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Hepatoctye growth factor pretreatment reduces apoptosis and mucosal damage after intestinal ischemia-reperfusion
Hepatoctye Growth Factor Pretreatment Reduces Apoptosis and Mucosal Damage After Intestinal Ischemia-Reperfusion By Keith A. Kuenzler, Philip Y. Pearson, and Marshall Z. Schwartz Wilmington, Delaware and Philadelphia, Pennsylvania
Background/Purpose: Ischemia-reperfusion (IR) injury to the intestine can result in mucosal damage and cellular death. This study was designed to evaluate the protective effects of pretreatment with hepatocyte growth factor (HGF) on intestine after moderate IR injury. Methods: Control animals (n ⫽ 7) received 48 hours of intravenous saline, and treatment animals (n ⫽ 7) received HGF (150 g/kg/d). After 35 minutes of mesenteric artery occlusion and 120 minutes of reperfusion, serum and jejunal mucosa samples were obtained. Fluorometric assays were performed for hexosaminidase A (HEX A) and -glucuronidase (GLUC), enzyme markers of enterocyte necrosis. Apoptosis was quantified by the TUNEL method. Transcription of tumor necrosis factor-␣ (TNF-␣) and interferon-␥ (IFN-␥) was assessed by multiplex reverse transcription polymerase chain reaction (RT-PCR) Statistical analysis was performed using the Student’s t test. Results: After HGF pretreatment, HEX A and GLUC activities
I
were reduced from 543 ⫾ 28 to 343 ⫾ 35 nmole/h/mL (P ⬍ .01) and 183 ⫾ 29 to 119 ⫾ 22 nmole/h/mL (P ⬍ .01), respectively. Pretreated animals had a reduced number of apoptotic cells per 10 crypts (33 ⫾ 11) compared with untreated rats (225 ⫾ 24) after IR injury (P ⬍ .01). Mean IFN-␥ band intensity was lower in HGF-pretreated animals (0.05 ⫾ 0.02) compared with controls (0.31 ⫾ 0.09; P ⬍ .05). Conclusions: Pretreatment with HGF reduces the severe crypt apoptosis and cellular necrosis after IR injury to the intestine. These data suggest that HGF may be beneficial in attenuating IR damage and thus may have significant clinical application. J Pediatr Surg 37:1093-1097. Copyright 2002, Elsevier Science (USA). All rights reserved. INDEX WORDS: Ischemia-reperfusion injury, hepatocyte growth factor, intestinal mucosa, intestinal ischemia, apoptosis, inflammatory mediators, tumor necrosis factor-alpha, interferon-gamma.
ISCHEMIA-REPERFUSION (IR) injury to the small intestine occurs in children in conditions such as midgut volvulus and resuscitation after shock states. The precise mechanisms of intestinal IR injury have not been elucidated completely, and there currently are no specific treatments once ischemia has taken place. Injury to the small intestine after IR injury appears to be mediated by several factors, including the cytotoxicity of oxygen radicals and the recruitment of inflammatory leukocytes.1 Necrosis has been assumed to be synonymous with epithelial cell death after an ischemic insult. However, more recent reports have noted that apoptosis is a significant, and perhaps the principal contributor to cell death after IR.2,3 In contrast to cell necrosis, apoptosis is an active, gene-directed process. Thus, understanding the sequence of events central to the apoptotic mechanism may lead investigators to potential means of modulating the sequelae of a variety of disease states. We and other investigators have shown that the administration of growth factors such as interleukin-11 (IL-11), glucagonlike-peptide-2 analogue (GLP-2␣), and heparin-binding epidermal growth factor (HB-EGF) after IR injury can result in increased protection of intestinal epithelium, acceleration of cellular renewal, and enhancement of function.4-7 Several studies have shown that hepatocyte growth factor (HGF) is another potent
trophic factor for the small intestine, but its potential effects in intestinal IR injury are unknown.8,9 The purpose of this study was to examine the benefit of systemic HGF pretreatment on mucosal necrosis and apoptosis after IR injury.
Journal of Pediatric Surgery, Vol 37, No 7 (July), 2002: pp 1093-1097
1093
MATERIALS AND METHODS
Animal Care All animal care and use complied with the institutional regulations set forth and approved by the Animal Care and Use Committee of the A. I. duPont Hospital for Children and the Nemours Research Programs (Wilmington, DE). Fourteen adult male Sprague-Dawley rats (Harlan Sprague Dawley, Indianapolis, IN) weighing 275 to 300 g were used in this study. Rats were kept in individual cages and fed standard rat chow and water ad libitum. After allowing the animals to acclimate to the
From the Department of Surgery, A.I. duPont Hospital for Children, Wilmington, DE, and Thomas Jefferson University Hospital, Philadelphia, PA. Presented at the 53rd Annual Meeting of the Section on Surgery of the American Academy of Pediatrics, San Francisco, California, October 19-21, 2001. Address reprint requests to Marshall Z. Schwartz, MD, A.I. duPont Hospital for Children, Department of Surgery, 1600 Rockland Rd, Wilmington, DE 19803. Copyright 2002, Elsevier Science (USA). All rights reserved. 0022-3468/02/3707-0033$35.00/0 doi:10.1053/jpsu.2002.33884
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environment for a period of 1 week, surgical procedures were performed using aseptic technique. General anesthesia was administered with an intramuscular injection of a combination of ketamine (70 mg/kg) and xylazine (14 mg/kg).
Animal Model Each rat underwent placement of a polyethylene jugular venous catheter that was connected to a subcutaneously placed osmotic pump designed to deliver its contents at a rate of 1 L/h (Alzet osmotic minipump, Model 1003D; Alza Scientific Products, Palo Alto, CA). Rats were divided into 2 groups as follows: control animals (n ⫽ 7) received intravenous saline, and treatment animals (n ⫽ 7) received intravenous recombinant human HGF (150 g/kg/d) (Genentech, San Francisco, CA) dissolved in saline. After 48 hours, each animal underwent a midline laparotomy and superior mesenteric artery occlusion for 35 minutes. After 120 minutes of reperfusion, serum and intestine samples were taken. After surgery, rats were killed.
Serum Lysosomal Enzyme Assays Serum was assayed for levels of -glucuronidase (GLUC) and hexosaminidase A (HEX A). GLUC and HEX A activities were quantified by fluorometric assays using 4MU--D-glucuronide and 4MU--D-NAcglucosamine sulfate, respectively, as substrates, according to the protocol of the Thomas Jefferson University Hospital Lysosomal Diseases-Testing Laboratory (Philadelphia, PA). Ten microliters of rat serum was incubated at 37°C with substrate in a final volume of 200 mL for 10 minutes, at which point the reaction was stopped by a glycine carbonate solution (pH ⫽ 10). Fluorometric readings were compared with known standards and expressed as nanomoles per hour per milliliter.
Multiplex RT-PCR to Detect Mucosal Expression of Inflammatory Mediators Midjejunal mucosal samples were harvested at operation and immediately stored at ⫺70°C for later semiquantitative reverse transcription polymerase chain reaction (RT-PCR) analysis. Total RNA was extracted from mucosa using the RNEasy Kit (Qiagen, Valencia, CA) and DNAse treated. RNA concentration and purity were determined by absorbency at 260 and 280 nm. One microgram of total RNA was reverse transcribed. The resultant cDNA was amplified using sense and antisense primers for tumor necrosis factor-␣ (TNF-␣), interferon-␥ (IFN-␥), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, internal standard). The temperature profile of the PCRs consisted of a melting step at 94°C followed by 30 cycles of 30 seconds at 60°C, 60 seconds at 65°C, 30 seconds at 94°C, with a final extension step of 5 minutes at 75°C. Independent experiments established that 30 cycles were within the linear range of the multiplex PCR assay. PCR products were separated on a 4% agarose gel and stained with ethidium bromide. Images were analyzed using EagleEye II analysis software and relative band intensities were calculated.
Histologic Analysis and Immunohistochemical Staining (TUNEL) Cross sections of jejunum 15 cm distal to duodenum were fixed immediately in formalin solution. Cut samples then were embedded in paraffin. Several sections per animal were stained with H&E for light microscopic examination. Areas in each of 4 quadrants of the intestinal cross sections, in 4 separate samples per animal, were graded by a blinded, independent reviewer using the Park/Chiu scoring system of bowel injury as described by Quaedackers et al.10 According to this system, observations of increased damage were assigned increasing points as follows: normal mucosa (0); subepithelial space at villus tips
Fig 1. Mean serum hexosaminidase A activity. HGF-pretreated animals have significantly lower levels after 35 minutes of intestinal ischemia and 120 minutes of reperfusion. ⴱⴱ ⴱⴱP < .01.
(1); extension of subepithelial space with moderate lifting (2); massive lifting, some denuded tips (3); denuded villi, dilated capillaries (4); disintegration of lamina propria (5); crypt layer injury (6); transmucosal infarction (7); and transmural infarction (8). Each animal was assigned an average score. Apoptotic crypt cells were identified using the terminal deoxynucleotidyl transferase (TdT)-mediated dUDP-biotin nick-end labeling (TUNEL) method using a standard laboratory detection kit (TdTFragEL, Oncogene Research Products, San Diego, CA). Samples were dewaxed, rehydrated in ethanol, and incubated with 3% hydrogen peroxide. Slides were treated with 100 L equilibration buffer for 30 minutes. Sections were then incubated for 1 hour at 37°C with 60 L TdT enzyme, followed by incubation for 30 minutes with 100 L conjugate solution, and detection with diaminobenzidine (DAB) solution. Sets of slides were counterstained alternately with either hematoxylin or methyl green. TUNEL-positive cells were counted by examining crypts of approximately equal size (70 m or 30 cells around) in 10 regions per slide per animal. Results were expressed as TUNEL ⫹ cells / 10 crypts.
Statistical Analysis Statistical analysis was performed using Student’s t test and expressed as mean ⫾ SEM.
RESULTS
Serum Lysosomal Enzyme Assays Mean serum HEX A in untreated control animals increased from 314 ⫾ 47 nmole/h/mL at baseline (before bowel ischemia) to 543 ⫾ 28 nmole/h/mL after 120 minutes of reperfusion (P ⬍ .01). In HGF-pretreated animals, mean HEX A levels also significantly rose with IR injury from 219 ⫾ 33 to 343 ⫾ 35 nmole/h/mL (P ⬍ .05), but these animals had HEX A values that were significantly lower after 120 minutes of reperfusion (P ⬍ .01; Fig 1). Mean serum GLUC in untreated control animals rose from 100 ⫾ 16 nmole/h/mL at baseline to 183 ⫾ 29 nmole/h/mL after 120 minutes of reperfusion (P ⬍ .01). In HGF-pretreated animals, mean GLUC levels also increased significantly with IR injury from 63 ⫾ 14 to 119 ⫾ 22 nmole/h/mL (P ⬍ .05), but these animals had GLUC values that were significantly lower after 120 minutes of reperfusion (P ⬍ .01; Fig 2).
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Fig 2. Mean serum -glucuronidase activity. HGF-pretreated animals have significantly lower levels after 35 minutes of intestinal ischemia and 120 minutes of reperfusion. **P < .01.
RT-PCR Average TNF-␣ relative band intensities after RTPCR were not significantly different in the control (0.74 ⫾ 0.06) and HGF-treated (0.85 ⫾ 0.07) groups. Mean IFN-␥ relative band intensities, however, were significantly reduced in treated animals (0.05 ⫾ 0.02) when compared with control animals (0.31 ⫾ 0.09; P ⬍ .05; Table 1). Histologic Analysis Portions of both animal groups showed varying damage to intestinal villi after 35 minutes of ischemia and 120 minutes of reperfusion (Figs 3A and 3B). Mean histologic injury scores were not significantly different between the untreated control group (2.67) and in the HGF pretreatment group (2.33). HGF-treated animals had significantly fewer TUNEL-positive cells per 10 crypts (33 ⫾ 11) than untreated control animals (225 ⫾ 24; P ⬍ .01) subjected to IR injury (Fig 4). DISCUSSION
Over the last 2 decades, our laboratory has shown that several gastrointestinal peptides can enhance intestinal growth and function in models of intestinal adaptation and inflammatory bowel disease.11-13 Among these peptides, HGF appears to show especially potent trophic effects.8,9 Because enterocyte cell death after IR injury is known to result from a combination of apoptosis and necrosis,2 we sought to examine the effects of pretreatment with HGF on preventing these modes of cell death. To evaluate the effects of IR and the potential benefit of HGF pretreatment, we chose to measure the release of
Fig 3. (A) Light micrograph of uninjured rat jejunal mucosa shows regular villus architecture and normal crypt depth. (H&E, original magnification ⴛ100.) (B) Light micrograph of rat jejunal mucosa after 35 minutes of intestinal ischemia and 120 minutes of reperfusion. Note the thickening, shortening and loss of villi, reduction of crypt depth, and inflammatory cell infiltrate. (H&E, original magnification ⴛ100.)
certain lysosomal enzymes and evaluate morphologic and apoptotic changes in the small intestine after this injury. Abe et al14 were the first investigators to show that -glucuronidase (GLUC), was released into the circulation after intestinal IR.14 Subsequently, Polson et al15 showed that hexosaminidase (HEX) increased in the
Table 1. Effect of HGF Pretreatment on Mucosal mRNA Expression After 35 Minutes of Intestinal Ischemia and 120 Minutes of Reperfusion
TNF-␣/GAPDH IFN-␥/GAPDH
IR
IR ⫹ HGF
P Value
0.7400 ⫾ .06 0.3116 ⫾ .09
0.8467 ⫾ .07 0.0506 ⫾ .02
.303 .018
NOTE. Relative band intensities after RT-PCR are shown for TNF-␣ and IFN-␥ compared with the GAPDH internal standard. *P ⬍ .05.
Fig 4. Mean number of TUNEL ⴙ cells per 10 crypts. HGF-pretreated animals show a significant reduction at 120 minutes of reperfusion. ⴱⴱ ⴱⴱP < .01.
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serum after IR injury.15 Further studies by Lobe et al16 and Maeda et al17 evaluated HEX in neonatal clinical settings and in an animal model of intestinal transplantation. Additional studies in our laboratory have been directed toward the potential role for growth factors in intestinal IR injury. Recently, we have shown that both GLP-2␣ and IL-11 can attenuate injury and enhance the recovery of absorptive function in a rat model of IR.4,7 In the current study, tissue samples were taken after 120 minutes of reperfusion because previous reports showed histologic evidence of high levels of crypt cell apoptosis in the early hours of reperfusion.24 Our results in the current study show that systemic infusion of HGF for 48 hours before IR injury result in a significant reduction of each of these lysosomal enzymes compared with small intestine subjected to IR injury without HGF pretreatment. We believe this reflects a “protective” effect of HGF on the mucosal cells of the small intestine. Further support for this conclusion is noted in our apoptosis studies. It should be noted that baseline levels of these enzymes (before injury) were not significantly different between the control and treated groups; thus, it appears that HGF pretreatment does not affect these enzymes before IR. Apoptosis is a significant mode of cell death in the intestine after IR injury.3 In healthy bowel, apoptosis allows continuous cell renewal as enterocytes are shed from villus tips. Normally, crypt cell apoptosis is rare (less than 1 cell per 10 crypts) in normal jejunum, but after IR it can be markedly increased in the first few hours of reperfusion.18 The major advantage of the TUNEL technique of identifying apoptotic cells in situ is the ability to localize which enterocytes are affected. In this study we showed an 85% decrease in apoptotic cells after HGF pretreatment. However, we were not able to show a benefit in the histologic injury score. The reason for this is not known. Because apoptosis is a gene-mediated cellular process, it is possible that growth factors such as HGF may have a role in affecting this phenomenon. Michalsky et al19
have recently observed inhibition of apoptosis using another growth factor, HB-EGF, in intestinal epithelial cell cultures stimulated by inflammatory mediators. In that study, both flow cytometric studies and “cell death” ELISAs confirmed that treatment with HB-EGF results in significant reductions in intestinal cell apoptosis. In an hepatic IR model, Ikegami et al20 have done experiments with the “deletion variant” of HGF, a splice variant whose 5 amino acid alterations render it more mitogenic toward hepatocytes specifically. These investigators showed reductions in both serum markers of hepatocyte death and in situ liver apoptosis in their HGF-treated rats. The exact mechanisms for accelerated apoptosis have not yet been confirmed. However, several studies have implicated inflammatory mediators such as TNF-␣ and IFN-␥ after hepatic, renal, and intestinal IR.21,22 Li et al23 recently have reported an increase in expression of mRNA for IFN-␥ in rats showing significant crypt cell apoptosis during small bowel transplant rejection. Our findings in this study confirm reduced transcription of this cytokine concurrent with reductions in cell death and crypt cell apoptosis. Although the specific mechanism for the apparent antiapoptotic effects of HGF have not yet been elucidated, we hypothesize that in the early period of reperfusion, HGF may be attenuating the mucosal injury that precedes apoptosis. Alternatively, HGF might be stimulating genetic antiapoptotic regulators such as Bcl-2, which have been shown to render transgenic mice resistant to IR-induced apoptosis.24 We postulate that in the future it may be appropriate to pretreat patients at risk for intestinal ischemia to attenuate IR damage, such as those with abdominal aortic aneurysms or intestinal transplantation, or at the time of ischemic injury, such as in necrotizing enterocolitis or midgut volvulus. ACKNOWLEDGMENTS The authors thank Juan P. Palazzo, MD, of the Department of Pathology, Thomas Jefferson University Hospital, for his teaching and help with histologic injury grading and David A. Wenger, PhD, of the Thomas Jefferson University Hospital Lysosomal Diseases Testing Laboratory, for his assistance with the enzyme assays.
REFERENCES 1. Schoenberg MH, Beger HG: Reperfusion injury after intestinal ischemia. Crit Care Med 21:1376-1386, 1993 2. Shah KA, Shurey S, Green CJ: Apoptosis after intestinal ischemia-reperfusion injury. Transplantation 64:1393-1397, 1997 3. Noda T, Iwakiri R, Fujimoto K, et al: Programmed cell death induced by ischemia-reperfusion in rat intestinal mucosa. Am J Physiol 274:G270-276, 1998 4. Prasad R, Alavi K, Schwartz MZ: Glucagonlike peptide-2 analogue enhances intestinal mucosal mass and absorptive function after ischemia-reperfusion injury. J Pediatr Surg 35:1537-1539, 2000 5. Du XX, Liu Q, Zhixiang Y, et al: Protective effects of interleukin-11 in a murine model of ischemic bowel necrosis. Am J Physiol 272:G545-552, 1997
6. Pillai SB, Hinman CE, Luquette MH, et al: Heparin-binding epidermal growth factor protects rat intestine from ischemia/reperfusion injury. J Surg Res 87:225-231, 1999 7. Kuenzler KA, Pearson PY, Schwartz MZ: Interleukin-11 enhances intestinal absorptive function after ischemia-reperfusion injury. J Pediatr Surg 37:457-459, 2002 8. Kato Y, Yu D, Schwartz MZ: Enhancement of intestinal adaptation by hepatocyte growth factor. J Pediatr Surg 33:235-239, 1998 9. Kato Y, Yu D, Lukish JR, et al: Hepatocyte growth factor enhances intestinal mucosal cell function and mass in vivo. J Pediatr Surg 32:991-994, 1997 10. Quaedackers JSLT, Beuk RJ, Bennet A, et al: An evaluation of
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methods for grading histologic injury following ischemia/reperfusion of the small bowel. Transplant Proc 32:1307-1310, 2000 11. Lukish JR, Yu D, Kato Y, et al: Persistent upregulation in intestinal function beyond intestinal adaptation following luminal growth factor perfusion. Surg Forum 47:675-678, 1996 12. Alavi K, Prasad R, Lundgren K, et al: Interleukin-11 enhances small intestinal absorptive function and mucosal mass after intestinal adaptation. J Pediatr Surg 35:371-374, 2000 13. Alavi K, Schwartz MZ, Palazzo JP, et al: Treatment of inflammatory bowel disease in a rodent model with the intestinal growth factor glucagon-like peptide-2. J Pediatr Surg 35:847-851, 2000 14. Abe H, Carballo J, Appert HE, et al: The release and fate of the intestinal lysosomal enzymes after acute ischemic injury of the intestine. Surg Gynecol Oncol 135:581-585, 1972 15. Polson H, Mowat C, Himal HS: Experimental and clinical studies of mesenteric infarction. Surg Gynecol Oncol 153:360-362, 1981 16. Lobe TE, Schwartz MZ, Richardson CJ, et al: Hexosaminidase: A marker for intestinal gangrene in necrotizing enterocolitis. J Pediatr Surg 18:449-452, 1983 17. Maeda K, Schwartz MZ, Bamberger MH, et al: A possible serum marker for rejection after small bowel transplantation. Am J Surg 153:68-74, 1987
18. Shah KA, Shurey S, Green CJ: Characterization of apoptosis in intestinal ischaemia-reperfusion injury—A light and electron microscopic study. Int J Exp Pathol 78:355-363, 1997 19. Michalsky MP, Kuhn A, Mehta V, et al: Heparin-binding EGF-like growth factor decreases apoptosis in intestinal epithelial cells in vitro. J Pediatr Surg 36:1130-1135, 2001 20. Ikegami T, Nishizaki T, Uchiyama H, et al: Experimental study of the effects of deletion variant of hepatocyte growth factor on hepatic ischaemia-reperfusion injury. Br J Surg 87:59-64, 2000 21. Sasaki H, Matsuna T, Tanaka N, et al: Activation of apoptosis during the reperfusion phase after rat liver ischemia. Transplant Proc 28:1908-1909, 1996 22. Daeman MARC, Heemskerk VH, van’tVeer C, et al: Functional protection by acute phase proteins ␣1-acid glycoprotein and ␣1-antitrypsin against ischemia/reperfusion injury by preventing apoptosis and inflammation. Circulation 102:1420-1426, 2000 23. Li XY, Li YS, Li BW, et al: Upregulated intragraft gene expression, ICAM-1 and IL-2R molecules, and apoptotic epithelial cells during rejection of rat small intestine allografts. Transpl Proc 32:1283-1286, 2000 24. Coopersmith CM, O’Donnell D, Gordon JI: Bcl-2 inhibits ischemia-reperfusion-induced apoptosis in the intestinal epithelium of mice. Am J Physiol 276:G677-G686, 1999
Discussion Dr Caty (Buffalo, NY): The dose that you use, 150 g, is that superphysiologic and, in particular, I what if you did a more physiologic experiment? You did a liver resection and then 8 days later or 2 days later you made the bowel ischemic and reperfusion. What if you had done that experiment, would you let us know what you found? If you haven’t, could you speculate what you might see? Is there physiologic cause of elevation of hepatocyte growth factor? K.A. Keunzler (response): To answer the second question, we have not done that. This is a superphysiologic dose, and I see it is recombinant human hepatocyte growth factor. So that is an excellent point. We probably should do that in the future. The way that we came up with this dose is that previous experiments in our laboratory in a short bowel model show this was an ideal dose of HGF for mucosal hyperplasia and for increased func-
tion of the bowel, so that is why we chose that particular dose for this experiment. Dr Gittes (Kansas City, MO): Do you know there has been shown, I think, overlap or convergence of the intracellular segue of HGF and interferon-gamma and to reject that system I believe? Do you know, in your cells, if that is also true, or have you looked at any possibility? It would make sense if there is an interplay there in the sense that one is being added due to the up and downregulation of the other? K.A. Keunzler (response): That’s an excellent point. We have not looked at that. I am excited to report that in our next set of experiments we will have DNA microassay data so instead of doing one gene at a time type of analysis we will be able to look at several thousand genes, and try to look at the various cascades. This might be one interplay we could look at.
Background/Purpose: Ischemia-reperfusion (IR) injury to the intestine can result in mucosal damage and cellular death. This study was designed to evaluate the protective effects of pretreatment with hepatocyte growth factor (HGF) on intestine after moderate IR injury. Methods: Control animals (n ⫽ 7) received 48 hours of intravenous saline, and treatment animals (n ⫽ 7) received HGF (150 g/kg/d). After 35 minutes of mesenteric artery occlusion and 120 minutes of reperfusion, serum and jejunal mucosa samples were obtained. Fluorometric assays were performed for hexosaminidase A (HEX A) and -glucuronidase (GLUC), enzyme markers of enterocyte necrosis. Apoptosis was quantified by the TUNEL method. Transcription of tumor necrosis factor-␣ (TNF-␣) and interferon-␥ (IFN-␥) was assessed by multiplex reverse transcription polymerase chain reaction (RT-PCR) Statistical analysis was performed using the Student’s t test. Results: After HGF pretreatment, HEX A and GLUC activities
I
were reduced from 543 ⫾ 28 to 343 ⫾ 35 nmole/h/mL (P ⬍ .01) and 183 ⫾ 29 to 119 ⫾ 22 nmole/h/mL (P ⬍ .01), respectively. Pretreated animals had a reduced number of apoptotic cells per 10 crypts (33 ⫾ 11) compared with untreated rats (225 ⫾ 24) after IR injury (P ⬍ .01). Mean IFN-␥ band intensity was lower in HGF-pretreated animals (0.05 ⫾ 0.02) compared with controls (0.31 ⫾ 0.09; P ⬍ .05). Conclusions: Pretreatment with HGF reduces the severe crypt apoptosis and cellular necrosis after IR injury to the intestine. These data suggest that HGF may be beneficial in attenuating IR damage and thus may have significant clinical application. J Pediatr Surg 37:1093-1097. Copyright 2002, Elsevier Science (USA). All rights reserved. INDEX WORDS: Ischemia-reperfusion injury, hepatocyte growth factor, intestinal mucosa, intestinal ischemia, apoptosis, inflammatory mediators, tumor necrosis factor-alpha, interferon-gamma.
ISCHEMIA-REPERFUSION (IR) injury to the small intestine occurs in children in conditions such as midgut volvulus and resuscitation after shock states. The precise mechanisms of intestinal IR injury have not been elucidated completely, and there currently are no specific treatments once ischemia has taken place. Injury to the small intestine after IR injury appears to be mediated by several factors, including the cytotoxicity of oxygen radicals and the recruitment of inflammatory leukocytes.1 Necrosis has been assumed to be synonymous with epithelial cell death after an ischemic insult. However, more recent reports have noted that apoptosis is a significant, and perhaps the principal contributor to cell death after IR.2,3 In contrast to cell necrosis, apoptosis is an active, gene-directed process. Thus, understanding the sequence of events central to the apoptotic mechanism may lead investigators to potential means of modulating the sequelae of a variety of disease states. We and other investigators have shown that the administration of growth factors such as interleukin-11 (IL-11), glucagonlike-peptide-2 analogue (GLP-2␣), and heparin-binding epidermal growth factor (HB-EGF) after IR injury can result in increased protection of intestinal epithelium, acceleration of cellular renewal, and enhancement of function.4-7 Several studies have shown that hepatocyte growth factor (HGF) is another potent
trophic factor for the small intestine, but its potential effects in intestinal IR injury are unknown.8,9 The purpose of this study was to examine the benefit of systemic HGF pretreatment on mucosal necrosis and apoptosis after IR injury.
Journal of Pediatric Surgery, Vol 37, No 7 (July), 2002: pp 1093-1097
1093
MATERIALS AND METHODS
Animal Care All animal care and use complied with the institutional regulations set forth and approved by the Animal Care and Use Committee of the A. I. duPont Hospital for Children and the Nemours Research Programs (Wilmington, DE). Fourteen adult male Sprague-Dawley rats (Harlan Sprague Dawley, Indianapolis, IN) weighing 275 to 300 g were used in this study. Rats were kept in individual cages and fed standard rat chow and water ad libitum. After allowing the animals to acclimate to the
From the Department of Surgery, A.I. duPont Hospital for Children, Wilmington, DE, and Thomas Jefferson University Hospital, Philadelphia, PA. Presented at the 53rd Annual Meeting of the Section on Surgery of the American Academy of Pediatrics, San Francisco, California, October 19-21, 2001. Address reprint requests to Marshall Z. Schwartz, MD, A.I. duPont Hospital for Children, Department of Surgery, 1600 Rockland Rd, Wilmington, DE 19803. Copyright 2002, Elsevier Science (USA). All rights reserved. 0022-3468/02/3707-0033$35.00/0 doi:10.1053/jpsu.2002.33884
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KUENZLER, PEARSON, AND SCHWARTZ
environment for a period of 1 week, surgical procedures were performed using aseptic technique. General anesthesia was administered with an intramuscular injection of a combination of ketamine (70 mg/kg) and xylazine (14 mg/kg).
Animal Model Each rat underwent placement of a polyethylene jugular venous catheter that was connected to a subcutaneously placed osmotic pump designed to deliver its contents at a rate of 1 L/h (Alzet osmotic minipump, Model 1003D; Alza Scientific Products, Palo Alto, CA). Rats were divided into 2 groups as follows: control animals (n ⫽ 7) received intravenous saline, and treatment animals (n ⫽ 7) received intravenous recombinant human HGF (150 g/kg/d) (Genentech, San Francisco, CA) dissolved in saline. After 48 hours, each animal underwent a midline laparotomy and superior mesenteric artery occlusion for 35 minutes. After 120 minutes of reperfusion, serum and intestine samples were taken. After surgery, rats were killed.
Serum Lysosomal Enzyme Assays Serum was assayed for levels of -glucuronidase (GLUC) and hexosaminidase A (HEX A). GLUC and HEX A activities were quantified by fluorometric assays using 4MU--D-glucuronide and 4MU--D-NAcglucosamine sulfate, respectively, as substrates, according to the protocol of the Thomas Jefferson University Hospital Lysosomal Diseases-Testing Laboratory (Philadelphia, PA). Ten microliters of rat serum was incubated at 37°C with substrate in a final volume of 200 mL for 10 minutes, at which point the reaction was stopped by a glycine carbonate solution (pH ⫽ 10). Fluorometric readings were compared with known standards and expressed as nanomoles per hour per milliliter.
Multiplex RT-PCR to Detect Mucosal Expression of Inflammatory Mediators Midjejunal mucosal samples were harvested at operation and immediately stored at ⫺70°C for later semiquantitative reverse transcription polymerase chain reaction (RT-PCR) analysis. Total RNA was extracted from mucosa using the RNEasy Kit (Qiagen, Valencia, CA) and DNAse treated. RNA concentration and purity were determined by absorbency at 260 and 280 nm. One microgram of total RNA was reverse transcribed. The resultant cDNA was amplified using sense and antisense primers for tumor necrosis factor-␣ (TNF-␣), interferon-␥ (IFN-␥), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, internal standard). The temperature profile of the PCRs consisted of a melting step at 94°C followed by 30 cycles of 30 seconds at 60°C, 60 seconds at 65°C, 30 seconds at 94°C, with a final extension step of 5 minutes at 75°C. Independent experiments established that 30 cycles were within the linear range of the multiplex PCR assay. PCR products were separated on a 4% agarose gel and stained with ethidium bromide. Images were analyzed using EagleEye II analysis software and relative band intensities were calculated.
Histologic Analysis and Immunohistochemical Staining (TUNEL) Cross sections of jejunum 15 cm distal to duodenum were fixed immediately in formalin solution. Cut samples then were embedded in paraffin. Several sections per animal were stained with H&E for light microscopic examination. Areas in each of 4 quadrants of the intestinal cross sections, in 4 separate samples per animal, were graded by a blinded, independent reviewer using the Park/Chiu scoring system of bowel injury as described by Quaedackers et al.10 According to this system, observations of increased damage were assigned increasing points as follows: normal mucosa (0); subepithelial space at villus tips
Fig 1. Mean serum hexosaminidase A activity. HGF-pretreated animals have significantly lower levels after 35 minutes of intestinal ischemia and 120 minutes of reperfusion. ⴱⴱ ⴱⴱP < .01.
(1); extension of subepithelial space with moderate lifting (2); massive lifting, some denuded tips (3); denuded villi, dilated capillaries (4); disintegration of lamina propria (5); crypt layer injury (6); transmucosal infarction (7); and transmural infarction (8). Each animal was assigned an average score. Apoptotic crypt cells were identified using the terminal deoxynucleotidyl transferase (TdT)-mediated dUDP-biotin nick-end labeling (TUNEL) method using a standard laboratory detection kit (TdTFragEL, Oncogene Research Products, San Diego, CA). Samples were dewaxed, rehydrated in ethanol, and incubated with 3% hydrogen peroxide. Slides were treated with 100 L equilibration buffer for 30 minutes. Sections were then incubated for 1 hour at 37°C with 60 L TdT enzyme, followed by incubation for 30 minutes with 100 L conjugate solution, and detection with diaminobenzidine (DAB) solution. Sets of slides were counterstained alternately with either hematoxylin or methyl green. TUNEL-positive cells were counted by examining crypts of approximately equal size (70 m or 30 cells around) in 10 regions per slide per animal. Results were expressed as TUNEL ⫹ cells / 10 crypts.
Statistical Analysis Statistical analysis was performed using Student’s t test and expressed as mean ⫾ SEM.
RESULTS
Serum Lysosomal Enzyme Assays Mean serum HEX A in untreated control animals increased from 314 ⫾ 47 nmole/h/mL at baseline (before bowel ischemia) to 543 ⫾ 28 nmole/h/mL after 120 minutes of reperfusion (P ⬍ .01). In HGF-pretreated animals, mean HEX A levels also significantly rose with IR injury from 219 ⫾ 33 to 343 ⫾ 35 nmole/h/mL (P ⬍ .05), but these animals had HEX A values that were significantly lower after 120 minutes of reperfusion (P ⬍ .01; Fig 1). Mean serum GLUC in untreated control animals rose from 100 ⫾ 16 nmole/h/mL at baseline to 183 ⫾ 29 nmole/h/mL after 120 minutes of reperfusion (P ⬍ .01). In HGF-pretreated animals, mean GLUC levels also increased significantly with IR injury from 63 ⫾ 14 to 119 ⫾ 22 nmole/h/mL (P ⬍ .05), but these animals had GLUC values that were significantly lower after 120 minutes of reperfusion (P ⬍ .01; Fig 2).
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Fig 2. Mean serum -glucuronidase activity. HGF-pretreated animals have significantly lower levels after 35 minutes of intestinal ischemia and 120 minutes of reperfusion. **P < .01.
RT-PCR Average TNF-␣ relative band intensities after RTPCR were not significantly different in the control (0.74 ⫾ 0.06) and HGF-treated (0.85 ⫾ 0.07) groups. Mean IFN-␥ relative band intensities, however, were significantly reduced in treated animals (0.05 ⫾ 0.02) when compared with control animals (0.31 ⫾ 0.09; P ⬍ .05; Table 1). Histologic Analysis Portions of both animal groups showed varying damage to intestinal villi after 35 minutes of ischemia and 120 minutes of reperfusion (Figs 3A and 3B). Mean histologic injury scores were not significantly different between the untreated control group (2.67) and in the HGF pretreatment group (2.33). HGF-treated animals had significantly fewer TUNEL-positive cells per 10 crypts (33 ⫾ 11) than untreated control animals (225 ⫾ 24; P ⬍ .01) subjected to IR injury (Fig 4). DISCUSSION
Over the last 2 decades, our laboratory has shown that several gastrointestinal peptides can enhance intestinal growth and function in models of intestinal adaptation and inflammatory bowel disease.11-13 Among these peptides, HGF appears to show especially potent trophic effects.8,9 Because enterocyte cell death after IR injury is known to result from a combination of apoptosis and necrosis,2 we sought to examine the effects of pretreatment with HGF on preventing these modes of cell death. To evaluate the effects of IR and the potential benefit of HGF pretreatment, we chose to measure the release of
Fig 3. (A) Light micrograph of uninjured rat jejunal mucosa shows regular villus architecture and normal crypt depth. (H&E, original magnification ⴛ100.) (B) Light micrograph of rat jejunal mucosa after 35 minutes of intestinal ischemia and 120 minutes of reperfusion. Note the thickening, shortening and loss of villi, reduction of crypt depth, and inflammatory cell infiltrate. (H&E, original magnification ⴛ100.)
certain lysosomal enzymes and evaluate morphologic and apoptotic changes in the small intestine after this injury. Abe et al14 were the first investigators to show that -glucuronidase (GLUC), was released into the circulation after intestinal IR.14 Subsequently, Polson et al15 showed that hexosaminidase (HEX) increased in the
Table 1. Effect of HGF Pretreatment on Mucosal mRNA Expression After 35 Minutes of Intestinal Ischemia and 120 Minutes of Reperfusion
TNF-␣/GAPDH IFN-␥/GAPDH
IR
IR ⫹ HGF
P Value
0.7400 ⫾ .06 0.3116 ⫾ .09
0.8467 ⫾ .07 0.0506 ⫾ .02
.303 .018
NOTE. Relative band intensities after RT-PCR are shown for TNF-␣ and IFN-␥ compared with the GAPDH internal standard. *P ⬍ .05.
Fig 4. Mean number of TUNEL ⴙ cells per 10 crypts. HGF-pretreated animals show a significant reduction at 120 minutes of reperfusion. ⴱⴱ ⴱⴱP < .01.
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serum after IR injury.15 Further studies by Lobe et al16 and Maeda et al17 evaluated HEX in neonatal clinical settings and in an animal model of intestinal transplantation. Additional studies in our laboratory have been directed toward the potential role for growth factors in intestinal IR injury. Recently, we have shown that both GLP-2␣ and IL-11 can attenuate injury and enhance the recovery of absorptive function in a rat model of IR.4,7 In the current study, tissue samples were taken after 120 minutes of reperfusion because previous reports showed histologic evidence of high levels of crypt cell apoptosis in the early hours of reperfusion.24 Our results in the current study show that systemic infusion of HGF for 48 hours before IR injury result in a significant reduction of each of these lysosomal enzymes compared with small intestine subjected to IR injury without HGF pretreatment. We believe this reflects a “protective” effect of HGF on the mucosal cells of the small intestine. Further support for this conclusion is noted in our apoptosis studies. It should be noted that baseline levels of these enzymes (before injury) were not significantly different between the control and treated groups; thus, it appears that HGF pretreatment does not affect these enzymes before IR. Apoptosis is a significant mode of cell death in the intestine after IR injury.3 In healthy bowel, apoptosis allows continuous cell renewal as enterocytes are shed from villus tips. Normally, crypt cell apoptosis is rare (less than 1 cell per 10 crypts) in normal jejunum, but after IR it can be markedly increased in the first few hours of reperfusion.18 The major advantage of the TUNEL technique of identifying apoptotic cells in situ is the ability to localize which enterocytes are affected. In this study we showed an 85% decrease in apoptotic cells after HGF pretreatment. However, we were not able to show a benefit in the histologic injury score. The reason for this is not known. Because apoptosis is a gene-mediated cellular process, it is possible that growth factors such as HGF may have a role in affecting this phenomenon. Michalsky et al19
have recently observed inhibition of apoptosis using another growth factor, HB-EGF, in intestinal epithelial cell cultures stimulated by inflammatory mediators. In that study, both flow cytometric studies and “cell death” ELISAs confirmed that treatment with HB-EGF results in significant reductions in intestinal cell apoptosis. In an hepatic IR model, Ikegami et al20 have done experiments with the “deletion variant” of HGF, a splice variant whose 5 amino acid alterations render it more mitogenic toward hepatocytes specifically. These investigators showed reductions in both serum markers of hepatocyte death and in situ liver apoptosis in their HGF-treated rats. The exact mechanisms for accelerated apoptosis have not yet been confirmed. However, several studies have implicated inflammatory mediators such as TNF-␣ and IFN-␥ after hepatic, renal, and intestinal IR.21,22 Li et al23 recently have reported an increase in expression of mRNA for IFN-␥ in rats showing significant crypt cell apoptosis during small bowel transplant rejection. Our findings in this study confirm reduced transcription of this cytokine concurrent with reductions in cell death and crypt cell apoptosis. Although the specific mechanism for the apparent antiapoptotic effects of HGF have not yet been elucidated, we hypothesize that in the early period of reperfusion, HGF may be attenuating the mucosal injury that precedes apoptosis. Alternatively, HGF might be stimulating genetic antiapoptotic regulators such as Bcl-2, which have been shown to render transgenic mice resistant to IR-induced apoptosis.24 We postulate that in the future it may be appropriate to pretreat patients at risk for intestinal ischemia to attenuate IR damage, such as those with abdominal aortic aneurysms or intestinal transplantation, or at the time of ischemic injury, such as in necrotizing enterocolitis or midgut volvulus. ACKNOWLEDGMENTS The authors thank Juan P. Palazzo, MD, of the Department of Pathology, Thomas Jefferson University Hospital, for his teaching and help with histologic injury grading and David A. Wenger, PhD, of the Thomas Jefferson University Hospital Lysosomal Diseases Testing Laboratory, for his assistance with the enzyme assays.
REFERENCES 1. Schoenberg MH, Beger HG: Reperfusion injury after intestinal ischemia. Crit Care Med 21:1376-1386, 1993 2. Shah KA, Shurey S, Green CJ: Apoptosis after intestinal ischemia-reperfusion injury. Transplantation 64:1393-1397, 1997 3. Noda T, Iwakiri R, Fujimoto K, et al: Programmed cell death induced by ischemia-reperfusion in rat intestinal mucosa. Am J Physiol 274:G270-276, 1998 4. Prasad R, Alavi K, Schwartz MZ: Glucagonlike peptide-2 analogue enhances intestinal mucosal mass and absorptive function after ischemia-reperfusion injury. J Pediatr Surg 35:1537-1539, 2000 5. Du XX, Liu Q, Zhixiang Y, et al: Protective effects of interleukin-11 in a murine model of ischemic bowel necrosis. Am J Physiol 272:G545-552, 1997
6. Pillai SB, Hinman CE, Luquette MH, et al: Heparin-binding epidermal growth factor protects rat intestine from ischemia/reperfusion injury. J Surg Res 87:225-231, 1999 7. Kuenzler KA, Pearson PY, Schwartz MZ: Interleukin-11 enhances intestinal absorptive function after ischemia-reperfusion injury. J Pediatr Surg 37:457-459, 2002 8. Kato Y, Yu D, Schwartz MZ: Enhancement of intestinal adaptation by hepatocyte growth factor. J Pediatr Surg 33:235-239, 1998 9. Kato Y, Yu D, Lukish JR, et al: Hepatocyte growth factor enhances intestinal mucosal cell function and mass in vivo. J Pediatr Surg 32:991-994, 1997 10. Quaedackers JSLT, Beuk RJ, Bennet A, et al: An evaluation of
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methods for grading histologic injury following ischemia/reperfusion of the small bowel. Transplant Proc 32:1307-1310, 2000 11. Lukish JR, Yu D, Kato Y, et al: Persistent upregulation in intestinal function beyond intestinal adaptation following luminal growth factor perfusion. Surg Forum 47:675-678, 1996 12. Alavi K, Prasad R, Lundgren K, et al: Interleukin-11 enhances small intestinal absorptive function and mucosal mass after intestinal adaptation. J Pediatr Surg 35:371-374, 2000 13. Alavi K, Schwartz MZ, Palazzo JP, et al: Treatment of inflammatory bowel disease in a rodent model with the intestinal growth factor glucagon-like peptide-2. J Pediatr Surg 35:847-851, 2000 14. Abe H, Carballo J, Appert HE, et al: The release and fate of the intestinal lysosomal enzymes after acute ischemic injury of the intestine. Surg Gynecol Oncol 135:581-585, 1972 15. Polson H, Mowat C, Himal HS: Experimental and clinical studies of mesenteric infarction. Surg Gynecol Oncol 153:360-362, 1981 16. Lobe TE, Schwartz MZ, Richardson CJ, et al: Hexosaminidase: A marker for intestinal gangrene in necrotizing enterocolitis. J Pediatr Surg 18:449-452, 1983 17. Maeda K, Schwartz MZ, Bamberger MH, et al: A possible serum marker for rejection after small bowel transplantation. Am J Surg 153:68-74, 1987
18. Shah KA, Shurey S, Green CJ: Characterization of apoptosis in intestinal ischaemia-reperfusion injury—A light and electron microscopic study. Int J Exp Pathol 78:355-363, 1997 19. Michalsky MP, Kuhn A, Mehta V, et al: Heparin-binding EGF-like growth factor decreases apoptosis in intestinal epithelial cells in vitro. J Pediatr Surg 36:1130-1135, 2001 20. Ikegami T, Nishizaki T, Uchiyama H, et al: Experimental study of the effects of deletion variant of hepatocyte growth factor on hepatic ischaemia-reperfusion injury. Br J Surg 87:59-64, 2000 21. Sasaki H, Matsuna T, Tanaka N, et al: Activation of apoptosis during the reperfusion phase after rat liver ischemia. Transplant Proc 28:1908-1909, 1996 22. Daeman MARC, Heemskerk VH, van’tVeer C, et al: Functional protection by acute phase proteins ␣1-acid glycoprotein and ␣1-antitrypsin against ischemia/reperfusion injury by preventing apoptosis and inflammation. Circulation 102:1420-1426, 2000 23. Li XY, Li YS, Li BW, et al: Upregulated intragraft gene expression, ICAM-1 and IL-2R molecules, and apoptotic epithelial cells during rejection of rat small intestine allografts. Transpl Proc 32:1283-1286, 2000 24. Coopersmith CM, O’Donnell D, Gordon JI: Bcl-2 inhibits ischemia-reperfusion-induced apoptosis in the intestinal epithelium of mice. Am J Physiol 276:G677-G686, 1999
Discussion Dr Caty (Buffalo, NY): The dose that you use, 150 g, is that superphysiologic and, in particular, I what if you did a more physiologic experiment? You did a liver resection and then 8 days later or 2 days later you made the bowel ischemic and reperfusion. What if you had done that experiment, would you let us know what you found? If you haven’t, could you speculate what you might see? Is there physiologic cause of elevation of hepatocyte growth factor? K.A. Keunzler (response): To answer the second question, we have not done that. This is a superphysiologic dose, and I see it is recombinant human hepatocyte growth factor. So that is an excellent point. We probably should do that in the future. The way that we came up with this dose is that previous experiments in our laboratory in a short bowel model show this was an ideal dose of HGF for mucosal hyperplasia and for increased func-
tion of the bowel, so that is why we chose that particular dose for this experiment. Dr Gittes (Kansas City, MO): Do you know there has been shown, I think, overlap or convergence of the intracellular segue of HGF and interferon-gamma and to reject that system I believe? Do you know, in your cells, if that is also true, or have you looked at any possibility? It would make sense if there is an interplay there in the sense that one is being added due to the up and downregulation of the other? K.A. Keunzler (response): That’s an excellent point. We have not looked at that. I am excited to report that in our next set of experiments we will have DNA microassay data so instead of doing one gene at a time type of analysis we will be able to look at several thousand genes, and try to look at the various cascades. This might be one interplay we could look at.