Cardiac-specific methylation patterns of circulating DNA for identification of cardiomyocyte death.

BACKGROUND: Correct detection of human cardiomyocyte death is essential for definitive diagnosis and appropriate management of cardiovascular diseases. Although current strategies have proven utility in clinical cardiology, they have some limitations. Our aim was to develop a new approach to monitor myocardial death using methylation patterns of circulating cell-free DNA (cf-DNA). METHODS: We first examined the methylation status of FAM101A in heart tissue and blood of individual donors using quantitative methylation-sensitive PCR (qMS-PCR). The concentrations and kinetics of cardiac cf-DNA in plasma from five congenital heart disease (CHD) children before and after they underwent cardiac surgery at serial time points were then investigated. RESULTS: We identified demethylated FAM101A specifically present in heart tissue. Importantly, our time course experiments demonstrated that the plasma cardiac cf-DNA level increased quickly during the early post-cardiac surgery phase, peaking at 4-6 h, decreased progressively (24 h) and returned to baseline (72 h). Moreover, cardiac cf-DNA concentrations pre- and post-operation were closely correlated with plasma troponin levels. CONCLUSIONS: We proposed a novel strategy for the correct detection of cardiomyocyte death, based on analysis of plasma cf-DNA carrying the cardiac-specific methylation signature. Our pilot study may lead to new tests for human cardiac pathologies.

An applied anatomical study of bronchial artery.

The aim of this study was to reveal the external features of the bronchial artery (BA) system, so as to provide morphological basis for clinic. The BAs in 48 adult cadavers were dissected and analyzed. The number of BAs in 48 cases was 118. The incidence of BA arising from thoracic aorta, right posterior intercostal artery, and right subclavian artery was 69.49, 27.12, and 3.39%, respectively. The origin of BAs in individual specimen might be single, two, or all of them, respectively. According to the different origin and/or origins of BAs, it could be divided into five categories. As for the course of BAs, in this study, all the left BAs arising from thoracic aorta passed forward around the left side of esophagus and then entered left pulmonary hilum; most (n = 15) of the right BAs arising from thoracic aorta passed forward around the left side of esophagus and then entered right pulmonary hilum; a few (n = 8) of the right BAs arising from thoracic passed forward the right side of esophagus and bronchus and then entered right pulmonary hilum. Besides, in our group, the special courses were that right intercostal-bronchial trunk (RICBT) arising from thoracic aorta passed between vertebra and esophagus and gave off BA which curved forward around the right side of esophagus and then entered right pulmonary hilum, common bronchial trunk (CBT) arising from thoracic aorta passed forward around the left side of esophagus laying anterior to bronchus or posterior to bronchus, then dividing into a left and a right BAs entering right and left pulmonary hilum, respectively. In 4 cadavers, the RICBT gave off the radiculomedullary artery and BA in turn, so radiculomedullary artery has the same origin with BA. Of all BAs, the mean diameter of right posterior intercostal artery, CBT, left BA, and right BA was 2.17 ± 0.84, 1.79 ± 0.57, 1.44 ± 0.50, and 1.39 ± 0.38 mm, respectively. The information gained from this study will be of value in clinic application.

Hyperosmotic induction of aquaporin expression in rat astrocytes through a different MAPK pathway.

Water homeostasis of the nervous system is important during neural signal transduction. Astrocytes are crucial in water transport in the central nervous system under both physiological and pathological conditions. To date, five aquaporins (AQP) have been found in rat brain astrocytes. Most studies have focused on AQP4 and AQP9, however, little is known about the expression of AQP3, -5, and -8 as well as their regulating mechanism in astrocytes. The expression patterns of AQP3, -5, and -8 in astrocytes exposed to hyperosmotic solutions were examined to clarify the roles of AQP3, -5, and -8 in astrocyte water movement. The expression of AQP4 and AQP9 under the same hyperosmotic conditions was also investigated. The AQP4 and AQP9 expressions continuously increased until 12 h after hyperosmotic solution exposure, whereas the AQP3, -5, and -8 expressions continued to increase until 6 h after hyperosmotic solution exposure. The different AQPs decreased at corresponding time points (24 h for AQP4 and AQP9; 12 h for AQP3, -5, and -8 after hyperosmotic solution exposure). The ERK inhibitor can attenuate the expression of AQP3, -5, and -8 after hyperosmotic solution exposure. The p38 inhibitor can inhibit the AQP4 and AQP9 expressions in cultured astrocytes. AQP expression is directly related to the extracellular hyperosmotic stimuli. Moreover, different AQPs can be regulated by a distinct MAPK signal transduction pathway.

Expression of aquaporin8 in human astrocytomas: correlation with pathologic grade.

Aquaporin8 (AQP8), a member of the aquaporin (AQP) protein family, is weakly distributed in mammalian brains. Previous studies on AQP8 have focused mainly on the digestive and the reproductive systems. AQP8 has a pivotal role in keeping the fluid and electrolyte balance. In this study, we investigated the expression changes of AQP8 in 75 cases of human brain astrocytic tumors using immunohistochemistry, Western blotting, and reverse transcription polymerase chain reaction. The results demonstrated that AQP8 was mainly distributed in the cytoplasm of astrocytoma cells. The expression levels and immunoreactive score of AQP8 protein and mRNA increased in low-grade astrocytomas, and further increased in high-grade astrocytomas, especially in glioblastoma. Therefore, AQP8 may contribute to the proliferation of astrocytomas, and may be a biomarker and candidate therapy target for patients with astrocytomas.

An applied anatomical study on the recurrent laryngeal nerve and inferior thyroid artery.

PURPOSE: The aim of this study was to provide some important information about the morphology and topography of the recurrent laryngeal nerve (RLN) and inferior thyroid artery (ITA), which significantly helps localize and protect the RLN in neck surgery, especially in thyroid surgery. METHODS: Eighty adult cadavers (160 sides) fixed with formalin were dissected, analyzed and measured. RESULTS: (1) 87.5% of the RLNs gave off multiple branches like a tree; the incidence of the RLN loop, connecting one branch to another was 3.125%; in 9.375%, one branch of RLN combined with cervical sympathetic chain (CSC) or superior laryngeal nerve (SLN). (2) A double RLN appeared in four sides, a non-recurrent inferior laryngeal nerve appeared in two cases. (3) In two cases, the RLN communicated with both of the SLN and the CSC near thyroid gland. (4) Most of the ITAs was derived from thyrocervical trunk, and divided into two or three branches before entering the thyroid gland. (5) Three ITAs gave off esophageal branch, one ITA gave off tracheal branch, one right ITA originated abnormally. (6) On the left side, the RLN was behind the ITA in 86.25% of the cases, in front of the artery in 7.5%, the nerve was between artery branches in 2.5%, the artery was between nerve branches in 1.25%, and was among the combined in 2.5%. On the right side, the RLN was in front of the artery in 75.0%, behind the artery in 10.0%, among the branches of the artery in 5.0%, 10.0% the branches of both nerves and artery were interlaced that the relationship between the branches of the nerve and the artery was uncertain. CONCLUSIONS: Because of the variability of the RLN and ITA and the complicated relationship between them, it is necessary to dissect and recognize the RLN to avoid mistaking, ignoring, and misligating of the nerve before ligating the ITA.

Superior laryngeal nerve loop: patterns and surgical implications.

This study clarifies the patterns of the superior laryngeal nerve loop (SLN loop), connecting the cervical sympathetic chain (CSC) and the SLN and its branches, so as to provide an anatomic basis for decreasing the risk of injury to the external laryngeal nerve (ELN) during neck surgery. Fifty Chinese adult human cadavers fixed with 4 % formalin were dissected, and their SLN loop patterns were analyzed and summarized. In 98 of 100 sides the CSC anastomosed with the SLN and its branches, forming a looped nerve structure which we called the SLN loop. The SLN loops could be divided into five types: e ( n ), t ( n ), i ( n ), t ( n ) e ( n ), and i ( n ) e ( n ) based on morphological variations. The results demonstrated that e ( n ) was most frequently found in the samples (82/100) followed by t ( n ) (9/100), i ( n ) (3/100), t ( n ) e ( n ) (2/100), and i ( n ) e ( n ) (2/100). Comparing with the previous work, we identified additional 18 subtypes of the SLN loop. The relations of the SLN loop to the surrounding structures were complicated, which brought more challenges to thyroidectomy. Thus, we do not advocate routine identification of ELN/ELN loop during the process of thyroidectomy, especially systematic identification of ELN during operation. However, this study introduces the possibility that nerve injury can be avoided by exposure of the nerve via careful dissection in the region of the superior pole of the thyroid gland to the extent that we can initiate individual ligation of the superior polar vessels, along with the help of neuromonitors, video monitors, and magnifying loupes.

Curcumin Ameliorates Memory Deficits by Enhancing Lactate Content and MCT2 Expression in APP/PS1 Transgenic Mouse Model of Alzheimer's Disease.

Curcumin is a natural product with several anti-Alzheimer's disease (AD) neuroprotective properties. This study aimed to investigate the effects of curcumin on memory deficits, lactate content, and monocarboxylate transporter 2 (MCT2) in APP/PS1 mouse model of AD. APP/PS1 transgenic mice and wild-type (WT) C57BL/6J mice were used in the present study. Spatial learning and memory of the mice was detected using Morris water-maze test. Cerebral cortex and hippocampus lactate contents were detected using lactate assay. MCT2 expression in the cerebral cortex and hippocampus was examined by immunohistochemistry and Western blotting. Results showed that spatial learning and memory deficits were improved in curcumin-treated APP/PS1 mouse group compared with those in APP/PS1 mice group. Brain lactate content and MCT2 protein level were increased in curcumin-treated APP/PS1 mice than in APP/PS1 mice. In summary, our findings indicate that curcumin could ameliorate memory impairments in APP/PS1 mouse model of AD. This phenomenon may be at least partially due to its improving effect on the lactate content and MCT2 protein expression in the brain. Anat Rec, 302:332-338, 2019. © 2018 Wiley Periodicals, Inc.

Role of rno-miR-124-3p in regulating MCT1 expression in rat brain after permanent focal cerebral ischemia.

This study aimed to assess the role of microRNAs (miRNAs) in regulating monocarboxylate transporter-1 (MCT1) expression in rat brain after permanent focal cerebral ischemia to identify a new target for early treatment of cerebral ischemia. Focal cerebral ischemia was induced by permanent middle cerebral artery occlusion (pMCAO) in rats. Morphology and protein expression levels of MCT1 were assessed by immunofluorescence and Western blotting. Using bioinformatics and double luciferase reporter assays, rno-miR-124-3p was selected as a direct target for rat MCT1. Expression of rno-miR-124-3p after pMCAO was detected. Then, rats were treated with rno-miR-124-3p agomir via lateral ventricle injection, and after 6 h or 24 h ischemia, rno-miR-124-3p expression and gene and protein expression of MCT-1 were detected by qRT-PCR and Western blotting. Brain infarction was identified by 2, 3, 5-triphenyltetrazolium chloride (TTC) staining. Results showed that pMCAO induced brain infarction and increased the expression of MCT1. The levels of rno-miR-124-3p after pMCAO were in contrast to those of MCT1 protein in ischemic region, while declined after 3, 6 and 12 h of pMCAO in ischemic penumbra. After administration of rno-miR-124-3p agomir, MCT1 mRNA and protein levels were increased after 6 h of pMCAO, while decreased after 24 h of pMCAO. Meanwhile, rno-miR-124-3p levels increased after both times. TTC staining showed treatment with rno-miR-124-3p agomir reduced brain infarction. The role of rno-miR-124-3p in regulating MCT1 was as a positive regulator after 6 h of pMCAO, while a negative regulator after 24 h of pMCAO, however, both activities had protective effects against cerebral ischemia.

Expression of Kir 4.1 in human astrocytic tumors: correlation with pathologic grade.

Inward rectifying potassium channels (Kir), and in particular Kir 4.1, are key regulators of glial functions, which in turn determine neuronal excitability and axonal conduction. A high expression of Kir 4.1 has been detected in in some pathological conditions. In this study, we investigated the expression of Kir 4.1 mRNA and protein in 80 cases of human astrocytic tumors by reverse transcription polymerase chain reaction, Western blot and immunohistochemistry, respectively. The correlation between Kir 4.1 expression and pathologic grade of astrocytic tumors was further analyzed. The results showed that the expression of Kir 4.1 mRNA and protein, as well as the Kir 4.1 immunoreactivity score (IRS), increased markedly with increasing pathologic grade. Therefore, Kir 4.1 may be a new biomarker for astrocytic tumors. It may also be an attractive therapy target for human astrocytic tumors.

Expression of livin and vascular endothelial growth factor in different clinical stages of human esophageal carcinoma.

AIM: To investigate the role of livin and vascular endothelial growth factor (VEGF) in human esophageal carcinoma, and analyze its relationship to clinical stages. METHODS: Expression of livin in fresh esophageal cancer tissues was detected by immunohistochemistry (IHC), Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR), and VEGF by Western blotting and RT-PCR. All statistical analyses were performed by SPSS version 11.0. RESULTS: Livin positivity was also significantly correlated with tumor stages, increasing with tumor progression. Expression of livin and VEGF increased with the process of esophageal carcinoma. In the fourth clinical stage, expression of livin and VEGF was the most significant. Expression of Livin was positively correlated with VEGF. CONCLUSION: Over-expression of livin and VEGF contributes to the pathogenesis of esophageal carcinoma.

[Apoptosis and caspase-12 expression in progressive compressive spinal cord injury: experiment with rats].

OBJECTIVE: To investigate the apoptosis and caspase-12 expression in progressive compression of spinal cord (PCSC). METHODS: 120 adult Wistar rats were randomly divided into 2 equal groups, experimental group, undergoing operation so as to establish PCSC models, and control group, undergoing sham operation. 1, 3, 7, 14, 21, and 28 days after the operation, 5 rats from each group were anesthetized with their spinal cords taken out. TUNNEL method was used to observe the apoptosis of the neurons in the compressed segments. Another 5 rats from each group at different time points were anesthetized with their spinal cords as well. Immunohistochemistry was used to detect the mRNA expression of caspase-12 in the compressed segments. Western blotting was used to detect the protein expression of caspase-12. RESULTS: The apoptotic rates of neurons and gliocytes 1, 3, 7, 14, 21, and 28 days after were 12.5% +/- 2.3%, 13.0% +/- 3.6%, 17.2% +/- 4.3%, 29.4% +/- 4.4%, 36.1% +/- 6.5%, and 2.3% +/- 7.9% respectively, with significant differences among the values 14, 21, and 28 days and those at other time points after the operation (all P < 0.05). Immunohistochemistry showed that the number of caspase-12 positive neurons increased since 1 day after, and became remarkably high 14, 21, and 28 days after with significant differences among different time points (all P < 0.05). Western blotting showed that the protein expression of caspase-12 was low 1, 3, and 7 days after, and peaked 14 days after, and then gradually decreased, however, the expression levels 21 and 28 days after were still significantly higher then those 1, 3, and 7 days after (all P < 0.05). CONCLUSION: Caspase-12 is involved in the apoptosis of neurons in PCSC.

[Changes in noradrenergic neurons in the rat spinal cord after brain injury].

OBJECTIVE: To investigate the impact of experimental brain injury on the noradrenergic (NA) neurons in the spinal cord, in order to explore possible mechanism of the changes in NA neuron expression. METHODS: Seventy healthy Wistar rats were randomly divided into two groups: normal group (n=10), brain injury (BI) group (n=60). The animals in BI group were subjected to BI with the modified Feeney's method. The spinal cords of rats were harvested at 3, 6, 12, 24, 48 and 72 hours after the injury, respectively. Immunohistochemistry (IHC) and reverse transcriptase-polymerase chain reaction (RT-PCR) were used to assess the changes in dopamine-beta-hydroxylase (DBH) expression. RESULTS: IHC showed a small number of DBH-positive neurons distributed in anterior horn of the cervical and lumbar enlargements in control group; a larger number of dark-stained noradrenergic neurons were found in anterior horn, and also in posterior and lateral horns of spinal cord in brain injury group at different time points after injury, as compared with control group (P<0.05 or P<0.01). RT-PCR showed that the expression of DBH mRNA in brain injury group was significantly higher than that in control group at different time points after injury (P<0.05 or P<0.01). CONCLUSION: A significant increase of NA neurons in spinal cord of brain injury group suggests that more NA is synthesized by the neurons, which is necessary to meet the demand of the organism under the stress condition, suggesting that NA plays a very important role in the course of brain injury.

p53-Mediated oligodendrocyte apoptosis initiates demyelination after compressed spinal cord injury by enhancing ER-mitochondria interaction and E2F1 expression.

Oligodendrocyte apoptosis mediated demyelination is a pathological change characteristic of compressed spinal cord injury (CSCI). However, the mechanism of demyelination due to oligodendrocyte apoptosis is not known. In this study, after successfully establishing a rat CSCI model using a custom-made compressor, we investigated the pathological changes, MBP expression, as well as apoptosis-related protein (p53, active caspase-3) expression to determine whether or not apoptosis and demyelination occurred after injury. To understand the possible mechanism of oligodendrocyte apoptosis, caspase-12 and cytochrome C were analyzed to explore the relationship between oligodendrocyte apoptosis and endoplasmic reticulum(ER)-mitochondria interaction. The transcription factor, E2F1, was also detected by immunofluorescence and Western blot assays. The results showed that CSCI increased the expression levels of p53, E2F1 and active caspase-3 followed by the swelling and breakdown of myelin sheaths. The number of myelinated nerve fibers also decreased with down-regulated expression of MBP. Expression levels of caspase-12 and cytochrome C were enhanced along with a reduction in the number of oligodendrocytes. After treatment of CSCI in rats with Pifithrin-µ(PFT-µ), a specific inhibitor of p53, pathomorphological changes of myelin sheath improved significantly. Expression levels of E2F1, active caspase-3, caspase-12 and cytochrome C were down-regulated, consistent with reduced the number of apoptotic oligodendrocytes. These results demonstrated that over-expression of p53 could mediate oligodendrocyte apoptosis thus resulting in demyelination in two ways; by enhancing ER-mitochondria interaction and by triggering the E2F1 mediated apoptosis pathway.

Anatomical Characteristics and Biomechanical Properties of the Oblique Popliteal Ligament.

This anatomical study sought to investigate the morphological characteristics and biomechanical properties of the oblique popliteal ligament (OPL). Embalmed cadaveric knees were used for the study. The OPL and its surrounding structures were dissected; its morphology was carefully observed, analyzed and measured; its biomechanical properties were investigated. The origins and insertions of the OPL were relatively similar, but its overall shape was variable. The OPL had two origins: one originated from the posterior surface of the posteromedial tibia condyle, merged with fibers from the semimembranosus tendon, the other originated from the posteromedial part of the capsule. The two origins converged and coursed superolaterally, then attached to the fabella or to the tendon of the lateral head of the gastrocnemius and blended with the posterolateral joint capsule. The OPL was classified into Band-shaped, Y-shaped, Z-shaped, Trident-shaped, and Complex-shaped configurations. The mean length, width, and thickness of the OPL were 39.54, 22.59, and 1.44 mm, respectively. When an external rotation torque (18 N·m) was applied both before and after the OPL was sectioned, external rotation increased by 8.4° (P = 0.0043) on average. The OPL was found to have a significant role in preventing excessive external rotation and hyperextension of the knee.

Lost Polarization of Aquaporin4 and Dystroglycan in the Core Lesion after Traumatic Brain Injury Suggests Functional Divergence in Evolution.

OBJECTIVE: To understand how aquaporin4 (AQP4) and dystroglycan (DG) polarized distribution change and their roles in brain edema formation after traumatic brain injury (TBI). METHODS: Brain water content, Evans blue detection, real-time PCR, western blot, and immunofluorescence were used. RESULTS: At an early stage of TBI, AQP4 and DG maintained vessel-like pattern in perivascular endfeet; M1, M23, and M1/M23 were increased in the core lesion. At a later stage of TBI, DG expression was lost in perivascular area, accompanied with similar but delayed change of AQP4 expression; expression of M1, M23, and DG and the ratio of M1/M2 were increased. CONCLUSION: At an early stage, AQP4 and DG maintained the polarized distribution. Upregulated M1 and M23 could retard the cytotoxic edema formation. At a later stage AQP4 and DG polarized expression were lost from perivascular endfeet and induced the worst cytotoxic brain edema. The alteration of DG expression could regulate that of AQP4 expression after TBI.

An applied anatomical study on the external laryngeal nerve loop and the superior thyroid artery in the neck surgical region.

This study was conducted to investigate the topographic relationship between the external laryngeal nerve (ELN) loop and the superior thyroid artery (STA), in order to provide the anatomical foundations for protecting the ELN during surgery. In the present study, 48 adult human cadavers were dissected and analyzed. For the 21 (21.9%) low-position ELN loops observed, the neurovascular relationship between the STA and the nerve was classified into four types: (1) the artery overlapped the nerve; (2) the artery passed through the ELN loop; (3) the muscular branch of the ELN loop and the laryngeal branch of the STA coursed together; and (4) the branches of the STA and the ELN loop were interlaced. Our study suggested that the patterns of ELN loops are so complicated that they have not been statistically defined in any previous study, which should be kept in mind when attempting to protect the nerve from injury. Also, because of the variable morphology of the ELN loop and its complicated topographic relationship to the STA, the vessels should be individually isolated and then ligated during thyroidectomy. When ligating the laryngeal branch of the STA during larynx surgery, special attention should be paid to avoiding damage to the muscular branch of the ELN/ELN loop.

Loss of AQP4 polarized localization with loss of ß-dystroglycan immunoreactivity may induce brain edema following intracerebral hemorrhage.

The aquaporin-4 (AQP4) water channel contributes to brain water homeostasis in perivascular and subpial membrane domains of astrocytes where it is concentrated. These membranes form the interface between the neuropil and the extracellular liquid spaces. The brain-selective deletion of the dystroglycan (DG) gene causes a disorganization of AQP4 on the astroglial endfeet. First, we analyzed the expression of AQP4, ß-DG in the brain following intracerebral hemorrhage (ICH) and correlated AQP4 expression with the expression pattern of the ß-DG, which is a component of dystrophin-dystroglycan complex (DDC). Besides, the vessels ultrastructure and brain water content were investigated at different time points post-ICH (day 1, day 3, day 7). We found that AQP4 polarity was disturbed in parallel with the loss of ß-DG in the perihematomal area post-ICH. At day 1 post-ICH, brain edema was obvious and the damage of vascular ultrastructure was the most severe. These results suggest a role for ß-DG in targeting and stabilizing AQP4 channel in astrocytic cells, which may be critical for water homeostasis in brain.

Internalization of aquaporin-4 after collagenase-induced intracerebral hemorrhage.

Brain edema formation following intracerebral hemorrhage (ICH) appears to be related with aquaporin-4 (AQP4), which is critically involved in brain volume homeostasis and water balance. Despite its importance, the regulation of AQP4 expression involved in transmembrane water movements still remains rudimentary. Many studies suggest that the internalization of several membrane-bound proteins, including AQP4, may occur with or without lysosomal degradation. Previously, we investigated the internalization of AQP4 in retinal ischemic-reperfusion model. Here, we test the hypothesis that AQP4 is internalized post-ICH and then degraded in the lysosome. The results demonstrated that both AQP4 and the mannose-6-phosphate receptor (MPR) co-localized in perihematomal region at 6 hr post-ICH. In addition, AQP4 and lysosomal-associated membrane protein 1 (LAMP1) also co-localized in perihematomal region, with co-expression increasing followed by a gradual decrease at different time windows post-ICH (6, 12, 24, 48, and 72 hr). After ICH, the Evans blue leakage happened very early at 1 hr and the brain swelling occurred at 3 hr. Moreover, we also found the AQP4 mRNA and AQP4 protein were increased post-ICH. These results suggest that AQP4 is internalized and the lysosome is involved in degrading the internalized AQP4 post-ICH. Both the AQP4 internalization and lysosomal degradation may provide biophysical insights regarding the potential of new treatments for brain edema.

A correlative study between AQP4 expression and the manifestation of DWI after the acute ischemic brain edema in rats.

OBJECTIVE: To investigate the rule of the aquaporin-4 (AQP4) expression in acute ischemic brain edema, and to study the correlation between AQP4 expression and diffusion-weighted imaging (DWI). METHODS: Thirty-six Wistar rats were divided into 2 groups randomly, control group (n = 12) and operation group (n = 24) in which right middle cerebral artery of each animal had been occluded unilaterally (MCAO) at interval times of: 15 minutes, 30 minutes, 1 hours, 3 hours, 6 hours and 24 hours, respectively. The operation process of the control group was the same as the operation group except for the MCAO. All groups were examined using DWI. The apparent diffusion coefficient (ADC), relative density (rd) and relative area (rs) of the biggest hyperintensity signal layer on DWI were measured. After that the animals were sacrificed and perfused with the mixture solution consisting of TTC. The biggest layers of the ischemic cerebral tissues in each rat corresponding to the DWI were stained with TTC and examined with immunochemistry (DeltaS), in situ hybridization (alpha) and histology. RESULTS: There was no significant change in the control group. In the operation group, a hyperintensity signal was found in the DWI of the right MAC territory at 15 minutes after MCAO. The ADC value decreased quickly within one hour after MCAO, while the AQP4 expression, rd-DWI and rs-DWI increased rapidly during this stage. As time progressed, the ADC value decreased further to (2.1 +/- 0.6) x 10(-4) mm(2)/s at 3 hours, and then began to increase slowly till 24 hours. But the AQP4 expression (DeltaS and alpha) and rd as well as the rs continuously increased slowly between 1 hour and 6 hours after MCAO, followed a peak after 6 hours. The AQP4 expression (alpha) showed a positive relationship with the rs-DWI, they all presented two peaks and a plateau. The corresponding sequential pathologic changes were a gradual increase of intracellular edema (within one hour), then an emergence of vasogenic edema (1-6 hours), and final necrosis and liquefaction (6 - 24 hours). CONCLUSIONS: Upregulated expression of AQP4 may play a significant role in acute ischemic brain edema, especially during the stages of intracellular edema and necrosis, but it has no correlation to vasogenic edema. Certainly, the high expression of AQP4 is perhaps one of the most important reasons of the decrease of ADC and hyperintensity on the DWI in the intracellular edema.

[Effect of hypotonic medium on expression of aquaporin-4 in astrocytes].

OBJECTIVE: To investigate the expression of aquaporin-4 (AQP4) and its role in cultured rat astrocytes after exposure to hypotonic medium. METHODS: Rat cerebral cortical astrocytes from 2 days newborn Wistar rats were undergone the pure culture. The cells were divided into control group and hypotonic groups randomly, in which group was examined at interval times of 3, 6, 12 and 24 h, respectively (n = 6), and its model of cell edema was established by exposed to hypotonic medium. The cells of the control group were cultured in normal culture medium. Inverted microscope and transmission electron microscope were used to observe the cells changes. The reactivity and rules of AQP4mRNA and protein expression of astrocyte to hypotonic medium were studied by RT-PCR, immunocytochemistry, the activity of LDH and imaging analysis. RESULTS: There was no significant change in the control group. After astrocytes cultured for 3, 6, 12, 24 h with hypotonic medium (268, 254, 240 mmol/L), the astrocytes showed typical features of cell edema, especially in lower osmolality group. The activity of LDH showed a significant change than those in the control group after astrocytes exposed to hypotonic medium (P < 0.05). RT-PCR and immunocytochemistry analysis confirmed that the expression of AQP4mRNA and protein was remarkably increased (P < 0.05), up-graduated expression of AQP4 was correlated with hypotonic medium and time dependent manner. The AQP4mRNA expression showed a positive relationship with the AQP4 protein expression during the astrocytes edema formation (r > 0.8369, P < 0.05). CONCLUSION: Hyponotic medium could induce cell edema and expression of AQP4mRNA and protein increased, and vibility of astrocytes inhibited. The AQP4 expression showed a positive relationship with the osmolality. The results suggest that AQP4 participated in the brain edema formation.