Toxicology > Animal Models

User Ratings

Your rating: None
Your rating: None
Your rating: None
 Add your comments

Assessment of Ischemic Vascular Damage

Viera Nosál'ová1,  Ruzena Sotníková1

1Institute of Experimental Pharmacology and Toxicology, Slovak Academy of Sciences, Bratislava, Slovak Republic

Publication Name: 
Current Protocols in Toxicology
Unit Number: 
Unit 21.4
DOI: 
10.1002/0471140856.tx2104s44
Online Posting Date: 
May, 2010
GO TO THE FULL TEXT:
PDF or HTML at Wiley Online Library
Are you the author of this protocol? Login or register and return to this page.

Abstract

This protocol describes a model convenient for acute experiments in anesthetized rats, performed by selective occlusion of the superior mesenteric artery. Such a model provides a means for assessing the role played by various pathophysiological mechanisms in the development of intestinal ischemic injury. It is especially suitable for studying different treatments, mainly pharmacological ones, to help cope with this problem in clinical practice. In the form of support protocols, this unit includes functional vascular and chemiluminescence studies, determination of vascular permeability and myeloperoxidase activity, transit time, and mortality. Curr. Protoc. Toxicol. 44:21.4.1-21.4.21. © 2010 by John Wiley & Sons, Inc.

Keywords: ischemia; reperfusion; reactive oxygen species; rat; superior mesenteric artery; ileum

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol: Intestinal Ischemia/Reperfusion Study
  • Support Protocol 1: Macroscopic Evaluation of the Extent of I/R Injury
  • Support Protocol 2: Microscopic/Histologic Evaluation of the Extent of I/R Injury
  • Support Protocol 3: Measurement of Vascular Function in Mesenteric I/R Model
  • Support Protocol 4: Measurement of ROS Production Using a Chemiluminescence Method
  • Support Protocol 5: Measurement of Vascular Permeability in Mesenteric I/R Model
  • Support Protocol 6: Cannulation of the Carotid Artery
  • Support Protocol 7: Cannulation of the Femoral Vein
  • Support Protocol 8: Measurement of Gastrointestinal Transit in Mesenteric I/R Model
  • Support Protocol 9: Measurement of Myeloperoxidase Activity in Mesenteric I/R Model
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol: Intestinal Ischemia/Reperfusion Study

 Materials
  • Male Wistar rats weighing ~250 to 280 g
  • Drugs to be studied (e.g., antioxidants: catalase, superoxide dismutase in concentrations of 10–4 to 10–5 mol/liter; antihistamines: loratadine, dithiaden, pheniramines 10 to 30 mg/kg; plant origin compounds: rosmarinic acid, arbutin, curcumin in concentration of 10–4 mol/liter)
  • Physiological saline: 0.9% (w/v) NaCl, ice cold
  • Thiopental (see recipe)
  • 70% (v/v) ethanol
  • Lamp
  • Pelleted rodent food
  • 2-ml syringes
  • 23-G needles
  • Animal scale
  • Electric shaver
  • Rat board
  • Surgical instruments including:
    • Scalpel
    • Hemostats
    • Jeweler's forceps, straight
    • Iridectomy scissors
    • Vessel clamp
    • Small straight anatomical forceps
    • Large straight surgical forceps
    • Scissors, sharp and blunt
  • Gauze
  • Sutures
  • Additional reagents and equipment sacrificing the rat (Donovan and Brown, 2006)

Support Protocol 1: Macroscopic Evaluation of the Extent of I/R Injury

 Additional Materials (also see Basic Protocol)
  • Male Wistar rats, control and treated
  • Gridded paper

Support Protocol 2: Microscopic/Histologic Evaluation of the Extent of I/R Injury

 Materials
  • Rat small intestine and other tissues, treated and control (see Basic Protocol)
  • 10% (w/v) formalin
  • Paraffin
  • Xylene
  • 50%, 70%, 80%, 90%, 95%, and 100% (v/v) ethanol
  • Acetone
  • Distilled water
  • Hematoxylin
  • Eosin
  • Xylene-based mounting medium
  • Embedding cassettes
  • Automated tissue processor (Autotechnikon AT-4, ZSSR)
  • Microtome with blades
  • Glass slides
  • 45° to 50°C oven
  • Coverslips
  • Microscope, e.g., Fluoval (Zeiss, DDR)

Support Protocol 3: Measurement of Vascular Function in Mesenteric I/R Model

 Additional Materials (also see Basic Protocol)
  • Rats, male Wistar, treated and control (Basic Protocol)
  • Physiological saline solution (PSS; see recipe)
  • Phenylephrine (see recipe)
  • Acetylcholine (see recipe)
  • NG-nitro-l-arginine methyl ester (L-NAME, 100 µM; see recipe)
  • Indomethacin (10 µM; see recipe)
  • 90-mm petri dishes
  • 10-ml jacketed organ bath (Experimetria)
  • Force transducer (Experimetria)
  • Graph paper or digital recorder
  • Precision balance

Support Protocol 4: Measurement of ROS Production Using a Chemiluminescence Method

 Additional Materials (also see Basic Protocol)
  • Male Wistar rats, treated and control (see Basic Protocol)
  • Physiological saline solution (PSS; see recipe), pre-oxygenated (95% O2 and 5% CO2)
  • Luminol (see recipe)
  • Beakers
  • Cuvettes
  • Lumi-aggregometer (model 500; Chrono-log)
  • Balance

Support Protocol 5: Measurement of Vascular Permeability in Mesenteric I/R Model

 Materials
  • Male Wistar rat
  • Thiopental (see recipe)
  • 1% Evans blue dye (see recipe)
  • Physiological saline: 0.9% (w/v) NaCl
  • Formamide (see recipe)
  • Animal scale
  • 2-ml syringes with 23-G needles
  • Filtration paper
  • 50°C shaking water bath
  • Spectrophotometer
  • Additional reagents and equipment for cannulating the carotid artery (Support Protocol 6) and femoral vein (Support Protocol 7) and sacrificing the rat (Donovan and Brown, 2006)

Support Protocol 6: Cannulation of the Carotid Artery

 Materials
  • Male Wistar rat
  • Thiopental (see recipe)
  • 70% (v/v) ethanol
  • Heparinized saline (50 IU/ml), 0.9% NaCl
  • Electric shaver
  • Rat board
  • Scissors
  • Blunt forceps
  • Suture
  • Clamps
  • Cannula

Support Protocol 7: Cannulation of the Femoral Vein

 Materials
  • Male Wistar rat
  • Thiopental (see recipe)
  • Electric shaver
  • Scissors
  • Rat board
  • Cannula (with a fine metallic needle in its tip)
  • Gauze

Support Protocol 8: Measurement of Gastrointestinal Transit in Mesenteric I/R Model

 Materials
  • Male Wistar rats
  • Thiopental (see recipe)
  • Evans blue (see recipe)
  • Physiological saline: 0.9% (w/v) NaCl
  • Balance
  • Orogastric cannula (metal probe)
  • Clamps
  • Additional reagents and equipment for isolating the small intestine (Basic Protocol)

Support Protocol 9: Measurement of Myeloperoxidase Activity in Mesenteric I/R Model

 Additional Materials (also see Basic Protocol)
  • Intestinal mucosa (see step 17 of the Basic Protocol)
  • Homogenization medium (see recipe)
  • o-dianisidine dihydrochloride reagent (see recipe)
  • Tissue homogenizer
  • Centrifuge
  • Spectrophotometer
Looking for materials?  Suppliers Guide
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

  • Figure 21.4.1
    Effects of different reperfusion durations on 60-min ischemia-induced changes in endothelial-dependent relaxation of the phenylephrine precontracted SMA. The most pronounced decrease of acetylcholine relaxation was induced by 60-min ischemia followed by 30-min reperfusion. Responses to acetylcholine of the phenylephrine precontracted SMA are obtained from sham-operated rats (––) after 60 min ischemia followed by reperfusion of 30 min (––), 60 min (––), and 90 min (–•–). Data are means ± SEM.

    View Image

Literature Cited

Literature Cited
    Bradley, P.P., Priebat, D.A., Christensen, R.D., and Rothstein, G. 1982. Measurement of cutaneous inflammation: Estimation of neutrophil content with an enzyme marker. J. Invest. Dermatol. 78:206-209.
    Chiu, C.J., McArdle, A.H., Brown, R., Scott, H.J., and Gurd, F.N. 1970. Intestinal mucosal lesion in low-flow states. I. A morphologic, hemodynamic and metabolic reappraisal. Arch. Surg. 101:478-483
    Cornet, A.D., Kingma, S.D., Trof, R.J., Wisselink, W., and Groeneveld, A.B. 2009. Hepatosplanchnic ischemia/reperfusion is a major determinant of lung vascular injury after aortic surgery. J. Surg. Res. 157:48-54.
    De Winter, B.Y., Boeckxstaens, G.E., De Man, J.G., Moreels, T.G., Herman, A.G., and Pelckmans, P.A. 1998. Differential effect of indomethacin and ketorolac on postoperative ileus in rats. Eur. J. Pharmacol. 344:71-76.
    Donovan, J. and Brown, P. 2006. Euthanasia. Curr. Protoc. Immunol. 73:1.8.1-1.8.4.
    Erickson, R.A., Chang, K., Lifrak, E., Rivera, N., and Stachura, J. 1992. 16, 16-dimethyl prostaglandin E2 reduces bile acid-mediated intestinal vascular injury in rats. Gastroenterology 102:1295-1305.
    Feinman, R.D., Lubowsky, J., Charo, I.F., and Zabinski, M.P. 1977. The lumi-aggregometer: A new instrument for simultaneous measurement of secretion and aggregation. J. Lab. Clin. Med. 90:125-129.
    Granger, D.N. and Barrowman, J.A. 1983. Microcirculaton of the alimentary tract. II. Pathophysiology of edema. Gastroenterology 84:1035-1049.
    Granger, D.N. and Korthuis, R.J. 1995. Physiologic mechanisms of postischemic tissue injury. Ann. Rev. Physiol. 57:311-332.
    Granger, D.N., Hollwarth, M.E., and Parks, D.A. 1986. Ischemia reperfusion injury. Role of oxygen derived free radicals. Acta Physiol. Scand. 548:47-63.
    Haglind, E., Haglund, S., Lundgren, O., Romanus, M., and Schersten, T. 1980. Graded intestinal vascular obstruction.: I. Description of an experimental shock model in the rat. Circ. Shock 7:83.
    Janinová, V., Nosál', R., Drábiková, K., and Danihelová, E. 2000. Inhibition of PMN leukocyte chemiluminescence by blood platelets-biological protection against destructive effects of reactive oxygen species Biologia 55:45-48.
    Janinová, V., Drábiková, K., Nosál', R., Raková, L., Májeková, M., and Fábryová, V. 2005. Chemiluminescence-a sensitive method to detect extra- and intracellular oxidants. Biologia 17:133-135.
    Kozuch, P.L. and Brandt, L.J. 2005. Review article: Diagnosis and management of mesenteric ischemia with an emphasis on pharmacotherapy. Aliment. Pharmacol. Ther. 21:211-215.
    Lange, S., Delbro, D.S., and Jennische, E. 1994. Evans blue permeation of intestinal mucosa in the rat. Scand. J. Gastroenterol. 29:38-46.
    Madara, J.L. 1990. Pathobiology of the intestinal epithelial barrier. Am. J. Pathol. 137:1273-1281.
    Malle, E., Furtmüller, P.G., Sattler, W., and Obinger, C. 2007. Myeloperoxidase: A target for new drug development Brit. J. Pharmacol. 152:838-854.
    Megison, S.M., Horton, J.W., Chao, He., and Walker, P.B. 1990. A new model of intestinal ischemia in the rat. J. Surg. Res. 49:168-173.
    Nakagawa, H., Tsunooka, N., Yamamoto, Y., Yoshida, M., Nakata, T., and Kawachi, K. 2009. Intestinal ischemia/reperfusion-induced bacterial translocation and lung injury in atherosclerotic rats with hypoadiponectinemia. Surgery 145:48-56.
    Nosál'ová, V., Navarová, J., Mihalová, D., and Sotníková, R. 2007. Mesenteric ischemia/reperfusion-induced intestinal and vascular damage: Effect of stobadine. Methods Find. Exp. Clin. Pharmacol. 29:39-45.
    Nosál'ová, V., Drábiková, K., Janinová, V., Peivová, J., Maiková, T., Petríková, M., Sotníková, R., and Nosál', R. 2008. Effect of H1 antihistamines in a model of mesenteric ischaemia/reperfusion. Inflamm. Res. 57:S55-S56.
    Nylander, O., Sababi, M., and Bark, J. 1991. Characterization of 51Cr-EDTA as a marker of duodenal mucosal permeability. Acta Physiol. Scand. 143:117-126.
    Park, P.O., Haglund, U., Bulkley, G.B., and Falt, K. 1990. The sequence of development of intestinal tissue injury following strangulation ischemia and reperfusion. Surgery 107:574-580.
    Schoenberg, M.H. and Beger H.G. 1993. Reperfusion injury after intestinal ischemia. Crit. Care Med. 21:1376-1386.
    Sotníková, R., Okruhlicová, L., and Noskovic, P. 1998. Endothelial protective effect of stobadine on ischaemia/reperfusion-induced injury. Gen. Physiol. Biophys. 17:253-264.
    Sotníková, R., Drábiková, K., Beová, M., Nosál'ová, V., Nosál', R., and Rajská, P. 2005. Increased chemiluminescence accompanies ischaemia/reperfusion-induced endothelial dysfunction of rat superior mesentery artery. Biologia 17:145-147.
    Souza, D.G., Cassali, G.D., Poole, S., and Teixeira, M.M. 2001. Effect of inhibitors of PDE4 and TNF alpha on local and remote injuries following ischemia and reperfusion injury. Br. J. Pharmacol. 134:985-994.
    Szocs, K., ilka, N., and Sotníková, R. 2004. "Endothelial dysfunction and superoxide production in superior mesenteric artery upon ischaemia/reperfusion". In Abstract Book of the Symposium New Frontiers in Basic Cardiovascular Research, Alterations of Muscular Systems during Cardiovascular Diseases. p. 44. La Grande-Motte, France.
    Talbert, D.J. and Tuggle, D.W. 1988. The effects of maturity and timed ischemia on survival rates in necrotizing enterocolitis. Curr. Surg. 45:307-309.
    van Dongen, J.J., Remie, R., Rensema, J.W., van Wunnik, G.H.J. (eds.) 1990. Manual of Microsurgery on the Laboratory Rat. Elsevier, Cambridge, Mass.
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library
Looking for Answers?
Do you have tips, tricks, or improvements to share?

Join the Conversation

Post new comment

The content of this field is kept private and will not be shown publicly.
CAPTCHA
This question is for testing whether you are a human visitor and to prevent automated spam submissions.