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The Inflammation in the Gut After Experimental Subarachnoid Hemorrhage
Journal of Surgical Research 137, 103–108 (2007) doi:10.1016/j.jss.2006.06.023
The Inflammation in the Gut After Experimental Subarachnoid Hemorrhage Meng-liang Zhou, M.D.,* Lin Zhu, M.D.,* Jian Wang, M.D.,† Chun-hua Hang, M.D., Ph.D.,* and Ji-xin Shi, M.D., Ph.D.*,1 *Department of Neurosurgery, †Department of General Surgery, Jinling Hospital, School of Medicine, Nanjing University, Nanjing, Jiangsu Province, China Submitted for publication March 7, 2006
Background. Gastrointestinal dysfunction could be frequently observed in the patients suffering from SAH. This study test the hypothesis that experimental SAH could induce histopathological changes and inflammatory response associating with NF-B activation pathway in the gut. Materials and methods. A total of 17 rabbits were randomly divided into two groups: control group (n ⴝ 8) and SAH group (n ⴝ 9). In the SAH group, the animals were subjected to experimental SAH according to the “two-hemorrhage” method. The histopathological study was performed to detect the intestinal mucosal morphological changes and immunohistochemical study was used to detect the TNF-␣ and ICAM-1 expressions. NF-B binding activity was measured using the electrophoretic mobility shift assay. Results. It was demonstrated that some damage changes and leukocytes infiltration occurred in the intestinal mucosa after SAH. More positive cells for TNF-␣ and ICAM-1 were observed in the SAH group. The NF-B binding activity in the intestines was significantly increased in the SAH group (P < 0.01). Conclusions. The results of the present study suggest that SAH in the rabbits could induce NF-B and proinflammatory cytokines activation in the intestine, which is associated with morphological changes. © 2007 Elsevier Inc. All rights reserved.
Key Words: subarachnoid hemorrhage; intestine; inflammation; nuclear factor-B. INTRODUCTION
Cerebrovascular diseases are the third leading cause of death in developed countries, behind only the coro1 To whom correspondence and reprint requests should be addressed at Department of Neurosurgery, Jinling Hospital, 305 East Zhongshan Road, Nanjing 210002, P. R. China. E-mail: [email protected].
nary artery disease and all type of cancers [1]. More than 20% of these deaths are because of aneurysmal subarachnoid hemorrhage (SAH) [2]. Cerebral vasospasm is the significant cause of morbidity and mortality in patients suffering from aneurysmal SAH. Cerebral vasospasm could result in brain ischemia and infarction which account for over one third of the total cases of disability and death [3]. In addition, non-neurologic complications also add to morbidity and mortality after SAH [3, 4]. Studies on nonneurologic complications associated with SAH in the literature concentrated on the pulmonary edema and pneumonia [5, 6], cardiac arrhythmia [7–11], electrolyte disturbance [12], and hematologic change [13, 14]. Besides those complications, gastrointestinal dysfunction could be frequently observed in the patients suffering from SAH. Dysfunction of the gastrointestinal tract leads to some symptoms such as gastrointestinal bleeding, gastric reflux, and decreased intestinal peristalsis, which could influence the outcome after SAH [15, 16]. Our previous study demonstrated that acute damage and inflammatory responses occurred in intestinal mucosa after traumatic brain injury and which might mediate mainly by up-regulation of nuclear factor-B (NF-B) pathway [17–19]. Also, some reports also indicated that the function of the intestines was influenced following the injury of the other organ because of many reasons, such as taking little food and stress after injury [20, 21]. The early enteral nutrition after brain injury, including after SAH, is necessary to maintain and improve the function of the intestines [22]. However, the effect of SAH on intestines has not been studied to date. NF-B is a pleiotropic transcription factor and plays a key role in the intestinal immune system [23]. NF-B
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TABLE 1
Tissue Harvest
Appetite Scores
On day 5, the rabbits were again anesthetized with an intramuscular injection of a mixture of ketamine (40 mg/kg) and droperidol (2.5 mg/kg) and killed by exsanguination. A 3-cm segment of the mid-ileum was taken and flushed with ice-cold saline. Half of it was immersed in the 10% buffered formalin for histopathological studies and the other was stored in liquid nitrogen immediately for electrophoretic mobility shift assay (EMSA).
The amount of the food eaten by the animal (standard food normally eaten by the animal in one day)
Score
1.0 ⬎0.5 ⬎0.2 ⬍0.2
3 2 1 0
members control transcriptional activity of various promoters of proinflammatory cytokines, cell surface receptors, transcription factors, and adhesion molecules that are involved in intestinal inflammation [23, 24]. In the present study, we investigated the alterations of intestinal mucosal morphology and the inflammation response involving NF-B pathway. MATERIALS AND METHODS Animal Preparation All procedures in animals were approved by the Animal Care and Use Committee of Nanjing University and conformed to Guide for the Care and Use of Laboratory Animals from National Institutes of Health. Seventeen adult male New Zealand White (NZW) rabbits weighing from 2.4 to 2.8 kg were purchased from the Animal Center of the Chinese Academy of Sciences (Shanghai, China). The rabbits were acclimated in a temperature and humidity-controlled room and maintained on the standard pellet diet at the Animal Center of Jinling Hospital for 10 days before the experiment. The temperature in both the feeding room and the operation room was maintained at about 25°C.
Rabbit Models of SAH The rabbits were randomly assigned to two groups. Animals in group 1 served as controls and intracisternal saline injection was performed (n ⫽ 8). The animals in group 2 were subjected to experimental SAH (n ⫽ 9). In groups 2, experimental SAH was produced according to the “two-hemorrhage” method. The rabbits were anesthetized with an intramuscular injection of a mixture of ketamine (25 mg/kg) and droperidol (1.0 mg/kg) on day 0. A 21-gauge butterfly needle was percutaneously placed in the cisterna magna. After withdrawal of 1.5 ml of the cerebrospinal fluid (CSF), 3 mL of non-heparinized fresh autologous auricular arterial blood was slowly injected into the cisterna magna for 1 min under aseptic technique. The animals were then kept in a 30 degree head-down position for 30 min. After recovery from anesthesia, they were returned to the feeding room. Forty-eight hours after the first SAH, a second one was produced in the same manner as the first. In the animals in the control group, the same technique was applied, with injection of sterile saline instead of blood.
Histopathological Examination The 10% buffered formalin-fixed jejunum was embedded in paraffin, sectioned at 4 m thickness with a microtome and stained with hematoxylin and eosin (H&E). The sections were examined under light microscope.
Immunohistochemical Assay The coronal sections were embedded in paraffin and sectioned again at 4 m thickness with a microtome. The antibodies (antiTNF-␣ and anti-ICAM-1, both purchased from Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were diluted with 1% bovine serum albumin/phosphate-buffered solution (BSA/PBS) (BSA, 1 g, pH 7.4, 0.01 mol/L, 100 mL) by a certain ratio (anti-TNF-␣ diluted 1:100 and anti-ICAM-1 diluted 1:200). The sections were incubated with the diluted antibodies overnight at 4°C respectively, washed, and blocked with 1.6% H 2O 2 in phosphate-buffered saline (PBS) for 10 min. After washing with PBS, sections were incubated with HRPconjugated goat anti-rabbit IgG (diluted 1:500) for 60 min at room temperature. DAB was used as chromogen and counterstaining was done with hematoxylin. Six views were selected randomly for each section and observed under a light microscope (⫻100). Then mean number of reactive cells in the six views was regarded as the data for each sample.
Nuclear Protein Extracts and EMSA Nuclear protein of intestinal tissue was extracted and quantified as described [17]. Briefly, frozen intestinal tissues were homogenized in 0.8 mL ice-cold Buffer A composed of 10 mM HEPES pH 7.9, 10 mM KCl, 2 mM MgCl 2, 0.1 mM EDTA, 1 mM dithiothreitol (DTT), and 0.5 mM phenylmethylsulfonyl fluoride (PMSF) (all from Sigma Chemical Co., St. Louis, MO). Then the homogenates were incubated on ice for 20 min, and vortexed for 30 s after addition of 50 L NP-40 (Sigma Chemical Co.). Then the mixture was centrifuged for 10 min (5,000 ⫻ g, 4°C). The pellet was then suspended in 50 L ice-cold buffer B [50 mM HEPES pH 7.9, 50 mM KCl, 300 mM NaCl, 0.1 mM EDTA, 1 mM
Appetite Evaluation Appetite scores were recorded by independent observations of a veterinarian who was blinded to the study. The scores of the animals in SAH group were recorded daily by using the modified scoring table (Table 1).
FIG. 1. Graph showing the appetite scores of the rabbits in SAH group. The scores were reduced after blood injection into the cisterna magna and peaked on the day 3. There is a significant difference between the scores on each day after first blood injection and the scores on day 0 (**P ⬍ 0.01).
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FIG. 2. Photomicrographs showing histopathological changes in the SAH group compared with control group. (A) The normal intestinal morphology of the control animals (H&E; original magnification, ⫻40). (B) The histopathoglogical changes in the intestines of the rabbits in the SAH group. These changes include shedding of the epithelial cell from the top of villi, epithelial cell necrosis, disarrangement of villi, fusion of adjacent villi into piece, mucosal atrophy, and leukocytes infiltration (H&E; original magnification, ⫻40). (Color version of figure is available online.)
DTT, 0.5 mM PMSF, with 10% (v/v) glycerol] and incubated on ice for 30 min with frequent mixing. After centrifugation (12,000 ⫻ g, 4°C) for 15 min, the supernatants were collected as nuclear extracts and stored at ⫺80°C until use. EMSA was performed using a commercial kit (Gel Shift Assay System; Promega, Madison, WI). Consensus oligonucleotide probe (5=-AGT TGA GGG GAC TTT CCC AGG C-3=) was end-labeled with [␥-32P]ATP (Free Biotech., Beijing, China) with T4polynucleotide kinase. Nuclear protein (30 g) was preincubated in a total volume of 9 L in a binding buffer, consisting of 10 mM Tris–HCl (pH 7.5), 4% glycerol, 1 mM MgCl 2 , 0.5 mM EDTA, 0.5 mM DTT, 0.5 mM NaCl, and 0.05 mg/mL poly(di-dc) for 10 min at room temperature. After addition of the 32P-labled oligonucleotide probe, the incubation was continued for 20 min at room temper-
ature. Reaction was stopped by adding 1 L of gel loading buffer and the mixture was subjected to nondenaturing 4% polyacrylamide gel electrophoresis in 0.5⫻TBE buffer (Tris-borate-EDTA). After electrophoresis was conducted at 390 V for 1 h, the gel was vacuum-dried and exposed to X ray film (Fuji Hyperfilm, Tokyo, Japan) at ⫺70°C with an intensifying screed.
Statistical Analysis Software SPSS 11.0 was used for the statistical analysis. To the appetite scores, the Mann-Whitney t-test was used to compare the scores of each day from day 1 to day 5 with day 0. The other measurements were analyzed by Student’s t-test. All data were presented as mean ⫾ SEM. The level of significance was P ⬍ 0.05.
FIG. 3. Photomicrographs showing representative findings of the immunohistochemical staining for TNF-␣ and ICAM-1 expression. It could be found that the brown positive cells for TNF-␣ (A) and ICAM-1 (C) were located in the lamina propia of the mucosa and the interstitial region in the control group. It was showed that the immunoreactivities of TNF-␣ (B) and ICAM-1 (D) were both stronger in the SAH group compared with control group (original magnification, ⫻200, Scale bars ⫽ 50 m). (Color version of figure is available online.)
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TABLE 2
DISCUSSION
The Number of Reactive Cells for TNF-␣ or ICAM-1 in the Intestine in Each View Under Light Microscope (Magnification, ⴛ100) Group
TNF-␣
ICAM-1
Control group SAH group
49.9 ⫾ 2.5 200.8 ⫾ 10.0*
50.4 ⫾ 3.4 184.9 ⫾ 5.5*
Values are expressed as means ⫾ SEM. * P ⬍ 0.001 versus the control group.
RESULTS General Observation
The mean baseline weights of rabbits in both control and SAH groups did not significantly differ. One rabbits in the SAH group was dead during the second blood injection. Appetite Evaluation
The Fig. 1 showed the change of appetite scores of the rabbits in the SAH group. The scores were decreased after blood injection, and peaked on day 3. There were significant difference between the scores of day 0 and each day after blood injection (P ⬍ 0.01).
The main findings of this study are that 1) alterations of the intestinal mucosal morphology occurred in rabbits after SAH; and 2) the inflammation response was triggered in the intestines after SAH, which associated with the activation of NF-B. Studies on intestinal mucosa structure after SAH have not been found to data. To better understand the alteration of the intestinal function and better conduct the efficacious enteral nutrition in the SAH patients, the study on the intestinal mucosa structure after SAH is urgent and warranted. In this study, we found that damage of intestinal mucosa occurred after SAH. The morphological alterations of intestinal mucosa including shedding of the epithelial cells, apoptosis of epithelial cells disarrangement of villi, fusion of adjacent villi, mucosal atrophy, and the inflammatory cells infiltration. The inflammatory cells infiltrated into the mucosa implied that the inflammation might take part in the damage of the intestinal mucosa after SAH. NF-B is one of the most important modulators of inflammatory gene expression which could subsequently
Histopathology
Histopathological examination showed that the morphology of intestinal mucosa was approximately normal in the control rabbits. In the rabbits subjected to SAH, The following changes could be observed: shedding of the epithelial cell from the top of villi, epithelial cell apoptosis, disarrangement of villi, fusion of adjacent villi into piece and mucosal atrophy. In addition, more severe inflammatory cells infiltrate emerges in the intestines in the SAH group than the control group (Fig. 2). Immunohistochemistry for TNF-␣ and ICAM-1
The immunohistochemical assay showed lower TNF-␣ and ICAM-1 immunoreactivities in the lamina propia of the mucosa and the interstitial region in the control group. And the immunoreactivities of the TNF-␣ and ICAM-1 were both significantly increased in the SAH group (Fig. 3, Table 2; P ⬍ 0.001). EMSA for NF-B
NF-B activation in the nuclear extracts was determined by EMSA. NF-B binding activity was detected at low level in control animals throughout the experiment. Compared with the control group, NF-B binding activity in the intestines was significantly increased (⬃150% increase) in the SAH group (Fig. 4A and 4B).
FIG. 4. Activation of NF-B in the intestines. (A) The representative EMSA picture indicating NF-B binding activity in the control and SAH groups, respectively. Group 1 indicates the SAH group 2 indicates the control group. (B) Graph showing the NF-B binding activity in the control and SAH groups. The figure indicates that the NF-B activity was increased significant in the SAH group (**P ⬍ 0.01).
ZHOU ET AL.: GUT AND SUBARACHNOID HEMORRHAGE
transcriptionally activate numerous genes encoding cytokines and adhesion molecules [25, 26]. NF-B could be activated by Gram-positive and Gram-negative bacteria and bacteria products, viruses and viral components, cytokines, free radicals, and oxidants [27, 28]. The activation of intestinal NF-B after traumatic brain injury was demonstrated by our laboratory [17, 19]. However, to data, no study in the literature has focused on the intestinal inflammation and NF-B pathway after SAH. Although the present study has shown that SAH could result in the up-regulation of NF-B in the intestines, the potential mechanism underlying the initial activation of intestinal NF-B following SAH remains unclear. Several factors might promote the activation of intestinal NF-B, such as cytokines, inflammatory mediators and hypoperfusion in the intestines [23, 29 –32]. Splanchnic hypoperfusion is a common phenomenon in trauma, especially in the condition of ruptured intracranial aneurysm-induced SAH, because of the sympathetic response and adaptive regulation of the blood supply of vital organs. Increased TNF-␣ in the intestines, as shown in this study, might also contribute to the activation NF-B. Whereas, further study in this field was warranted to understand completely the mechanism on the activation of intestinal inflammation and NF-B. Another pronounced problem is the animal model chosen to study the secondary organ damage after the SAH. The endovascular perforation model may be the preferred model for its severe symptom after SAH in the past. However, we chose the method of blood injection into the cisterna magna but not endovascular perforation method because of the lower mortality rate of the blood injection method. In addition, the blood injection model might be more similar in pathophysiology with that of human SAH. However, some improvements were made in the model in the present study. We injected the blood into the cisterna magna twice and added the amount of the blood to 3 ml into cisterna magna to produce a relatively severe SAH model. In conclusion, the results of the present study suggest that SAH in the rabbits could induce NF-B and proinflammatory cytokines activation in the intestinal epithelium, which is associated with morphological changes.
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The unchanging pattern of subarachnoid hemorrhage in a community. Neurology 1980;30:1034. Solenski NJ, Haley EC, Jr., Kassell NF, et al. Medical complications of aneurysmal subarachnoid hemorrhage: A report of the multicenter, cooperative aneurysm study. Participants of the Multicenter Cooperative Aneurysm Study. Crit Care Med 1995;23:1007. Gruber A, Reinprecht A, Illievich UM, et al. Extracerebral organ dysfunction and neurologic outcome after aneurysmal subarachnoid hemorrhage. Crit Care Med 1999;27:505. Weir BK. Pulmonary edema following fatal aneurysm rupture. J Neurosurg 1978;49:502. Schell AR, Shenoy MM, Friedman SA, Patel AR. Pulmonary edema associated with subarachnoid hemorrhage. Evidence for a cardiogenic origin. Arch Intern Med 1987;147:591. Svigelj V, Grad A, Kiauta T. Heart rate variability, norepinephrine and ECG changes in subarachnoid hemorrhage patients. Acta Neurol Scand 1996;94:120. Marion DW, Segal R, Thompson ME. Subarachnoid hemorrhage and the heart. Neurosurgery 1986;18:101. Di Pasquale G, Pinelli G, Andreoli A, Manini G, Grazi P, Tognetti F. Holter detection of cardiac arrhythmias in intracranial subarachnoid hemorrhage. Am J Cardiol 1987;59:596. Asplin BR, White RD. Subarachnoid hemorrhage: Atypical presentation associated with rapidly changing cardiac arrhythmias. Am J Emerg Med 1994;12:370. Estanol Vidal B, Badui Dergal E, Cesarman E, et al. Cardiac arrhythmias associated with subarachnoid hemorrhage: Prospective study. Neurosurgery 1979;5:675. Dooling E, Winkelman C. Hyponatremia in the patient with subarachnoid hemorrhage. J Neurosci Nurs 2004;36:130. Spallone A, Acqui M, Pastore FS, Guidetti B. Relationship between leukocytosis and ischemic complications following aneurysmal subarachnoid hemorrhage. Surg Neurol 1987;27:253. Parkinson D, Stephensen S. Leukocytosis and subarachnoid hemorrhage. Surg Neurol 1984;21:132. Davenport RJ, Dennis MS, Warlow CP. Gastrointestinal hemorrhage after acute stroke. Stroke 1996;27:421. Tanaka S, Mori T, Ohara H, Takaku A, Suzuki J. Gastrointestinal bleeding in cases of ruptured cerebral aneurysms. Acta Neurochir (Wien) 1979;48:223. Hang CH, Shi JX, Li JS, Li WQ, Wu W. Expressions of intestinal NF-kappaB, TNF-alpha, and IL-6 following traumatic brain injury in rats. J Surg Res 2005;123:188.
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Hang CH, Shi JX, Li JS, Wu W, Yin HX. Alterations of intestinal mucosa structure and barrier function following traumatic brain injury in rats. World J Gastroenterol 2003;9:2776.
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Hang CH, Shi JX, Li JS, Li WQ, Yin HX. Up-regulation of intestinal nuclear factor kappa B and intercellular adhesion molecule-1 following traumatic brain injury in rats. World J Gastroenterol 2005;11:1149.
ACKNOWLEDGMENTS
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The authors thank Dr. Gen-bao Feng and Bo Wu for their technical assistance. This work was supported by grants from Jinling Hospital of China.
D’Ancona G, Baillot R, Poirier B, et al. Determinants of gastrointestinal complications in cardiac surgery. Tex Heart Inst J 2003;30:280.
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Frick TW, Kach K, Sulser H, Hailemariam S, Largiader F, Glinz W. Intestinal infarction after nonabdominal trauma; association with cerebral trauma. J Trauma 1992;33:870.
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Neurath MF, Becker C, Barbulescu K. Role of NF-kappaB in immune and inflammatory responses in the gut. Gut 1998;43:856.
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Souza DG, Vieira AT, Pinho V, et al. NF-kappaB plays a major role during the systemic and local acute inflammatory response following intestinal reperfusion injury. Br J Pharmacol 2005; 145:246.
The Inflammation in the Gut After Experimental Subarachnoid Hemorrhage Meng-liang Zhou, M.D.,* Lin Zhu, M.D.,* Jian Wang, M.D.,† Chun-hua Hang, M.D., Ph.D.,* and Ji-xin Shi, M.D., Ph.D.*,1 *Department of Neurosurgery, †Department of General Surgery, Jinling Hospital, School of Medicine, Nanjing University, Nanjing, Jiangsu Province, China Submitted for publication March 7, 2006
Background. Gastrointestinal dysfunction could be frequently observed in the patients suffering from SAH. This study test the hypothesis that experimental SAH could induce histopathological changes and inflammatory response associating with NF-B activation pathway in the gut. Materials and methods. A total of 17 rabbits were randomly divided into two groups: control group (n ⴝ 8) and SAH group (n ⴝ 9). In the SAH group, the animals were subjected to experimental SAH according to the “two-hemorrhage” method. The histopathological study was performed to detect the intestinal mucosal morphological changes and immunohistochemical study was used to detect the TNF-␣ and ICAM-1 expressions. NF-B binding activity was measured using the electrophoretic mobility shift assay. Results. It was demonstrated that some damage changes and leukocytes infiltration occurred in the intestinal mucosa after SAH. More positive cells for TNF-␣ and ICAM-1 were observed in the SAH group. The NF-B binding activity in the intestines was significantly increased in the SAH group (P < 0.01). Conclusions. The results of the present study suggest that SAH in the rabbits could induce NF-B and proinflammatory cytokines activation in the intestine, which is associated with morphological changes. © 2007 Elsevier Inc. All rights reserved.
Key Words: subarachnoid hemorrhage; intestine; inflammation; nuclear factor-B. INTRODUCTION
Cerebrovascular diseases are the third leading cause of death in developed countries, behind only the coro1 To whom correspondence and reprint requests should be addressed at Department of Neurosurgery, Jinling Hospital, 305 East Zhongshan Road, Nanjing 210002, P. R. China. E-mail: [email protected].
nary artery disease and all type of cancers [1]. More than 20% of these deaths are because of aneurysmal subarachnoid hemorrhage (SAH) [2]. Cerebral vasospasm is the significant cause of morbidity and mortality in patients suffering from aneurysmal SAH. Cerebral vasospasm could result in brain ischemia and infarction which account for over one third of the total cases of disability and death [3]. In addition, non-neurologic complications also add to morbidity and mortality after SAH [3, 4]. Studies on nonneurologic complications associated with SAH in the literature concentrated on the pulmonary edema and pneumonia [5, 6], cardiac arrhythmia [7–11], electrolyte disturbance [12], and hematologic change [13, 14]. Besides those complications, gastrointestinal dysfunction could be frequently observed in the patients suffering from SAH. Dysfunction of the gastrointestinal tract leads to some symptoms such as gastrointestinal bleeding, gastric reflux, and decreased intestinal peristalsis, which could influence the outcome after SAH [15, 16]. Our previous study demonstrated that acute damage and inflammatory responses occurred in intestinal mucosa after traumatic brain injury and which might mediate mainly by up-regulation of nuclear factor-B (NF-B) pathway [17–19]. Also, some reports also indicated that the function of the intestines was influenced following the injury of the other organ because of many reasons, such as taking little food and stress after injury [20, 21]. The early enteral nutrition after brain injury, including after SAH, is necessary to maintain and improve the function of the intestines [22]. However, the effect of SAH on intestines has not been studied to date. NF-B is a pleiotropic transcription factor and plays a key role in the intestinal immune system [23]. NF-B
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TABLE 1
Tissue Harvest
Appetite Scores
On day 5, the rabbits were again anesthetized with an intramuscular injection of a mixture of ketamine (40 mg/kg) and droperidol (2.5 mg/kg) and killed by exsanguination. A 3-cm segment of the mid-ileum was taken and flushed with ice-cold saline. Half of it was immersed in the 10% buffered formalin for histopathological studies and the other was stored in liquid nitrogen immediately for electrophoretic mobility shift assay (EMSA).
The amount of the food eaten by the animal (standard food normally eaten by the animal in one day)
Score
1.0 ⬎0.5 ⬎0.2 ⬍0.2
3 2 1 0
members control transcriptional activity of various promoters of proinflammatory cytokines, cell surface receptors, transcription factors, and adhesion molecules that are involved in intestinal inflammation [23, 24]. In the present study, we investigated the alterations of intestinal mucosal morphology and the inflammation response involving NF-B pathway. MATERIALS AND METHODS Animal Preparation All procedures in animals were approved by the Animal Care and Use Committee of Nanjing University and conformed to Guide for the Care and Use of Laboratory Animals from National Institutes of Health. Seventeen adult male New Zealand White (NZW) rabbits weighing from 2.4 to 2.8 kg were purchased from the Animal Center of the Chinese Academy of Sciences (Shanghai, China). The rabbits were acclimated in a temperature and humidity-controlled room and maintained on the standard pellet diet at the Animal Center of Jinling Hospital for 10 days before the experiment. The temperature in both the feeding room and the operation room was maintained at about 25°C.
Rabbit Models of SAH The rabbits were randomly assigned to two groups. Animals in group 1 served as controls and intracisternal saline injection was performed (n ⫽ 8). The animals in group 2 were subjected to experimental SAH (n ⫽ 9). In groups 2, experimental SAH was produced according to the “two-hemorrhage” method. The rabbits were anesthetized with an intramuscular injection of a mixture of ketamine (25 mg/kg) and droperidol (1.0 mg/kg) on day 0. A 21-gauge butterfly needle was percutaneously placed in the cisterna magna. After withdrawal of 1.5 ml of the cerebrospinal fluid (CSF), 3 mL of non-heparinized fresh autologous auricular arterial blood was slowly injected into the cisterna magna for 1 min under aseptic technique. The animals were then kept in a 30 degree head-down position for 30 min. After recovery from anesthesia, they were returned to the feeding room. Forty-eight hours after the first SAH, a second one was produced in the same manner as the first. In the animals in the control group, the same technique was applied, with injection of sterile saline instead of blood.
Histopathological Examination The 10% buffered formalin-fixed jejunum was embedded in paraffin, sectioned at 4 m thickness with a microtome and stained with hematoxylin and eosin (H&E). The sections were examined under light microscope.
Immunohistochemical Assay The coronal sections were embedded in paraffin and sectioned again at 4 m thickness with a microtome. The antibodies (antiTNF-␣ and anti-ICAM-1, both purchased from Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were diluted with 1% bovine serum albumin/phosphate-buffered solution (BSA/PBS) (BSA, 1 g, pH 7.4, 0.01 mol/L, 100 mL) by a certain ratio (anti-TNF-␣ diluted 1:100 and anti-ICAM-1 diluted 1:200). The sections were incubated with the diluted antibodies overnight at 4°C respectively, washed, and blocked with 1.6% H 2O 2 in phosphate-buffered saline (PBS) for 10 min. After washing with PBS, sections were incubated with HRPconjugated goat anti-rabbit IgG (diluted 1:500) for 60 min at room temperature. DAB was used as chromogen and counterstaining was done with hematoxylin. Six views were selected randomly for each section and observed under a light microscope (⫻100). Then mean number of reactive cells in the six views was regarded as the data for each sample.
Nuclear Protein Extracts and EMSA Nuclear protein of intestinal tissue was extracted and quantified as described [17]. Briefly, frozen intestinal tissues were homogenized in 0.8 mL ice-cold Buffer A composed of 10 mM HEPES pH 7.9, 10 mM KCl, 2 mM MgCl 2, 0.1 mM EDTA, 1 mM dithiothreitol (DTT), and 0.5 mM phenylmethylsulfonyl fluoride (PMSF) (all from Sigma Chemical Co., St. Louis, MO). Then the homogenates were incubated on ice for 20 min, and vortexed for 30 s after addition of 50 L NP-40 (Sigma Chemical Co.). Then the mixture was centrifuged for 10 min (5,000 ⫻ g, 4°C). The pellet was then suspended in 50 L ice-cold buffer B [50 mM HEPES pH 7.9, 50 mM KCl, 300 mM NaCl, 0.1 mM EDTA, 1 mM
Appetite Evaluation Appetite scores were recorded by independent observations of a veterinarian who was blinded to the study. The scores of the animals in SAH group were recorded daily by using the modified scoring table (Table 1).
FIG. 1. Graph showing the appetite scores of the rabbits in SAH group. The scores were reduced after blood injection into the cisterna magna and peaked on the day 3. There is a significant difference between the scores on each day after first blood injection and the scores on day 0 (**P ⬍ 0.01).
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FIG. 2. Photomicrographs showing histopathological changes in the SAH group compared with control group. (A) The normal intestinal morphology of the control animals (H&E; original magnification, ⫻40). (B) The histopathoglogical changes in the intestines of the rabbits in the SAH group. These changes include shedding of the epithelial cell from the top of villi, epithelial cell necrosis, disarrangement of villi, fusion of adjacent villi into piece, mucosal atrophy, and leukocytes infiltration (H&E; original magnification, ⫻40). (Color version of figure is available online.)
DTT, 0.5 mM PMSF, with 10% (v/v) glycerol] and incubated on ice for 30 min with frequent mixing. After centrifugation (12,000 ⫻ g, 4°C) for 15 min, the supernatants were collected as nuclear extracts and stored at ⫺80°C until use. EMSA was performed using a commercial kit (Gel Shift Assay System; Promega, Madison, WI). Consensus oligonucleotide probe (5=-AGT TGA GGG GAC TTT CCC AGG C-3=) was end-labeled with [␥-32P]ATP (Free Biotech., Beijing, China) with T4polynucleotide kinase. Nuclear protein (30 g) was preincubated in a total volume of 9 L in a binding buffer, consisting of 10 mM Tris–HCl (pH 7.5), 4% glycerol, 1 mM MgCl 2 , 0.5 mM EDTA, 0.5 mM DTT, 0.5 mM NaCl, and 0.05 mg/mL poly(di-dc) for 10 min at room temperature. After addition of the 32P-labled oligonucleotide probe, the incubation was continued for 20 min at room temper-
ature. Reaction was stopped by adding 1 L of gel loading buffer and the mixture was subjected to nondenaturing 4% polyacrylamide gel electrophoresis in 0.5⫻TBE buffer (Tris-borate-EDTA). After electrophoresis was conducted at 390 V for 1 h, the gel was vacuum-dried and exposed to X ray film (Fuji Hyperfilm, Tokyo, Japan) at ⫺70°C with an intensifying screed.
Statistical Analysis Software SPSS 11.0 was used for the statistical analysis. To the appetite scores, the Mann-Whitney t-test was used to compare the scores of each day from day 1 to day 5 with day 0. The other measurements were analyzed by Student’s t-test. All data were presented as mean ⫾ SEM. The level of significance was P ⬍ 0.05.
FIG. 3. Photomicrographs showing representative findings of the immunohistochemical staining for TNF-␣ and ICAM-1 expression. It could be found that the brown positive cells for TNF-␣ (A) and ICAM-1 (C) were located in the lamina propia of the mucosa and the interstitial region in the control group. It was showed that the immunoreactivities of TNF-␣ (B) and ICAM-1 (D) were both stronger in the SAH group compared with control group (original magnification, ⫻200, Scale bars ⫽ 50 m). (Color version of figure is available online.)
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TABLE 2
DISCUSSION
The Number of Reactive Cells for TNF-␣ or ICAM-1 in the Intestine in Each View Under Light Microscope (Magnification, ⴛ100) Group
TNF-␣
ICAM-1
Control group SAH group
49.9 ⫾ 2.5 200.8 ⫾ 10.0*
50.4 ⫾ 3.4 184.9 ⫾ 5.5*
Values are expressed as means ⫾ SEM. * P ⬍ 0.001 versus the control group.
RESULTS General Observation
The mean baseline weights of rabbits in both control and SAH groups did not significantly differ. One rabbits in the SAH group was dead during the second blood injection. Appetite Evaluation
The Fig. 1 showed the change of appetite scores of the rabbits in the SAH group. The scores were decreased after blood injection, and peaked on day 3. There were significant difference between the scores of day 0 and each day after blood injection (P ⬍ 0.01).
The main findings of this study are that 1) alterations of the intestinal mucosal morphology occurred in rabbits after SAH; and 2) the inflammation response was triggered in the intestines after SAH, which associated with the activation of NF-B. Studies on intestinal mucosa structure after SAH have not been found to data. To better understand the alteration of the intestinal function and better conduct the efficacious enteral nutrition in the SAH patients, the study on the intestinal mucosa structure after SAH is urgent and warranted. In this study, we found that damage of intestinal mucosa occurred after SAH. The morphological alterations of intestinal mucosa including shedding of the epithelial cells, apoptosis of epithelial cells disarrangement of villi, fusion of adjacent villi, mucosal atrophy, and the inflammatory cells infiltration. The inflammatory cells infiltrated into the mucosa implied that the inflammation might take part in the damage of the intestinal mucosa after SAH. NF-B is one of the most important modulators of inflammatory gene expression which could subsequently
Histopathology
Histopathological examination showed that the morphology of intestinal mucosa was approximately normal in the control rabbits. In the rabbits subjected to SAH, The following changes could be observed: shedding of the epithelial cell from the top of villi, epithelial cell apoptosis, disarrangement of villi, fusion of adjacent villi into piece and mucosal atrophy. In addition, more severe inflammatory cells infiltrate emerges in the intestines in the SAH group than the control group (Fig. 2). Immunohistochemistry for TNF-␣ and ICAM-1
The immunohistochemical assay showed lower TNF-␣ and ICAM-1 immunoreactivities in the lamina propia of the mucosa and the interstitial region in the control group. And the immunoreactivities of the TNF-␣ and ICAM-1 were both significantly increased in the SAH group (Fig. 3, Table 2; P ⬍ 0.001). EMSA for NF-B
NF-B activation in the nuclear extracts was determined by EMSA. NF-B binding activity was detected at low level in control animals throughout the experiment. Compared with the control group, NF-B binding activity in the intestines was significantly increased (⬃150% increase) in the SAH group (Fig. 4A and 4B).
FIG. 4. Activation of NF-B in the intestines. (A) The representative EMSA picture indicating NF-B binding activity in the control and SAH groups, respectively. Group 1 indicates the SAH group 2 indicates the control group. (B) Graph showing the NF-B binding activity in the control and SAH groups. The figure indicates that the NF-B activity was increased significant in the SAH group (**P ⬍ 0.01).
ZHOU ET AL.: GUT AND SUBARACHNOID HEMORRHAGE
transcriptionally activate numerous genes encoding cytokines and adhesion molecules [25, 26]. NF-B could be activated by Gram-positive and Gram-negative bacteria and bacteria products, viruses and viral components, cytokines, free radicals, and oxidants [27, 28]. The activation of intestinal NF-B after traumatic brain injury was demonstrated by our laboratory [17, 19]. However, to data, no study in the literature has focused on the intestinal inflammation and NF-B pathway after SAH. Although the present study has shown that SAH could result in the up-regulation of NF-B in the intestines, the potential mechanism underlying the initial activation of intestinal NF-B following SAH remains unclear. Several factors might promote the activation of intestinal NF-B, such as cytokines, inflammatory mediators and hypoperfusion in the intestines [23, 29 –32]. Splanchnic hypoperfusion is a common phenomenon in trauma, especially in the condition of ruptured intracranial aneurysm-induced SAH, because of the sympathetic response and adaptive regulation of the blood supply of vital organs. Increased TNF-␣ in the intestines, as shown in this study, might also contribute to the activation NF-B. Whereas, further study in this field was warranted to understand completely the mechanism on the activation of intestinal inflammation and NF-B. Another pronounced problem is the animal model chosen to study the secondary organ damage after the SAH. The endovascular perforation model may be the preferred model for its severe symptom after SAH in the past. However, we chose the method of blood injection into the cisterna magna but not endovascular perforation method because of the lower mortality rate of the blood injection method. In addition, the blood injection model might be more similar in pathophysiology with that of human SAH. However, some improvements were made in the model in the present study. We injected the blood into the cisterna magna twice and added the amount of the blood to 3 ml into cisterna magna to produce a relatively severe SAH model. In conclusion, the results of the present study suggest that SAH in the rabbits could induce NF-B and proinflammatory cytokines activation in the intestinal epithelium, which is associated with morphological changes.
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ACKNOWLEDGMENTS
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The authors thank Dr. Gen-bao Feng and Bo Wu for their technical assistance. This work was supported by grants from Jinling Hospital of China.
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