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Brain damage related to hemorrhagic transformation following cerebral ischemia and the role of KATP channels
BR A I N R ES E A RC H 1 2 4 1 ( 2 00 8 ) 1 6 8 –17 5
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
Brain damage related to hemorrhagic transformation following cerebral ischemia and the role of KATP channels Yi Yang, Xiang-jian Zhang⁎, Jing Yin, Li-tao Li Department of Neurology, Second Hospital of Hebei Medical University, Shijiazhuang 050000, China
A R T I C LE I N FO
AB S T R A C T
Article history:
Background: Hemorrhagic transformation (HT) is a major factor limiting the use of
Accepted 21 August 2008
thrombolytic treatment for stroke. Animal model can help us to understand HT. This
Available online 10 September 2008
study is to establish a HT model in rats to compare HT with uncomplicated cerebral infarction in neurobehavioral deficit, brain edema, brain adenosine triphosphatase (ATPase)
Keywords:
activity and succinic dehydrogenase (SDH) activity, and to investigate its pathology changes
Hemorrhagic transformation
as well as the impact of, Glibenclamide, a ATP-sensitive K+ channel (KATP channel) blocker,
Model
on the pathogenesis of HT. Methods: Male, Sprague–Dawley rats were randomly assigned to
Brain edema
four groups: hemorrhagic transformation (HT), cerebral infarction (CI), Glibenclamide + HT
Transmission electron microscope
(GH) and a control. To create HT model, right middle cerebral artery occlusion (MCAO) was
ATP-sensitive K+ channel
conducted with intraluminal thread technique; 30 min after MCAO, 50 μL arterial blood was injected into the caudate nucleus where the infarction occurred. Neurologic deficit was evaluated by Longa test, Berderson test and Beam test. Brain water content, brain ATPase activity and SDH activity were measured. Histology was examined using light microscope and transmission electron microscope. Results: No significant difference in neurobehavioral deficit and brain water content was observed between HT and CI groups in all time points (P N 0.05). Brain ATPase activity 12 h and 24 h after operation and brain SDH activity 24 h after the operation in HT group were both significantly increased compared with those in CI group (P b 0.05); the increase of brain ATPase and SDH activity in HT group could be prevented by Glibenclamide. Neuronal degeneration and tissue edema in HT group, swollen neuropil and loosen intercellular substance in CI group were revealed by histology study. Ultrastructural changes including swollen mitochondria and interstitial edema were also observed in both HT and CI groups. Conclusions: The results demonstrated that moderate hemorrhagic transformation does not significantly aggravate cerebral infarction, and that KATP channels have an important role in energy metabolism. © 2008 Elsevier B.V. All rights reserved.
1.
Introduction
Thrombolysis is an effective treatment for early cerebral infarction (CI) (The ATLANTIS, ECASS and NIND rt-PA Study Group Investigators, 2004). However, the probability of hemor-
rhagic transformation (HT), a complication of cerebral infarction occurred in 10–43% of the CI patients, could be increased 2–3 times in CI patients treated by thrombolysis (Berger et al., 2001; Gilligan et al., 2002; Motto et al., 1999). It showed that opening of adenosine triphosphate sensitive potassium (KATP)
⁎ Corresponding author. Fax: +86 0311 66002915. E-mail address: [email protected] (X. Zhang). 0006-8993/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2008.08.083
BR A I N R ES E A RC H 1 2 4 1 ( 2 00 8 ) 1 6 8 – 1 75
channels played a protective role at early stage in ischemic brain injury. The damage caused by CI complicated with HT was different from that caused by CI only. Whether KATP channels are involved in CI complicated with HT remains unclear. HT refers to hemorrhage in ischemic area after CI. There are many questions in HT, such as its pathological process, its impact on neuronal dysfunction, the effect of autologous blood on ischemic area. Understanding these questions will certainly help neurologists to explore possible therapies for HT. The objectives of this study are to establish a CI model, to develop a HT model through the combination of CI with hemorrhage, and to imitate the effects of autologous blood on ischemic tissue using the HT model. The study also investigated the impacts of HT on neuronal dysfunction, brain edema, the adenosine triphosphatase (ATPase) activity, succinic dehydrogenase (SDH) activity, and the function of KATP channels in the HT pathological process.
2.
Results
2.1.
Neurological examination
Left palsy and right Horner symptoms were observed in rats in both HT group and CI group in Longa test, Berderson test as well as Beam test. Although no significant difference was found between rats in HT group and those in CI group (P N 0.05), the scores in both HT and CI groups were significantly higher than those in the control (P b 0.05) (Fig. 1 Parts 1–3).
2.2.
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Brain water content
Brain water content was found significantly increased at each time point in the rats of HT and CI groups compared with those in the control (P b 0.05), but no significant difference was observed between the rats in HT group and those in CI group at any time point (P N 0.05) (Fig. 1 Part 4).
2.3.
ATPase activity and SDH activity
Assay of brain ATPase activity showed that the difference between HT and CI rats was not significant at time points 3 h and 6 h (P N 0.05). However, ATPase activity in HT group was higher than that in CI group 12 h and 24 h (P b 0.05); ATPase activity in GH group was significantly lower than that in HT group 6 h, 12 h and 24 h (P b 0.05) (Fig. 2 Part 1). A significant decrease of brain SDH activity was observed in HT and CI groups at each time point compared to the control; the SDH activity in HT group was significantly higher than that in CI group at 24 h (P b 0.05), but no significant difference in SDH activity was found between HT and CI groups at 3 h, 6 h and 12 h (P N 0.05). Additionally, SDH activity in GH group was significantly lower than that in HT group at 6 h, 12 h and 24 h (P b 0.05) (Fig. 2 Part 2).
2.4.
TTC staining
In contrast to normal brain tissue stained red in triphenyltetrazolium chloride (TTC), ischemic area was pale in both CI group and HT group at 24 h (Fig. 3 Parts 1–2). In addition, a
Fig. 1 – Behavioral test and brain water content. (Parts 1–3 Behavioral test) At each time point, the behavioral scores were significantly higher in HT and CI groups than that in the control, but no significant difference was found between HT and CI groups. (Part 4 Brain water content) At each time point, brain water content was significantly higher in HT and CI groups than that in the control (aP < 0.05), however, no significant difference was found between HT and CI groups (P > 0.05).
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Fig. 2 – Brain ATPase activity and brain SDH activity. (Part 1 Brain ATPase activity) Brain ATPase activity was significantly decreased in CI group compared with that in HT group at time points 12 h and 24 h (bP < 0.05). Glibenclamide significantly decreased the brain ATPase activity in GH rats at time points 6 h, 12 h and 24 h (cP < 0.05). (Part 2 Brain SDH activity) Brain SDH activity was significantly decreased in HT and CI groups compared with control group at each time point (aP < 0.05), and SDH activity in CI rats was significantly lower than that in HT rats at 24 h (bP < 0.05). Glibenclamide significantly decreased brain SDH activity in GH rats compared with HT rats at 6 h, 12 h and 24 h (cP < 0.05). hematoma was observed in the infarction in HT brain tissue (Fig. 3 Part 2).
2.5.
HE staining
The infarction focus was observed in the middle cerebral artery region in both CI group and HT group. Swollen vacuolated neuropil, necrotized neurons, loosened intercellular substance and gathered spongiocyte (Fig. 4 Part 1) were found in CI tissue. In HT group, erythrocyte aggregation was found in the infarction and its surroundings where spongiocyte proliferation and nucleus degeneration in neurons were also observed (Fig. 4 Part 2).
2.6.
Ultrastructural changes of nerve cells
The impact of CI on the ultrastructure of nerve cells appeared 24 h post cerebral infarction, as indicated by low electron concentration neuraxon of medullated nerve fiber, swollen spongiocyte, neurons cytolymph edema, and swollen mitochondria with few mussy crista on the rim at thalamencephon-basal nucleus, and by high electron concentration neurons and intercellular substance edema in cortex (Fig. 5).
Swollen mitochondria with vague crista, slightly puffed endothelial cell of the vessel and significantly expanded gapping place were also observed (Fig. 5). The effect of HT on the ultrastructure of brain tissue and cells was also found at 24 h after HT. Swollen nerve tracts, medullated nerve fibers, interstitial edema, cell pyknosis and peripheral edema were found in the tissue around hematoma at thalamencephon-basal nucleus. Additionally, cytolymph edema, blurred nuclear membrane, liquified nuclear, dilated endoplasmic reticulum and balloon-like mitochondria with crista on the rim were observed in neurons; blurred nuclear membrane, dilated endoplasmic reticulum and un-swollen mitochondria were found in the neurons of cortex. Furthermore, endothelial conjunction was normal, and gliocyte has end-feet edema (Fig. 6).
3.
Discussion
3.1.
Animal model
As far as we know, no well recognized HT model matching clinical feature has been reported yet. We used intraluminal
Fig. 3 – Brain from CI and HT stained by TTC. Normal brain tissue was stained red, while ischemic area were stained pale in both CI group and HT group at time point 24 h. Hematoma in the infarction was visible in HT brain tissue.
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Fig. 4 – Pathology of CI and HT (HE stain 40×). (Part 1 Pathology of CI) Swollen neuropil was vacuolated, the intercellular substance was loosened, spongiocyte was gathered. (Part 2 Pathology of HT) Surrounding the erythrocyte aggregation was spongiocyte proliferation and nucleus degeneration in neurons.
thread technique to occlude MCA, followed by injecting autologous blood into the ischemia to mimic the pathophysiologic processes of HT. Compared to techniques such as photochemical method, craniotomy and blood clot emboli, intraluminal thread is regarded as a Golden Standard technique with minimum injury since it does not need to open cranium. In addition, this technique has a standard operation spot and a controlled ischemic time. Injection of autologous whole blood, collagenase digestion of blood vessels, balloon inflation and spontaneous hemorrhage are the main techniques used to create intracerebral hemorrhage. Autologous whole blood injection is superior to other techniques since the blood quantity injected could be controlled and the model can be scaled up according to the amount of blood injected. Because the blood injected is
unheparinized, the effect of vaso-active substance on brain tissue can be evaluated during the coagulation process. Blood injection is comparable to intracerebral hemorrhage in patients, preferably used to investigate natural process and pathological changes of intracerebral hemorrhage. The HT model developed by combining intraluminal thread and autologous whole blood injection was used to imitate the pathological process resulted from cerebral infarction followed by its complicated hemorrhage. The model was practical and reliable, which allowed us to monitor the time used for the injection and the amount of hemorrhage, and to investigate the effects of a variety of time periods used for blood injection and different amounts of hemorrhage on HT and CI. The blood from tail did not affect limb function, which allowed us to evaluate neurofunction accurately. In contrast,
Fig. 5 – Ultrastructural changes of nerve cells in CI tissue. (Part 1) The swollen medullated nerve fibers and spongiocyte (2000×). (A) medullated nerve fibers. (B) spongiocyte. (Part 2) Neurons with swollen mitochondria in thalamencephon-basal nucleus (15K×). (A) nucleus. (B) mitochondria with crista on the rim. (Part 3) The high electron concentration neurons and intercellular substance edema in cortex (20K×). (A) nucleus. (B) mitochondria. (Part 4) The endothelial cell of the vessel was swelled slightly, and the gapping place was expanded obviously (6000×). (A) the endothelial cell.
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Fig. 6 – Ultrastructural changes of nerve cells in HT tissue. (Part 1) Neurons of thalamencephon-basal nucleus (4000×). (A) liquified nucleus. (B) balloon-like mitochondria with crista on the rim. (Part 2) The swollen nerve tracts and medullated nerve fibers and interstitial edema (2000×). (A) medullated nerve fibers. (Part 3) The neurons of cortex: blurred nuclear membrane and dilated endoplasmic reticulum, but no evident swollen mitochondria (20K×). (A) nucleus. (B) mitochondria. (C) endoplasmic reticulum. (Part 4) The endothelial conjunction kept the normal structure and end-feet edema of gliocyte (6000×). (A) the endothelial conjunction. (B) end-feet edema of gliocyte.
clinical manifestations and prognosis are often diverse due to different hemorrhagic amount and various time length of HT.
3.2. The mechanism of brain damage in HT and the role of KATP channels on HT The results showed that the neurofunction was impaired in CI group and in CI complicated with HT group, such as left palsy and right Horner sign. However, no significant difference was found between the damages caused by CI and that by CI complicated with HT, suggesting that moderate bleeding did not aggravate neurofunctional deficit. The water content is an objective index of brain edema, it showed that the brain edema was significantly worse 12–24 h post CI and HT than that in the control. Additionally, it showed erythrocyte aggregation in infarct area in HE stain, obvious edema in peripheral tissues in HT group, and brain edema in infarct area in CI group. Swollen mitochondria found in the tissue of both CI and HT groups was an indication of the impairment in energy (ATP) supply and damage of Na+–K+ pump function, which resulted in the accumulation of sodium and water in mitochondria and brain cells, i.e., edema. No significant difference in brain damage and edema was observed between CI and HT rats, suggesting that HT did not aggravate brain edema. ATPase is sensitive to hypoxia brain injury, it catalyzes decomposition of adenosine triphosphate (ATP) to generate energy for the cell. SDH activity is an indication of mitochondria function. Damage in cerebral ischemia could result in decreasing of ATP molecules, accumulation of acidic meta-
bolite and epicyte injury, leading to the reduction of both ATPase activity and SDH activity. The decline progress of ATPase and SDH activity in HT group was slower than that in CI group, and KATP channel blocker Glibenclamide significantly reduced the ATPase and SDH activity in GH group. These results suggested that the opening of KATP channels was required for maintaining a relatively higher ATPase and SDH activity in HT than that in early stage of CI. Additionally, the opening status of the KATP channels in rats of HT group was much longer than that in rats of CI group. It was the activated KATP channels that postponed the brain damage after HT at early stage, and the blood in the infarction that played a similar role as the KATP channel opening stimulator. However, the damage in both CI and HT tissue was irreversible because of the persistence of ischemia. KATP channels were voltage independent, ligand-gated ion channels initially reported in Guinea pig cardiomyocytes (Noma, 1983), and were found present in the membrane of variety of cells, such as β-cells, brain neuron, skeletal muscle, kidney and smooth muscle, and in the inner membrane of mitochondrion. KATP channels could be blocked by sulfonamides (such as tobutamide and glibenclamide), but activated by KATP channel openers (Inagaki et al., 1996). Usually KATP channels are closed, however, they could be activated indirectly by brain ischemia via reducing the ATP molecules and changing the pH in the cell. The activated KATP channels further couple cellular metabolism, membrane potential and excitatory, playing important roles in neuron excitatory, neurotransmitter releasing and postsynaptic membrane function. Activation of KATP channels is a mechanism of self-
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defending for ischemia, but the channels would decay as the ischemia continues, resulting in the loss of metabolic substrates (especially oxygen and glucose) and trans-membranes ion gradients which depolarize the cell membranes. The depolarized neuron membranes favor calcium influx, freeradical generation, lipid peroxidation and glutamate release. Furthermore, the opening of KATP channels can lead to membrane hyperpolarization, which further result in the closure of voltage-dependent calcium channels, the reduction of calcium influx, and the release of glutamate (Ishida et al., 2001; Mitsushige et al., 2001; Yasushi et al., 2003). In contrast, the activation of KATP channels stimulates glutamate transporters for uptaking glutamate (Dai et al., 2006), induces HSP70 expression (Blondeau et al., 2000) but inhibits the expression of some the genes induced by ischemia (Jiang et al., 2004; Liu et al., 2002; Yasushi et al., 2003). The study also found that the invasion of blood in the infarcted territory had a similar role as KATP channel opener by temporarily postponing the brain injury in HT. At early stage of ischemia, the neurobehavioral deficit and brain injury was not intensified by a moderate volume of HT after CI, however, they both were affected by KATP channels. An early report showed that of the 89 CI patients who received Intra-Arterial thrombolysis, only 6 patients (7%) had major symptomatic HT, while 29 patients (33%) had asymptomatic HT (Kidwell et al., 2002). It is the physicians' concern whether HT can change the clinical processes and prognosis. According to CT image, 5 categories were assigned for HT: (1) no hemorrhagic transformation, (2) HI1 (small petechiae), (3) HI2 (more confluent petechiae), (4) PH1 (b30% of the infarcted area with some mild space-occupying effect) and (5) PH2 (N30% of the infarcted area with significant space-occupying effect, or clot remote from infarcted area) (Fiorelli et al., 1999; Trouillas and von Kummer, 2006). Here HI was defined as a petechial infarction without space-occupying effect, and PH was defined as a hemorrhage (coagulum) with mass effect. Compared with no hemorrhagic transformation, the results showed that HI1, HI2 and PH1 did not modify the risk of early neurological deterioration, death and disability, while in placebo group and recombinant tissue plasminogen activator group, PH2 had a devastating impact on early neurological course. Our data was consistent with documented clinical studies, suggested that large hematoma is the only hemorrhagic transformation that may alter the clinical course of ischemic stroke, and moderate hemorrhage after CI did not aggravate the disease.
4.
Experimental procedures
4.1.
Experimental groups
A total of 102 male Sprague–Dawley rats 250 to 280 g in weight were provided by the Hebei Medical University and used in this study. The protocol was approved by the Institutional Animal Care and Use Committee and the local experimental ethics committee. Seventy-eight rats were randomly assigned to four groups. (1) HT group (n = 24 rats): 50 μL autologous blood was injected
173
into the ischemic area 30 min after middle cerebral artery occlusion (MCAO); (2) CI group (n = 24 rats): permanent MCAO was to create ischemia; (3) GH (Glibenclamide + HT) group (n = 24 rats): 8 μL Glibenclamide (10 μmol/L) was injected into paracele 30 min before HT; (4) control (n = 6 rats). In experimental group HT, CI and GH, six rats were further randomly assigned to 4 subgroups at time points 3 h, 6 h, 12 h and 24 h after operation. Twelve HT rats and twelve CI rats were used for TTC staining, light microscope and transmission electron microscope observation.
4.2.
Rat models
4.2.1.
Cerebral infarction (CI) model
Rat middle cerebral artery was occluded to create rat CI model as described (Longa et al., 1989). Briefly, in anesthetized rats, the right common carotid artery (CCA), the external carotid artery (ECA) and the internal carotid artery (ICA) were dissected from connective tissue through a midline neck incision. The ECA was ligated and cut, leaving a stump about 3–4 mm attached to the CCA. Next, a curved microvascular clip was placed across CCA and ICA, a 0.2 mm incision was made on the ECA stump. A 5 cm nylon suture with a round end and a mark at 18 mm was introduced into the ECA lumen through the incision. A silk suture at the ECA stump was tightened around the intraluminal nylon suture to prevent bleeding, then the microvascular clip was removed, and the nylon suture was gently advanced from the ECA to the ICA lumen. The suture in the ICA lumen was visible as the mark at 18 mm was reaching the branch of CCA, resistance could be felt when the suture tip reached anterior cerebral and occluded the middle cerebral artery. The suture was then curved slightly, and the wound was sewed up.
4.2.2.
Hemorrhagic transformation (HT) model
30 min after CI, 50 μL arterial blood was injected stereotaxically into the caudate nucleus as illustrated (Hua et al., 2002). After MCAO, rat was positioned in a stereotaxic frame (SR-6N, Japan), a midline incision was made on rat head to expose the Bregma. Then a cranial burr hole (1 mm) was drilled on the skull (coordinates: 0.2 mm anterior, 5.5 mm ventral and 3.5 mm lateral to Bregma). A needle was fixed on the stereotaxic frame to the hole in a vertical position, a midline incision was made on the root segment of the tail to isolate tail artery, 50 μL blood was phlebotomized from the artery and the needle was removed. Then the autologous blood was slowly and stereotactically injected into the infarcted territory through the skull hole in 2 min, to prevent blood flowing back the needle was removed 10 min after injection. The burr hole was sealed with bone max and the wound was sutured, and the animal was placed in a warm box with free access to food and water.
4.2.3.
Glibenclamide + HT (GH) model
30 min before the operation, Glibenclamide was injected slowly into the right lateral ventricle for 1 min. The needle was inserted 4.5 mm in depth at a position 1.2 mm back of and 2 mm right of the middle line of Bregma, then the model was developed as the HT model described above.
174 4.3.
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Neurological examination
Three motor functional tests were evaluated at time points 3 h, 6 h, 12 h and 24 h after operation. (1) Longa behavioral test (Longa et al., 1989) was used to evaluate spontaneous contralateral circling and tumble, the results were graded from 0 (no circling) to 4 points (unconsciousness). (2) Berderson behavioral test (Bederson et al., 1986) was used to measure the palsy of contralateral limbs, the score was assigned from 0 (no palsy) to 3 points (circling of the contralateral). (3) Beam walking test (Altumbabic et al., 1998) was employed to exam the ability to walk on a wood beam 80 cm in length and 2.5 cm in width, the data was expressed from 0 (traversed the beam) to 5 points (unable to move or fell off the beam).
4.4.
Water content, ATPase and SDH activity test
After neurofunctional study, rats were sacrificed by overdose of intravenous (i.v.) pentobarbital, rat brain was immediately removed and cut along the blood injection track level which was about 7 mm from the forehead pole. Anterior 2 mm brain coronal slices were placed in preweighed crucibles to obtain their wet weight (WW), followed by incubation for 48 h at 100 °C to determine their dry weight (DW), water content (percentage of water) in the samples were obtained by using the formula (WW − DW) / WW × 100%. The posterior 2 mm brain coronal slices frozen in liquid nitrogen were homogenized in 9 volume cold normal saline on ice, after centrifugation at 1000 rpm for 5 min, the supernatant was used for ATPase and SDH activity test according to the manufacturer's instruction (Nanjing Jiancheng Bioengineering Institute, China). ATPase activity unit was defined as generating 1 μmol phosphorus per milligram protein per hour. SDH activity unit was defined as OD decrease 0.01 per milligram protein per minute. Protein was measured according to Coomassie Brilliant Blue method.
4.5.
Light microscope examination
The anterior 2 mm brain coronal slices were fixed in 4% paraformaldehyde, embedded in paraffin, cut into 6 μm slices, dehydrated in alcohol, stained in hematoxylin and eosin (HE stain) and observed under light microscope.
4.6.
The preparation for TTC staining
2 mm brain coronal slices were incubated at 37 °C for 30 min in 2% TTC in 25 mmol/L potassium phosphate-buffered saline, then fixed and photographed to identify the infarcted and hemorrhagic region.
4.7.
Transmission electron microscope examination
Rats were anesthetized with pentobarbital, perfused with fixing solution (2% paraform and 2.5% glutaraldehyde). Thalamencephalon-basal nucleus slices and temporal-parietal cortex slices were collected to generate ultrathin sections (600–800 nm). Ultrathin sections were mounted to single-hole copper grids and multi-hole grids, stained in uranyl acetate
and citric acid lead, and examined under transmission electron microscope.
4.8.
Statistical analysis
Using software SPSS10.0, Kruskal–Wallis H test and Q test were carried out for neurobehavioral deficit data comparisons, ANOVA and q test were carried out for brain water content, ATPase activity and SDH activity. The significance level was set at P b 0.05.
Acknowledgments This work was funded by Hebei Province, No.:C2006000915; we thank Dr. Zhao-duo Zhang and Dr. Hong-qun Liu for reading the manuscript and providing valuable suggestions.
REFERENCES
Altumbabic, M., Peeling, J., Bigio, M.R.D., 1998. Intracerebral hemorrhage in the rat: effects of hematoma aspiration. Stroke 29, 1917–1923. Bederson, J.B., Pitts, L.H., Daves, R.L., 1986. Rat middle cerebral artery occlusion: evaluation of the model and development of a neurologic examination. Stroke 17, 472–476. Berger, C., Fiorelli, M., Steiner, T., 2001. Hemorrhagic transformation of ischemic brain tissue: asymptomatic or symptomatic? Stroke 32, 1330–1335. Blondeau, N., Plamondon, H., Richelme, C., 2000. K (ATP) channel openers, adenosine agonists and epileptic preconditioning are stress signals inducing hippocampal neuroprotection. Neuroscience 100, 465–474. Dai, C.P., Zeng, X.N., Sun, X.L., 2006. Diazoxide increases glutamate uptake by astrocytes via activating mitochondrial ATP-sensitive potassium channels. Chin. J. Clin. Pharmacol. Ther. 11, 398–401. Fiorelli, M., Bastianello, S., von Kummer, R., 1999. Hemorrhagic transformation within 36 hours of a cerebral infarct relationships with early clinical deterioration and 3-month outcome in the European Cooperative Acute Stroke Study I (ECASS I) cohort. Stroke 30, 2280–2284. Gilligan, A.K., Markus, R., Read, S., 2002. Baseline blood pressure but not early computed tomography changes predicts major hemorrhage after streptokinase in acute ischemic stroke. Stroke 33, 2236–2242. Hua, Y., Schallert, T., Keep, R.F., 2002. Behavioral test after intracerebal hemorrhage in the rat. Stroke 33, 2478–2484. Inagaki, N., Gonoi, T., Clement, J.P., 1996. A family of sulfonylurea receptors determines the pharmacological properties of ATP-sensitive K+ channels. Neuron 16, 1011–1017. Ishida, H., Hirota, Y., Genka, C., 2001. Opening of mitochondrial KATP channels attenuates the ouabain-induced calcium overload in mitochondria. Circ. Res. 89, 856–858. Jiang, K.W., Zhao, Z.Y., Shui, Q.X., 2004. Electro-acupuncture preconditioning abrogates the elevation of c-Fos and c-Jun expression in neonatal hypoxic-ischemic rat brains induced by glibenclamide, an ATP-sensitive potassium channel blocker. Brain Res. 998, 13–19. Kidwell, C.S., Saver, J.L., Carneado, J., 2002. Predictors of hemorrhagic transformation in patients receiving infra-arterial thrombolysis. Stroke 33, 717–724. Liu, D., Lu, C., Wan, R., 2002. Activation of mitochondrial ATP-dependent potassium channels protects neurons against
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Noma, A., 1983. ATP-regulated K+ channels in cardiac muscle. Nature 305, 147–148. The ATLANTIS, ECASS and NIND rt-PA Study Group Investigators, 2004. Association of outcome with early stroke treatment: pooled analysis of ATLANTIS, ECASS and NIND rt-PA stroke trials. Lancet 363, 768–774. Trouillas, P., von Kummer, R., 2006. Classification and pathogenesis of cerebral hemorrhages after thrombolysis in ischemic stroke. Stroke 37, 556–561. Yasushi, T., Masaharu, A., Ronald, A.L., 2003. Mitochondrial ATP-sensitive potassium channel activation protects cerebellar granule neurons from apoptosis induced by oxidative stress. Stroke 34, 1796–1802.
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
Brain damage related to hemorrhagic transformation following cerebral ischemia and the role of KATP channels Yi Yang, Xiang-jian Zhang⁎, Jing Yin, Li-tao Li Department of Neurology, Second Hospital of Hebei Medical University, Shijiazhuang 050000, China
A R T I C LE I N FO
AB S T R A C T
Article history:
Background: Hemorrhagic transformation (HT) is a major factor limiting the use of
Accepted 21 August 2008
thrombolytic treatment for stroke. Animal model can help us to understand HT. This
Available online 10 September 2008
study is to establish a HT model in rats to compare HT with uncomplicated cerebral infarction in neurobehavioral deficit, brain edema, brain adenosine triphosphatase (ATPase)
Keywords:
activity and succinic dehydrogenase (SDH) activity, and to investigate its pathology changes
Hemorrhagic transformation
as well as the impact of, Glibenclamide, a ATP-sensitive K+ channel (KATP channel) blocker,
Model
on the pathogenesis of HT. Methods: Male, Sprague–Dawley rats were randomly assigned to
Brain edema
four groups: hemorrhagic transformation (HT), cerebral infarction (CI), Glibenclamide + HT
Transmission electron microscope
(GH) and a control. To create HT model, right middle cerebral artery occlusion (MCAO) was
ATP-sensitive K+ channel
conducted with intraluminal thread technique; 30 min after MCAO, 50 μL arterial blood was injected into the caudate nucleus where the infarction occurred. Neurologic deficit was evaluated by Longa test, Berderson test and Beam test. Brain water content, brain ATPase activity and SDH activity were measured. Histology was examined using light microscope and transmission electron microscope. Results: No significant difference in neurobehavioral deficit and brain water content was observed between HT and CI groups in all time points (P N 0.05). Brain ATPase activity 12 h and 24 h after operation and brain SDH activity 24 h after the operation in HT group were both significantly increased compared with those in CI group (P b 0.05); the increase of brain ATPase and SDH activity in HT group could be prevented by Glibenclamide. Neuronal degeneration and tissue edema in HT group, swollen neuropil and loosen intercellular substance in CI group were revealed by histology study. Ultrastructural changes including swollen mitochondria and interstitial edema were also observed in both HT and CI groups. Conclusions: The results demonstrated that moderate hemorrhagic transformation does not significantly aggravate cerebral infarction, and that KATP channels have an important role in energy metabolism. © 2008 Elsevier B.V. All rights reserved.
1.
Introduction
Thrombolysis is an effective treatment for early cerebral infarction (CI) (The ATLANTIS, ECASS and NIND rt-PA Study Group Investigators, 2004). However, the probability of hemor-
rhagic transformation (HT), a complication of cerebral infarction occurred in 10–43% of the CI patients, could be increased 2–3 times in CI patients treated by thrombolysis (Berger et al., 2001; Gilligan et al., 2002; Motto et al., 1999). It showed that opening of adenosine triphosphate sensitive potassium (KATP)
⁎ Corresponding author. Fax: +86 0311 66002915. E-mail address: [email protected] (X. Zhang). 0006-8993/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2008.08.083
BR A I N R ES E A RC H 1 2 4 1 ( 2 00 8 ) 1 6 8 – 1 75
channels played a protective role at early stage in ischemic brain injury. The damage caused by CI complicated with HT was different from that caused by CI only. Whether KATP channels are involved in CI complicated with HT remains unclear. HT refers to hemorrhage in ischemic area after CI. There are many questions in HT, such as its pathological process, its impact on neuronal dysfunction, the effect of autologous blood on ischemic area. Understanding these questions will certainly help neurologists to explore possible therapies for HT. The objectives of this study are to establish a CI model, to develop a HT model through the combination of CI with hemorrhage, and to imitate the effects of autologous blood on ischemic tissue using the HT model. The study also investigated the impacts of HT on neuronal dysfunction, brain edema, the adenosine triphosphatase (ATPase) activity, succinic dehydrogenase (SDH) activity, and the function of KATP channels in the HT pathological process.
2.
Results
2.1.
Neurological examination
Left palsy and right Horner symptoms were observed in rats in both HT group and CI group in Longa test, Berderson test as well as Beam test. Although no significant difference was found between rats in HT group and those in CI group (P N 0.05), the scores in both HT and CI groups were significantly higher than those in the control (P b 0.05) (Fig. 1 Parts 1–3).
2.2.
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Brain water content
Brain water content was found significantly increased at each time point in the rats of HT and CI groups compared with those in the control (P b 0.05), but no significant difference was observed between the rats in HT group and those in CI group at any time point (P N 0.05) (Fig. 1 Part 4).
2.3.
ATPase activity and SDH activity
Assay of brain ATPase activity showed that the difference between HT and CI rats was not significant at time points 3 h and 6 h (P N 0.05). However, ATPase activity in HT group was higher than that in CI group 12 h and 24 h (P b 0.05); ATPase activity in GH group was significantly lower than that in HT group 6 h, 12 h and 24 h (P b 0.05) (Fig. 2 Part 1). A significant decrease of brain SDH activity was observed in HT and CI groups at each time point compared to the control; the SDH activity in HT group was significantly higher than that in CI group at 24 h (P b 0.05), but no significant difference in SDH activity was found between HT and CI groups at 3 h, 6 h and 12 h (P N 0.05). Additionally, SDH activity in GH group was significantly lower than that in HT group at 6 h, 12 h and 24 h (P b 0.05) (Fig. 2 Part 2).
2.4.
TTC staining
In contrast to normal brain tissue stained red in triphenyltetrazolium chloride (TTC), ischemic area was pale in both CI group and HT group at 24 h (Fig. 3 Parts 1–2). In addition, a
Fig. 1 – Behavioral test and brain water content. (Parts 1–3 Behavioral test) At each time point, the behavioral scores were significantly higher in HT and CI groups than that in the control, but no significant difference was found between HT and CI groups. (Part 4 Brain water content) At each time point, brain water content was significantly higher in HT and CI groups than that in the control (aP < 0.05), however, no significant difference was found between HT and CI groups (P > 0.05).
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Fig. 2 – Brain ATPase activity and brain SDH activity. (Part 1 Brain ATPase activity) Brain ATPase activity was significantly decreased in CI group compared with that in HT group at time points 12 h and 24 h (bP < 0.05). Glibenclamide significantly decreased the brain ATPase activity in GH rats at time points 6 h, 12 h and 24 h (cP < 0.05). (Part 2 Brain SDH activity) Brain SDH activity was significantly decreased in HT and CI groups compared with control group at each time point (aP < 0.05), and SDH activity in CI rats was significantly lower than that in HT rats at 24 h (bP < 0.05). Glibenclamide significantly decreased brain SDH activity in GH rats compared with HT rats at 6 h, 12 h and 24 h (cP < 0.05). hematoma was observed in the infarction in HT brain tissue (Fig. 3 Part 2).
2.5.
HE staining
The infarction focus was observed in the middle cerebral artery region in both CI group and HT group. Swollen vacuolated neuropil, necrotized neurons, loosened intercellular substance and gathered spongiocyte (Fig. 4 Part 1) were found in CI tissue. In HT group, erythrocyte aggregation was found in the infarction and its surroundings where spongiocyte proliferation and nucleus degeneration in neurons were also observed (Fig. 4 Part 2).
2.6.
Ultrastructural changes of nerve cells
The impact of CI on the ultrastructure of nerve cells appeared 24 h post cerebral infarction, as indicated by low electron concentration neuraxon of medullated nerve fiber, swollen spongiocyte, neurons cytolymph edema, and swollen mitochondria with few mussy crista on the rim at thalamencephon-basal nucleus, and by high electron concentration neurons and intercellular substance edema in cortex (Fig. 5).
Swollen mitochondria with vague crista, slightly puffed endothelial cell of the vessel and significantly expanded gapping place were also observed (Fig. 5). The effect of HT on the ultrastructure of brain tissue and cells was also found at 24 h after HT. Swollen nerve tracts, medullated nerve fibers, interstitial edema, cell pyknosis and peripheral edema were found in the tissue around hematoma at thalamencephon-basal nucleus. Additionally, cytolymph edema, blurred nuclear membrane, liquified nuclear, dilated endoplasmic reticulum and balloon-like mitochondria with crista on the rim were observed in neurons; blurred nuclear membrane, dilated endoplasmic reticulum and un-swollen mitochondria were found in the neurons of cortex. Furthermore, endothelial conjunction was normal, and gliocyte has end-feet edema (Fig. 6).
3.
Discussion
3.1.
Animal model
As far as we know, no well recognized HT model matching clinical feature has been reported yet. We used intraluminal
Fig. 3 – Brain from CI and HT stained by TTC. Normal brain tissue was stained red, while ischemic area were stained pale in both CI group and HT group at time point 24 h. Hematoma in the infarction was visible in HT brain tissue.
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Fig. 4 – Pathology of CI and HT (HE stain 40×). (Part 1 Pathology of CI) Swollen neuropil was vacuolated, the intercellular substance was loosened, spongiocyte was gathered. (Part 2 Pathology of HT) Surrounding the erythrocyte aggregation was spongiocyte proliferation and nucleus degeneration in neurons.
thread technique to occlude MCA, followed by injecting autologous blood into the ischemia to mimic the pathophysiologic processes of HT. Compared to techniques such as photochemical method, craniotomy and blood clot emboli, intraluminal thread is regarded as a Golden Standard technique with minimum injury since it does not need to open cranium. In addition, this technique has a standard operation spot and a controlled ischemic time. Injection of autologous whole blood, collagenase digestion of blood vessels, balloon inflation and spontaneous hemorrhage are the main techniques used to create intracerebral hemorrhage. Autologous whole blood injection is superior to other techniques since the blood quantity injected could be controlled and the model can be scaled up according to the amount of blood injected. Because the blood injected is
unheparinized, the effect of vaso-active substance on brain tissue can be evaluated during the coagulation process. Blood injection is comparable to intracerebral hemorrhage in patients, preferably used to investigate natural process and pathological changes of intracerebral hemorrhage. The HT model developed by combining intraluminal thread and autologous whole blood injection was used to imitate the pathological process resulted from cerebral infarction followed by its complicated hemorrhage. The model was practical and reliable, which allowed us to monitor the time used for the injection and the amount of hemorrhage, and to investigate the effects of a variety of time periods used for blood injection and different amounts of hemorrhage on HT and CI. The blood from tail did not affect limb function, which allowed us to evaluate neurofunction accurately. In contrast,
Fig. 5 – Ultrastructural changes of nerve cells in CI tissue. (Part 1) The swollen medullated nerve fibers and spongiocyte (2000×). (A) medullated nerve fibers. (B) spongiocyte. (Part 2) Neurons with swollen mitochondria in thalamencephon-basal nucleus (15K×). (A) nucleus. (B) mitochondria with crista on the rim. (Part 3) The high electron concentration neurons and intercellular substance edema in cortex (20K×). (A) nucleus. (B) mitochondria. (Part 4) The endothelial cell of the vessel was swelled slightly, and the gapping place was expanded obviously (6000×). (A) the endothelial cell.
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Fig. 6 – Ultrastructural changes of nerve cells in HT tissue. (Part 1) Neurons of thalamencephon-basal nucleus (4000×). (A) liquified nucleus. (B) balloon-like mitochondria with crista on the rim. (Part 2) The swollen nerve tracts and medullated nerve fibers and interstitial edema (2000×). (A) medullated nerve fibers. (Part 3) The neurons of cortex: blurred nuclear membrane and dilated endoplasmic reticulum, but no evident swollen mitochondria (20K×). (A) nucleus. (B) mitochondria. (C) endoplasmic reticulum. (Part 4) The endothelial conjunction kept the normal structure and end-feet edema of gliocyte (6000×). (A) the endothelial conjunction. (B) end-feet edema of gliocyte.
clinical manifestations and prognosis are often diverse due to different hemorrhagic amount and various time length of HT.
3.2. The mechanism of brain damage in HT and the role of KATP channels on HT The results showed that the neurofunction was impaired in CI group and in CI complicated with HT group, such as left palsy and right Horner sign. However, no significant difference was found between the damages caused by CI and that by CI complicated with HT, suggesting that moderate bleeding did not aggravate neurofunctional deficit. The water content is an objective index of brain edema, it showed that the brain edema was significantly worse 12–24 h post CI and HT than that in the control. Additionally, it showed erythrocyte aggregation in infarct area in HE stain, obvious edema in peripheral tissues in HT group, and brain edema in infarct area in CI group. Swollen mitochondria found in the tissue of both CI and HT groups was an indication of the impairment in energy (ATP) supply and damage of Na+–K+ pump function, which resulted in the accumulation of sodium and water in mitochondria and brain cells, i.e., edema. No significant difference in brain damage and edema was observed between CI and HT rats, suggesting that HT did not aggravate brain edema. ATPase is sensitive to hypoxia brain injury, it catalyzes decomposition of adenosine triphosphate (ATP) to generate energy for the cell. SDH activity is an indication of mitochondria function. Damage in cerebral ischemia could result in decreasing of ATP molecules, accumulation of acidic meta-
bolite and epicyte injury, leading to the reduction of both ATPase activity and SDH activity. The decline progress of ATPase and SDH activity in HT group was slower than that in CI group, and KATP channel blocker Glibenclamide significantly reduced the ATPase and SDH activity in GH group. These results suggested that the opening of KATP channels was required for maintaining a relatively higher ATPase and SDH activity in HT than that in early stage of CI. Additionally, the opening status of the KATP channels in rats of HT group was much longer than that in rats of CI group. It was the activated KATP channels that postponed the brain damage after HT at early stage, and the blood in the infarction that played a similar role as the KATP channel opening stimulator. However, the damage in both CI and HT tissue was irreversible because of the persistence of ischemia. KATP channels were voltage independent, ligand-gated ion channels initially reported in Guinea pig cardiomyocytes (Noma, 1983), and were found present in the membrane of variety of cells, such as β-cells, brain neuron, skeletal muscle, kidney and smooth muscle, and in the inner membrane of mitochondrion. KATP channels could be blocked by sulfonamides (such as tobutamide and glibenclamide), but activated by KATP channel openers (Inagaki et al., 1996). Usually KATP channels are closed, however, they could be activated indirectly by brain ischemia via reducing the ATP molecules and changing the pH in the cell. The activated KATP channels further couple cellular metabolism, membrane potential and excitatory, playing important roles in neuron excitatory, neurotransmitter releasing and postsynaptic membrane function. Activation of KATP channels is a mechanism of self-
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defending for ischemia, but the channels would decay as the ischemia continues, resulting in the loss of metabolic substrates (especially oxygen and glucose) and trans-membranes ion gradients which depolarize the cell membranes. The depolarized neuron membranes favor calcium influx, freeradical generation, lipid peroxidation and glutamate release. Furthermore, the opening of KATP channels can lead to membrane hyperpolarization, which further result in the closure of voltage-dependent calcium channels, the reduction of calcium influx, and the release of glutamate (Ishida et al., 2001; Mitsushige et al., 2001; Yasushi et al., 2003). In contrast, the activation of KATP channels stimulates glutamate transporters for uptaking glutamate (Dai et al., 2006), induces HSP70 expression (Blondeau et al., 2000) but inhibits the expression of some the genes induced by ischemia (Jiang et al., 2004; Liu et al., 2002; Yasushi et al., 2003). The study also found that the invasion of blood in the infarcted territory had a similar role as KATP channel opener by temporarily postponing the brain injury in HT. At early stage of ischemia, the neurobehavioral deficit and brain injury was not intensified by a moderate volume of HT after CI, however, they both were affected by KATP channels. An early report showed that of the 89 CI patients who received Intra-Arterial thrombolysis, only 6 patients (7%) had major symptomatic HT, while 29 patients (33%) had asymptomatic HT (Kidwell et al., 2002). It is the physicians' concern whether HT can change the clinical processes and prognosis. According to CT image, 5 categories were assigned for HT: (1) no hemorrhagic transformation, (2) HI1 (small petechiae), (3) HI2 (more confluent petechiae), (4) PH1 (b30% of the infarcted area with some mild space-occupying effect) and (5) PH2 (N30% of the infarcted area with significant space-occupying effect, or clot remote from infarcted area) (Fiorelli et al., 1999; Trouillas and von Kummer, 2006). Here HI was defined as a petechial infarction without space-occupying effect, and PH was defined as a hemorrhage (coagulum) with mass effect. Compared with no hemorrhagic transformation, the results showed that HI1, HI2 and PH1 did not modify the risk of early neurological deterioration, death and disability, while in placebo group and recombinant tissue plasminogen activator group, PH2 had a devastating impact on early neurological course. Our data was consistent with documented clinical studies, suggested that large hematoma is the only hemorrhagic transformation that may alter the clinical course of ischemic stroke, and moderate hemorrhage after CI did not aggravate the disease.
4.
Experimental procedures
4.1.
Experimental groups
A total of 102 male Sprague–Dawley rats 250 to 280 g in weight were provided by the Hebei Medical University and used in this study. The protocol was approved by the Institutional Animal Care and Use Committee and the local experimental ethics committee. Seventy-eight rats were randomly assigned to four groups. (1) HT group (n = 24 rats): 50 μL autologous blood was injected
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into the ischemic area 30 min after middle cerebral artery occlusion (MCAO); (2) CI group (n = 24 rats): permanent MCAO was to create ischemia; (3) GH (Glibenclamide + HT) group (n = 24 rats): 8 μL Glibenclamide (10 μmol/L) was injected into paracele 30 min before HT; (4) control (n = 6 rats). In experimental group HT, CI and GH, six rats were further randomly assigned to 4 subgroups at time points 3 h, 6 h, 12 h and 24 h after operation. Twelve HT rats and twelve CI rats were used for TTC staining, light microscope and transmission electron microscope observation.
4.2.
Rat models
4.2.1.
Cerebral infarction (CI) model
Rat middle cerebral artery was occluded to create rat CI model as described (Longa et al., 1989). Briefly, in anesthetized rats, the right common carotid artery (CCA), the external carotid artery (ECA) and the internal carotid artery (ICA) were dissected from connective tissue through a midline neck incision. The ECA was ligated and cut, leaving a stump about 3–4 mm attached to the CCA. Next, a curved microvascular clip was placed across CCA and ICA, a 0.2 mm incision was made on the ECA stump. A 5 cm nylon suture with a round end and a mark at 18 mm was introduced into the ECA lumen through the incision. A silk suture at the ECA stump was tightened around the intraluminal nylon suture to prevent bleeding, then the microvascular clip was removed, and the nylon suture was gently advanced from the ECA to the ICA lumen. The suture in the ICA lumen was visible as the mark at 18 mm was reaching the branch of CCA, resistance could be felt when the suture tip reached anterior cerebral and occluded the middle cerebral artery. The suture was then curved slightly, and the wound was sewed up.
4.2.2.
Hemorrhagic transformation (HT) model
30 min after CI, 50 μL arterial blood was injected stereotaxically into the caudate nucleus as illustrated (Hua et al., 2002). After MCAO, rat was positioned in a stereotaxic frame (SR-6N, Japan), a midline incision was made on rat head to expose the Bregma. Then a cranial burr hole (1 mm) was drilled on the skull (coordinates: 0.2 mm anterior, 5.5 mm ventral and 3.5 mm lateral to Bregma). A needle was fixed on the stereotaxic frame to the hole in a vertical position, a midline incision was made on the root segment of the tail to isolate tail artery, 50 μL blood was phlebotomized from the artery and the needle was removed. Then the autologous blood was slowly and stereotactically injected into the infarcted territory through the skull hole in 2 min, to prevent blood flowing back the needle was removed 10 min after injection. The burr hole was sealed with bone max and the wound was sutured, and the animal was placed in a warm box with free access to food and water.
4.2.3.
Glibenclamide + HT (GH) model
30 min before the operation, Glibenclamide was injected slowly into the right lateral ventricle for 1 min. The needle was inserted 4.5 mm in depth at a position 1.2 mm back of and 2 mm right of the middle line of Bregma, then the model was developed as the HT model described above.
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Neurological examination
Three motor functional tests were evaluated at time points 3 h, 6 h, 12 h and 24 h after operation. (1) Longa behavioral test (Longa et al., 1989) was used to evaluate spontaneous contralateral circling and tumble, the results were graded from 0 (no circling) to 4 points (unconsciousness). (2) Berderson behavioral test (Bederson et al., 1986) was used to measure the palsy of contralateral limbs, the score was assigned from 0 (no palsy) to 3 points (circling of the contralateral). (3) Beam walking test (Altumbabic et al., 1998) was employed to exam the ability to walk on a wood beam 80 cm in length and 2.5 cm in width, the data was expressed from 0 (traversed the beam) to 5 points (unable to move or fell off the beam).
4.4.
Water content, ATPase and SDH activity test
After neurofunctional study, rats were sacrificed by overdose of intravenous (i.v.) pentobarbital, rat brain was immediately removed and cut along the blood injection track level which was about 7 mm from the forehead pole. Anterior 2 mm brain coronal slices were placed in preweighed crucibles to obtain their wet weight (WW), followed by incubation for 48 h at 100 °C to determine their dry weight (DW), water content (percentage of water) in the samples were obtained by using the formula (WW − DW) / WW × 100%. The posterior 2 mm brain coronal slices frozen in liquid nitrogen were homogenized in 9 volume cold normal saline on ice, after centrifugation at 1000 rpm for 5 min, the supernatant was used for ATPase and SDH activity test according to the manufacturer's instruction (Nanjing Jiancheng Bioengineering Institute, China). ATPase activity unit was defined as generating 1 μmol phosphorus per milligram protein per hour. SDH activity unit was defined as OD decrease 0.01 per milligram protein per minute. Protein was measured according to Coomassie Brilliant Blue method.
4.5.
Light microscope examination
The anterior 2 mm brain coronal slices were fixed in 4% paraformaldehyde, embedded in paraffin, cut into 6 μm slices, dehydrated in alcohol, stained in hematoxylin and eosin (HE stain) and observed under light microscope.
4.6.
The preparation for TTC staining
2 mm brain coronal slices were incubated at 37 °C for 30 min in 2% TTC in 25 mmol/L potassium phosphate-buffered saline, then fixed and photographed to identify the infarcted and hemorrhagic region.
4.7.
Transmission electron microscope examination
Rats were anesthetized with pentobarbital, perfused with fixing solution (2% paraform and 2.5% glutaraldehyde). Thalamencephalon-basal nucleus slices and temporal-parietal cortex slices were collected to generate ultrathin sections (600–800 nm). Ultrathin sections were mounted to single-hole copper grids and multi-hole grids, stained in uranyl acetate
and citric acid lead, and examined under transmission electron microscope.
4.8.
Statistical analysis
Using software SPSS10.0, Kruskal–Wallis H test and Q test were carried out for neurobehavioral deficit data comparisons, ANOVA and q test were carried out for brain water content, ATPase activity and SDH activity. The significance level was set at P b 0.05.
Acknowledgments This work was funded by Hebei Province, No.:C2006000915; we thank Dr. Zhao-duo Zhang and Dr. Hong-qun Liu for reading the manuscript and providing valuable suggestions.
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