect on in vivo and in vitro rat ischemia models via the reduction of mitochondrial calcium overload and activation of P2 Y receptors [16]. P2 Y receptors are G protein-coupled receptors. P2 Y2 and P2 Y4 receptors are activated by UTP [7]. UTP is an important excitomo- tor of P2 Y receptor, and it was found to be elevated as a result of ischemia [4]. Besides heart IR, renal and hepatic injuries were also reduced by nucleotides via P2 Y receptors [3]. Thus, UTP might benefit cerebral IR through mitochondria and P2 Y receptors. It was reported that UTP had a vasodilator effect via the activation of a specific P2 Y purinoceptors and it dilated the rat MCA through the release of endothelium-derived relaxing fac- tor/nitric oxide (EDRF/NO) [18]. In MCAs of rats after 2 h of ischemia followed by 24 h of reperfusion, dilations were elicited due to the upregulation of the endothelium-derived hyperpolarizing fac- tor (EDHF) [10]. It is possible that UTP administration 30 min after ischemia vasodilated the vascular bed, mainly collaterals, afforded an increased blood flow to the brain, consequently low- ered the necrotic area and brain edema. Thus, the protective effect of UTP on cerebral IR may be partly due to its dilated effect on artery after cerebral IR. The speculative mechanisms of how UTP protect ischemic stroke requires experimental valida- tion.
In summary, UTP has a dose-dependent protective effect on cerebral IR. The possible therapeutic application of UTP in the patients at risk of cerebral ischemic damage is promising. However, how UTP benefits cerebral IR remains further studies.
Acknowledgements
We are grateful to Tie-Jun Li, Fu-Ming Shen, for their help in undertaking the experiment and Yan Zhang for her help in English revision.
This study was supported by the fund of National Nature Science Foundation of China (30772092). References
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