PURPOSE: The pancreatic capillary blood flow (PCBF) was studied to determine its alterations during caerulein-induced pancreatitis in rats. METHODS: Twenty rats were divided in groups: control and caerulein. A laser-Doppler flowmeter to measure PCBF continuously was used. Blood pressure (BP) and heart rate (HR) were monitored. Serum biochemistry analyses were determined. Histopathological study was performed. RESULTS: The PCBF measured a mean of 109.08 ± 14.54% and 68.24 ± 10.47% in control group and caerulein group, respectively. Caerulein group had a mean decrease of 31.75 ± 16.79%. The serum amylase was 1323.70 ± 239.10U.I-1 and 2184.60 ± 700.46U.I-1 in control and caerulein groups, respectively. There was a significant difference in the PCBF (p<0.05) and serum amylase (p<0.05) when compared to control and caerulein groups. Although micro and microvacuolization were seen in 30% in caerulein group, no significant difference was seen between the groups. CONCLUSION: A decrease in the PCBF may be one of the leading events and it is present before histopathological tissue injury had been established in this model of acute pancreatitis.
Blood flow; Caerulein; Pancreatitis; Laser-Doppler
OBJETIVO: O fluxo capilar pancreático (FCP) foi estudado para determinar suas alterações durante a pancreatite aguda induzida por ceruleína, em ratos. MÉTODOS: Vinte ratos foram divididos em grupo controle e grupo ceruleína. Um laser-Doppler fluxímetro foi empregado para determinar, continuamente, o FCP durante 120 minutos. A pressão arterial média (PAM) e a freqüência cardíaca (FC) foram determinadas, durante o experimento. Análise bioquímica sérica e estudo histopatológico, por microscopia ótica, do tecido pancreático foram realizados, ao final do experimento. RESULTADOS: O FCP foi em média 109,08 ± 2,17% e 68,24 ± 16,79% nos grupos controle e ceruleína , respectivamente. No grupo ceruleína, houve uma diminuição média de 31,75 ± 16,79%. Os níveis de amilase sérica foram de 1323,70 ± 239,10U.I-1 e 2184,60 ± 700,46U.I-1 nos grupos controle e ceruleína, respectivamente. Houve diferença significante (p<0,05) no FCP e na amilasemia, quando comparado o grupo controle com o grupo ceruleína. Embora micro e macrovacuolização estivessem presentes no grupo ceruleína, não houve diferença histológica entre os grupos. CONCLUSÃO: A diminuição do FCP parece um evento precoce, antecedendo o aparecimento de alterações histopatológicas, por microscopia ótica, que caracterizam este modelo de pancreatite edematosa aguda.
Fluxo sanguíneo; Ceruleína; Pancreatite; Laser-Doppler
Pancreatic capillary blood flow during caerulein-induced pancreatitis evaluated by a laser-doppler flowmeter in rats
Estudo das alterações do fluxo capilar pancreático após infusão de ceruleína avaliado por laser-Doppler em ratos
Roberto Ferreira Meirelles Jr.I; Reginaldo CenevivaII; José Liberato Ferreira CabocloIII; Michael M. EisenbergIV
IPós-graduando da área de Clínica Cirúrgica do Departamento de Cirurgia e Anatomia da FMRP-USP e Prof. Assistente do Departamento de Cirurgia da FAMERP- S.P.
IIProfessor Titular do Departamento de Cirurgia e Anatomia da FMRP-USP
IIIProfessor Titular do Departamento de Cirurgia da FAMERP- S.P
IVProfessor Adjunto do Departamento de Cirurgia da Cornell University Medical College, NY, EUA
Correspondence Correspondence to Dr. Roberto Ferreira Meirelles Junior Faculdade de Medicina de São José do Rio Preto, S.P. Departamento de Cirurgia Av. Brigadeiro Faria Lima, 5544 Cep; 15090-000 São José do Rio Preto, S.P. e-mail: firstname.lastname@example.org
PURPOSE: The pancreatic capillary blood flow (PCBF) was studied to determine its alterations during caerulein-induced pancreatitis in rats.
METHODS: Twenty rats were divided in groups: control and caerulein. A laser-Doppler flowmeter to measure PCBF continuously was used. Blood pressure (BP) and heart rate (HR) were monitored. Serum biochemistry analyses were determined. Histopathological study was performed.
RESULTS: The PCBF measured a mean of 109.08 ± 14.54% and 68.24 ± 10.47% in control group and caerulein group, respectively. Caerulein group had a mean decrease of 31.75 ± 16.79%. The serum amylase was 1323.70 ± 239.10U.I-1 and 2184.60 ± 700.46U.I-1 in control and caerulein groups, respectively. There was a significant difference in the PCBF (p<0.05) and serum amylase (p<0.05) when compared to control and caerulein groups. Although micro and microvacuolization were seen in 30% in caerulein group, no significant difference was seen between the groups.
CONCLUSION: A decrease in the PCBF may be one of the leading events and it is present before histopathological tissue injury had been established in this model of acute pancreatitis.
Key Words: Blood flow. Caerulein. Pancreatitis. Laser-Doppler.
OBJETIVO: O fluxo capilar pancreático (FCP) foi estudado para determinar suas alterações durante a pancreatite aguda induzida por ceruleína, em ratos.
MÉTODOS: Vinte ratos foram divididos em grupo controle e grupo ceruleína. Um laser-Doppler fluxímetro foi empregado para determinar, continuamente, o FCP durante 120 minutos. A pressão arterial média (PAM) e a freqüência cardíaca (FC) foram determinadas, durante o experimento. Análise bioquímica sérica e estudo histopatológico, por microscopia ótica, do tecido pancreático foram realizados, ao final do experimento.
RESULTADOS: O FCP foi em média 109,08 ± 2,17% e 68,24 ± 16,79% nos grupos controle e ceruleína , respectivamente. No grupo ceruleína, houve uma diminuição média de 31,75 ± 16,79%. Os níveis de amilase sérica foram de 1323,70 ± 239,10U.I-1 e 2184,60 ± 700,46U.I-1 nos grupos controle e ceruleína, respectivamente. Houve diferença significante (p<0,05) no FCP e na amilasemia, quando comparado o grupo controle com o grupo ceruleína. Embora micro e macrovacuolização estivessem presentes no grupo ceruleína, não houve diferença histológica entre os grupos.
CONCLUSÃO: A diminuição do FCP parece um evento precoce, antecedendo o aparecimento de alterações histopatológicas, por microscopia ótica, que caracterizam este modelo de pancreatite edematosa aguda.
Descritores: Fluxo sanguíneo. Ceruleína. Pancreatite. Laser-Doppler.
Acute pancreatitis (AP) is an acute inflammatory process of the pancreas with variable involvement of other regional tissues or remote organ systems1,2. Its clinical features are severe upper abdominal pain associated with vomiting, leukocytoses and increase of pancreatic enzymes1,2,3.
Pancreatic blood flow seems to be involved in the pathogenesis of AP. Pancreatic blood flow impairment is an important mechanism in the transition of edematous to necrohemorrhagic pancreatitis4. Although changes in the pancreatic blood flow during AP are well reported4,5,6,7, the initial mechanisms involving its pathogenesis remains poorly understood8. Besides, it is unclear whether the pancreatic capillary blood flow (PCBF) changes are cause or consequence of the AP5.
The purpose of this experiment was to study pancreatic capillary blood flow PCBF changes, using a laser-Doppler flowmeter, (LDF) during caerulein-induced pancreatitis.
Surgical preparation: Twenty Sprague-Dawley male rats weighing between 320 and 410 g were used. All rats were starved for 18 hours prior to the experiment, except for water ad libitum. A single subcutaneous injection of 25% urethan anesthetic (1.75 g of urethane/ 1000 g body weight; Urethane, Sigma, St. Louis, MO) was used. The body temperature during the experiment was kept between 36.4 -36.6o C using a thermo controller (made by Béla Kurucz, E.E., Maglód, Hungary). An arterial and venous line were obtained via the right iliac artery and left iliac vein that were isolated and cannulated with heparinized PE-50 polyethylene tubing. The abdominal wall was opened by a mid-line incision extending from the xiphoid to the suprapubic region. The pancreas was isolated and two gauze sponges were placed between the posterior abdominal wall and the pancreas. The laser-Doppler probe was placed on the anterior surface of the body of the pancreas.
Measurement of PCBF, Blood pressure (BP) and heart rate (HR): PCBF measurement was performed with a laser-Doppler Capillary Perfusion Monitor (model LD- 6000, Medpacific Corp., Seattle, W A) (9). The laser-Doppler flowmeter was connected to a computer (IBM PS/2 model 50Z, Armonk, NY) equipped with an appropriate software package (LD-6000 Data Collection; written by Howard Amols, Ph.D., Columbia University, New York, NY) that collected, recorded and stored six data points per second of PCBF.
BP and HR were monitored throughout the experiment (Weco VT -1, Winston Electronics Co., Millbrae, CA) via the right iliac artery.
After a 20 minutes stability period, the baseline of the PCBF, BP and HR was determined during the next 10 minutes; the means were considered 100%. PCBF was measured continuously over 120 minutes with recordings of the mean and standard deviation taken every 5 minutes. BP and HR were recorded every 5 minutes throughout the experiment.
Pancreatitis model and spin-trapping nitrone solution: Acute pancreatitis was induced using 5 X 10-6 g/ 1000 g body weight/ h of caerulein (Sigma, St. Louis, MO) i.v. infusion10. This infusion began immediately after the baseline measurements.
The PBN (Sigma, St. Louis, MO) compound was used in a dose of 150 mg/ 1000 g body weight. Special precautions were taken during handling the PBN, since it is inactivated by light and air. The PBN was diluted in dimethylsulfate (DMS; Sigma, St. Louis, MO).
Experimental protocol: The animals were divided in two groups of ten. All groups received 0.9% sodium chloride intravenously (0.083 ml/ 1000 g body weight/ min.; Syringe infusion Pump 22, Harvard Apparatus, South Natick, MA) to compensate for insensible losses. This infusion began when the laser-Doppler probe was placed on the pancreas.
Group control: Animals in the control group received DMS 20 minutes before baseline and 0.9% sodium chloride i.v. after baseline.
Group caerulein: Animals in the caerulein-induced pancreatitis received DMS 20 minutes before baseline and caerulein solution after baseline.
Blood collection, biopsy and analysis: At the end of the experiment arterial blood samples were taken to determine gases (288 Blood Gas System, Ciba-Corning Diagnostics Corp., Medfield, MA) at the Blood Gas Laboratory, Lenox Hill Hospital, New York, NY. Venous blood samples were taken to determine serum amylase, glucose, calcium, sodium, potassium and chloride (Kodak Ektachem 700 XR Analyzer, Eastman Kodak, Rochester, NY) at Lenox Hill Hospital Laboratory, New York, NY.
Pancreatic biopsies were taken from the pancreatic tissue underlying the laser-Doppler probe.
Histophatological analysis: The pancreatic biopsies from forty rats were fixed in Bouin's solution, paraffin embedded and sectioned at 4 microns and then stained with hematoxilin phloxin safran stain. The slides were examined randomly and blindly by two pathologists using a Optiphot Labpot Nikon microscope (Yokohama, Japan). The slides were screened for vacuolation, piknosis and ballooning degeneration. The vacuolation was characterized by the presence of micro and macrovacuolation of the cytoplasm that was normal in color and the granules were distinct. Piknosis and ballooning degeneration was characterized by small foci of piknosis of the nuclei and distention of the cytoplasm becoming pale pink in color with loss of granules.
Statistical analysis: Statistical analysis was performed using PC Statistical Software, (Human Systems Dynamics, Northridge, CA). The results are described as the mean ± standard deviation. Student's t-test was employed to make comparison between group means. P values less than 0.05 were considered significant. All PCBF, BP and HR results were expressed in percentage.
PCBF measured a mean of 109.08 ± 14.54%, 68.24 ± 10.47% in control and caerulein groups, respectively. The PCBF measurement did not change significantly (p>0.05) in control group throughout the experiment. The PCBF decreased over time a mean of 31.75 ± 16.79% in caerulein group. These PCBF decreases were statistically (p<0.05) significant after 20 minutes following baseline for caerulein group when compared with control group. (Fig.1).
The BP measured a mean of 93.11 ± 9.85%, 108.81 ± 16.43% in control and caerulein groups, respectively. The BP increased (p<0.05) throughout the experiment only in caerulein group and it was significant (p<0.05) when compared with control group.
The HR measured a mean of 102.88 ± 14.80%, 121.42 ± 12.26% in control and caerulein groups, respectively. The HR increased significantly (p<0.05) throughout the experiment only in caerulein group and it was significant (p<0.05) when compared with control group.
The serum amylase was 1323.70 ± 239.10 U/l, 2184.60 ± 700.46 U/l in control and caerulein groups, respectively. There was significant increase (p<0.01) when comparing control group with caerulein group. (Table I).
The histopathological study reviewed no qualitative changes in 30% of all slides. Vacuolation of the cytoplasm in the acinar cells was found in 3 slides in caerulein group. These lesions were isolated in some cases, and multifocal in others. Small foci of piknosis and ballooning degenaration were seen in 6 and 1 slide(s) in control and caerulein groups, respectively. No qualitative difference was seen between groups.
The importance of blood flow in the pathogenesis of acute pancreatitis has been investigated since 1862 by Panum when pancreatic hemorrhage was induced by injecting small particles of wax into the pancreatic artery of experimental animals11. Caerulein-induced pancreatitis is characterized by acinar cell damage and interstitial edema10,12. Enclosure of intracellular organelles within autophagic vacuoles with subsequent lysosomal degradation appears to contribute to the destruction of the acinar cells in this model of acute pancreatitis12,13. During caerulein-induced pancreatitis there is a collapse in the pancreatic capillaries and surface blebbing, formation of cytoplasmatic vacuoles, edema, and swollen mitochondria in the endothelial cells14,15,16. These structural alterations were present early in the experimental edematous pancreatitis14. Overall, experimental17 and clinical5 studies have found that during acute pancreatitis there is a decrease in the blood flow to the pancreas. In our experiment, the PCBF decreased significantly a mean of 31% after 20 minutes of caerulein infusion. It leads us to assume that the PCBF impairment may allow the pancreas to become subject and susceptible to ischemia in this model of pancreatitis. Whether ischemia is a cause or an effect during the course of acute pancreatitis is still controversial. Nevertheless, the important relationship between pancreatic blood flow and the complications following acute pancreatitis should not be underestimated5. Ischemia seems to play a key role in the transition from pancreatic edema to necrosis and improvement of capillary perfusion has been shown to be an efficient therapeutic tool4,18. Ischemia can serve as an important co-factor to potentiate pancreatitis and convert an incipient insult to the pancreas into a frank pancreatitis8. During caerulein-induced pancreatitis, sympathetic excitation induced by water-immersion precipitated hemorrhagic pancreatitis19 and phenylephrine exacerbated the acute pancreatitis20. In this study, supramaximal caerulein infusion (5 X 10-6 g.kg-1 body weight.h-1) caused serum amylase and calcium increase with hipoglicemia. The PCBF decreased associated with increased BP and HR. Early findings of vacuolation were present in 30% of the caerulein group.
The caerulein effects in the pancreas are dosage and time related20. In caerulein-induced AP the PBF determined by LDF it is decreased from 43% to 51% after 2-5 hours of caerulein infusion in rats1,22. When H2-clearence technique is used to determine PBF, caerulein doses at 0.5, 10 and 40 mg.kg-1.h-1 there is also a decrease in PBF by 30 to 40% after 5 hours of AP in rats23,24,25,26. However, doses of 5 and 20 mg.kg-1.h-1 showed an increase in PBF23. In that study, there was a volemic replacement with a crystalloid infusion rate three times higher than our study. Besides, normovolemia does not represent the AP natural clinical evolution where there is intravascular fluid depletion due to increased capillary permeability14.
Nevertheless, experimental studies using dogs presented increased PBF after caerulein-induced AP27,28,29 and increase18 as well decrease30 in PBF when rabbits.
The impairment of PBF associated to increased BP and HR might be an adrenergic response due to caerulein infusion, the caerulein direct effect in the pancreas or vasoactive substances released from the inflamed pancreas. The caerulein circulatory effect is known and it is specie dependent. In rats, there is an increase in BP, as seen in the caerulein group. In humans, there is arterial hypotension following caerulein infusion31. In our study, both groups were submitted to the same method, differing only by the caerulein infusion, thus the adrenergic response may not be directly responsible by decreasing PCBF. Furthermore, there was no correlation over time among increasing BP and HR with decreasing PCBF. BP and HR remained increased from the beginning of the experiment while PCBF had a progressive decrease after 20 minutes of caerulein infusion.
The beginning of PCBF decrease is in accordance with early acinar intracellular changes seen after 15 minutes of caerulein infusion32. Electron microscopy showed endothelial injury such as vascular degeneration, disorganized intracellular junctions, perivascular edema and vascular occlusion during caerulein-incuced AP. Early edema formation is due to increased capillary permeability in caerulein-induced AP14.
Platelet activation factor (PAF) seems to be directly involved in caerulein-induced AP pathogenesis. PAF inhibitors attenuate hyperamylasemia, edema formation and improve histopathological changes in this AP model33. PAF impairs pancreatic microcirculation in early phases of caerulein-induced AP. PCBF decreased before we could identify edema and leukocyte infiltration in the histopathological study.
The decreased glucose serum levels seen after AP induction is a caerulein modulating insulin output effect in the pancreatic beta-cells34. Caerulein promotes insulin releasing by interaction with beta-cells receptors35,36.
In conclusion, a decrease in the PCBF may be one of the leading events and it is present before histopathological tissue injury had been established in this model of acute pancreatitis. Future experimental investigations should address what are the causes leading to early PCBF impairment such as PAF, free radicals, TNF-a, and nitric oxide.
- 1. Bradley III, EL. A clinical based classification system for acute pancreatitis. Summary of the international symposium on acute pancreatitis, Arch Surg1993; 128(5): 586-90.
- 2. Cunha, J.E.M. Pancreatite aguda: uma classificação com base clínica.Arq Gastroenterol1994; 31(2): 52-6.
- 3. Howard, A.R. Pancreas. In: Schwartz, SI., Shires GT, Spencer FC et al.(ed.). Principles of Surgery1995:1406.
- 4. Klar E. Etiology and pathogenesis of acute pancreatitis. Helv Chir Acta 1992; 59 (1):7-16.
- 5. Clavien PA, Hauser H, Meyer P et al. Value of contrast-enhanced computerized tomography in the early diagnosis and prognosis of acute pancreatitis. A prospective study of 202 patients. Am J Surg 1988;155 (3):457-66.
- 6. Klar E, Messmer K, Warshaw AL et al. Pancreatic ischemia in experimental acute pancreatitis: mechanism, significance and therapy. Br J Surg 1990; 77(11):1205-10.
- 7. Klar E, Rattner DW, Compton C et al. Adverse effect of therapeutic vasoconstrictors in experimental acute pancreatitis. Ann Surg 1991; 214(2):168-74.
- 8. Prinz RA. Mechanisms of acute pancreatitis. Int J Pancreatol 1991; 9:31-8.
- 9. Dib JA, Cooper-Vastola SA, Meirelles Jr. RF et al. Acute effects of ethanol and ethanol plus furosemide on pancreatic capillary blood flow in rats. Am J Surg1993; 166(1):18-23.
- 10. Lampel M, Kern HF. Acute interstitial pancreatitis in the rat induced by excessive doses of a pancreatic segretagogue. Virchows Arch Pathol Anat Histol 1977; 373(2): 97-117.
- 11. Panum PL. Experimentelle butrage zur lehre von der embolie. Virchows Arch Anat Pathol Histol 1862; 25: 308.
- 12. Watanabe O, Baccino FM, Steer ML et al. Supramaximal caerulein stimulation and ultrastructure of rat pancreatic acinar cell: early morphological changes during development of experimental pancreatitis. Am J Phsyiol 1984; 246(4): 457-67.
- 13. Saluja AK, Saito I, Saluja, M et al. In vivo rat pancreatic acinar cell function during supramaximal stimulation with caerulein. Am J Physiol 1985; 249(6): 702-10.
- 14. Gress TM, Arnold R, Adler G. Structural alterations of pancreatic microvasculature in cerulein-induced pancreatitis in the rat. Res Exp Med 1990;190(6): 401-12.
- 15. Mcentee GP, Leahy A, Cotell D et al. A three dimensional morphological study of the pancreatic microvasculature in caerulein induced experimental pancreatitis. Br J Surg 1989; 76(8): 853-5.
- 16. Kelly MD, McEntee GP, McGeeney KF et al. Microvasculature of the pancreas, liver, and kidney in cerulein-induced pancreatitis. Arch Surg 1993;128(3): 293-5.
- 17. Klar E, Endrich B, Messmer K. Microcirculation of the pancreas. A quantitative study of physiology and changes in pancreatitis. Int J Microcirc Clin Exp 1990; 9(1): 85-101.
- 18. Klar E, Schratt W, Foitzik T et al. Impact of microcirculatory flow pattern changes on the development of acute edematous and necrotizing pancreatitis in rabbit pancreas. Dig Dis Sci 1994; 39(12): 2639-44.
- 19. Yamaguchi H, Kimura T, Nawata H. Does stress play a role in the development of severe pancreatitis in rats?. Gastroenterology 1990; 98(6): 682-8.
- 20. Fujimura K, Kubota Y, Ogura M et al. Role of endogenous platelet-activating factor in caerulein-induced acute pancreatitis in rats: protective effects of a PAF-antagonist. J Gastroenterol Hepatol 1992; 7(2): 199-202.
- 21. Konturek SJ, Dembinski A, Konturek PJ et al. Role of platelet activating factor in pathogenesis of acute pancreatitis in rats. Gut 1992; 33(9): 1268-74.
- 22. Tomaszewska R, Dembinski A, Warzecha Z et al. Platelet activating factor (PAF) inhibitor (TVC-309) reduces caerulein- and PAF-induced pancreatitis. A morphologic and functional study in the rat. J Physiol Pharmacol 1992; 43(4): 345-52.
- 23. Satoh A, Shimosegawa T, Abe T et al. Role of nitric oxide in the pancreatic blood flow response to caerulein. Pancreas 1994; 9(5): 574-9.
- 24. Furukawa M, Kimura T, Sumii T et al. Role of local pancreatic blood flow in development of hemorrhagic pancreatitis induced by stress in rats. Pancreas 1993; 8(4): 499-505.
- 25. Furukawa M, Kimura T, Yamaguchi H et al. Role of oxygen-derived free radicals in hemorrhagic pancreatitis induced by stress and cerulein in rats. Pancreas 1994; 9(1): 67-72.
- 26. Liu XH. Kimura T, Ishikawa H et al. Effect of endothelin-1 on the development of hemorrhagic pacreatitis in the rats. Scand. J. Gastroenterol; 30(3): 276-82.
- 27. Dorigotti L, Glasser AH. Comparative effects of caerulein, pancreozymin and secretin on pancreatic blood flow. Experientia, Basel 1968; 24(8): 806-7.
- 28. Papp M, Feher S, Varga, B et al. Humoral influences on local blood flow and external secretion of the resting pancreas. Acta Med Acad Sci Hung 1977; 34(4):185-8.
- 29. Homma T, Malik KU. Effect of secretin and caerulein in canine pancreas: relation to prostaglandins. Am J Physiol 1983; 244(6): 660-7.
- 30. Yotsumoto F, Manabe T, Kyogoku T et al. Platelet-activating factor involvement in the aggravation of acute pancreatitis in rabbits. Digestion Basel 1994; 55(4): 260-7.
- 31. Erspamer V, Roseghini M, Endean R et al. Biogenic amines and active polypeptides in the skin of Australian amphibians. Nature 1966; 212 (58): 204.
- 32. Adler G, Rohr G, Kern HF. Alteration of membrane fusion as a cause of acute pancreatitis in the rat. Dig Dis Sci 1982; 27(11): 993-1002.
- 33. Alonso R, Montero A, Arvalo M et al. Platelet-activating factor mediates pancreatic function derangement in caerulein-induced pancreatitis in rats. Clin Sci 1994; 87(1): 85-90.
- 34. Rossetti L, Shulman GI, Zawalich WS. Physiological role of cholecystokinin in meal-induced insulin secretion in concious rats. Studies with L 364718, a specific inhibitor of CCK-receptor binding. Diabetes 1987; 36(10): 1212-5.
- 35. Sakamoto C, Golfine JD, Roach E et al. Localization of saturable CCK binding sites in rat pancreatic islets by light and electron microscope antoradiography. Diabetes 1985; 34(4): 390-4.
- 36. Verspohl EJ, Ammon HPT, Willians JA et al. Evidence that cholecystokinin interacts with specific receptors and regulates insulin release in isolated rat islets of Langerhans. Diabetes, 1986; 35 (1): 38-43.
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