Name: A Model of Chronic Nutrient Infusion in the Rat (Video) | JoVE Journal
Uploaded: 2013-08-14T12:00:00.000+00:00
Duration: 8 min 18 s
Description: Learn a chronic nutrient infusion protocol in rats to study glucose homeostasis and metabolic health effects. This method uses jugular vein and carotid artery catheterization with glucose and Intralipid solutions.

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5 minutes

This work was supported by the National Institutes of Health (R01DK58096 to Vincent Poitout). Vincent Poitout holds the Canada Research Chair in Diabetes and Pancreatic Beta-cell Function. Bader Zarrouki received post-doctoral fellowships from Merck and Eli Lilly. Ghislaine Font&eacute;s was supported by a post-doctoral fellowship from the Canadian Diabetes Association.

Overview: The procedure consists of catheterizing the right jugular vein and left carotid artery under general anesthesia; allowing a 7-day recuperation period; connecting the catheters to the pumps using a swivel and counterweight system that enables the animal to move freely in the cage; and infusing glucose and/or Intralipid (a soybean oil emulsion which generates a mixture of approximately 80% unsaturated/20% saturated fatty acids when infused with heparin 9) for 72 hr.
1. Canulation of the Right Jugular Vein and the Left Carotid Artery
<ol>
	<li>Sterilize surgical instruments. Canulation tubing must also be cold-sterilized using a liquid sterilizing agent (2.6% glutaraldehyde) prior to the procedure. Immerse the tubing in an autoclaved container for 16-24 hr. Rinse and flush thoroughly with sterile distilled water to remove all traces of glutaraldehyde before use.</li>
	<li>Weigh the rat to calculate drug dosages: 
	Carprofen 5 mg/kg: dilution 1/10 = Body weight (g) x 0.001 ml SC (analgesic) 
	Glycopyrrolate 0.01 mg/kg : dilution 1/10 = BW (g) X 0.0012 ml SC (anticholinergic)</li>
	<li>Anesthetize the rat using isoflurane and oxygen.</li>
	<li>Place the rat on its stomach. Shave the area behind the ears to the base of the shoulders. Lay the rat on its back. Shave the region under the neck to the front paws.</li>
	<li>Prep the surgical site with chlorhexidine, alcohol, and iodine. Administer drugs.</li>
	<li>Transfer the rat to the surgical field.</li>
	<li>Using aseptic technique, canulate the right jugular vein and the left carotid artery with a PE-50 canula attached to a 1 ml syringe filled with 5 U of heparinized saline. Flush the canulas with 50 U of heparinized saline to avoid clotting during the recovery period. Use blunted 23G needles. Close each canula with a 23G pin.</li>
	<li>After the surgery, trim approximately 2.5 mm off the bottom incisors and place the rat in an infusion jacket to prevent the canulas from being eaten.</li>
	<li>Provide oxygen (1 L/min for 10 min) to help evacuate isoflurane.</li>
	<li>Place the rat in a heated cage until it is fully awake.</li>
	<li>Operate four rats to use one per infusion condition (Table 1).</li>
</ol>
2. Post-operative Care (Post-surgical Treatment and Connection of the Catheters)
<ol>
	<li>Weigh the rats on day 1 and day 2 post-surgery.</li>
	<li>Administer glycopyrrolate (BW(g) x 0.00048 ml) SC twice on day 1 post-surgery and once on day 2 post-surgery.</li>
	<li>Additional supportive treatments can be administered if necessary: fluids, heating pad, wet diet, oxygen therapy, analgesics, anticholinergics.</li>
	<li>On day 7 post-surgery, weigh the rats to calculate the flow rates for infusion.</li>
	<li>Connect each rat to an infusion system using a tether and swivel mounted on a cage grill top (Figure 1).</li>
	<li>Flush the canulas with 5 U of heparinized saline to remove clots. Flush the canulas once more with 50 U of heparinized saline to prevent clotting.</li>
	<li>Allow the rats to acclimate to the tether and swivel for at least 24 hr before starting the infusion.</li>
</ol>
3. Infusion
<ol>
	<li>Draw 0.15 ml of blood from the carotid of each rat and measure glycemia. Flush the jugular canulas. Use 50 U of heparinized saline to prevent clotting in both canulas of each rat.</li>
	<li>Transfer the blood sample to a 0.5 ml collection tube containing 2% EDTA. Centrifuge at 10,000 rpm for 2 min and freeze the plasma at -20 &deg;C.</li>
	<li>Fill two 60 ml syringes for each of the infusion conditions listed below. Place Syringe 1 on the front position of the pump and place Syringe 2 on the back position of the pump.</li>
	<li>Join each pair of solutions together with y-connectors and CO-EX T22 tubing that has been sterilized. Place the syringes on a Harvard 33 dual syringe pump.</li>
	<li>Change the cage bottom and remove all food from the cage grill top.</li>
	<li>Weigh 150 g of standard chow and place on the cage grill top.</li>
	<li>Connect the syringes to the swivel on the cage grill. Flush the syringes properly to remove trapped air from the lines.</li>
	<li>Calculate the infusion flow rates using the body weight that was taken prior to connecting the rat to the infusion system. Rates are calculated with a Microsoft Excel file that converts the glucose infusion rate (GIR) into ml/h.</li>
	<li>Set the pump to administer flow rates for 60 ml syringes according to the manufacturer's settings. Enter the rate for Syringe 1 (front syringe) and the rate for Syringe 2 (back syringe).</li>
	<li>Start the pump.</li>
</ol>
4. Monitoring
<ol>
	<li>After starting the pump, verify that there is no leaking from the swivel or from the canulas and that infusion tubing is not twisted. Also verify that there are no air bubbles in the tubing.</li>
	<li>After 3 hr of infusion, take a blood sample to monitor glycemia. Repeat after 6, 24, 48, and 72 hr of infusion. As a precaution, glycemia is also monitored after 30, 34, 54, and 60 hr of the infusion. Blood glucose can be measured using one drop of whole blood using a portable glucose monitor. This limits the amount of blood drawn during the infusion and therefore avoids significantly altering blood volume and/or hematocrit.</li>
	<li>The rate for Syringe 1 is modified based on glycemia values to maintain blood glucose at 220-250 mg/dl. The rate for Syringe 2 does not change during the 72 hr of infusion because free fatty acids are maintained at 1 mmol/L.</li>
	<li>Infusion conditions are paired so that the volume received for the control animals is equivalent to the volume received for the test animals (Table 1).</li>
	<li>After 24 hr of infusion, change the cage bottom and weigh the food remaining in the cage grill. Return the uneaten portion to the cage grill. Repeat after 48 hr.</li>
	<li>Refill syringes daily as needed during the 72 hr of infusion.</li>
	<li>During the infusion, always observe the rat for any signs of inflammation or discomfort. Administer appropriate care if needed.</li>
</ol>
5. Post-infusion Euthanasia
<ol>
	<li class="jove_content">After 72 hr of infusion, rats are anesthetized intravenously with 0.5 ml of a ketamine/xylazine cocktail (182 mg/kg of ketamine and 11.6 mg/kg of xylazine diluted 1:2 with 0.9% NaCl).</li>
	<li class="jove_content">The toe-pinch reflex is used to verify the level of anesthesia. Additional anesthetic is administered as needed.</li>
	<li class="jove_content">When the rat is anesthetized, the abdominal cavity is opened with surgical scissors. The rat is exsanguinated by drawing 10-15 ml of blood in a syringe from the aorta or the vena cava.</li>
</ol>

<ol><li>Poitout, V., Robertson, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Glucolipotoxicity%3A+fuel+excess+and+beta-cell+dysfunction%5BTitle%5D)+AND+%22Endocr.+Rev%22%5BJournal%5D)">Glucolipotoxicity: fuel excess and beta-cell dysfunction.</a> Endocr. Rev. 29, 351-366 (2008).</li>
<li>Poitout, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Glucolipotoxicity+of+the+pancreatic+beta+cell%5BTitle%5D)+AND+%22Biochim.+Biophys.+Acta%22%5BJournal%5D)">Glucolipotoxicity of the pancreatic beta cell.</a> Biochim. Biophys. Acta. 1801, 289-298 (2010).</li>
<li>Unger, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Minireview%3A+weapons+of+lean+body+mass+destruction%3A+the+role+of+ectopic+lipids+in+the+metabolic+syndrome%5BTitle%5D)+AND+%22Endocrinology%22%5BJournal%5D)">Minireview: weapons of lean body mass destruction: the role of ectopic lipids in the metabolic syndrome.</a> Endocrinology. 144, 5159-5165 (2003).</li>
<li>Harmon, J. S., Gleason, C. E., Tanaka, Y., Poitout, V., Robertson, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antecedent+hyperglycemia%2C+not+hyperlipidemia%2C+is+associated+with+increased+islet+triacylglycerol+content+and+decreased+insulin+gene+mRNA+level+in+Zucker+diabetic+fatty+rats%5BTitle%5D)+AND+%22Diabetes%22%5BJournal%5D)">Antecedent hyperglycemia, not hyperlipidemia, is associated with increased islet triacylglycerol content and decreased insulin gene mRNA level in Zucker diabetic fatty rats.</a> Diabetes. 50, 2481-2486 (2001).</li>
<li>Bachar, E., Ariav, Y., Cerasi, E., Kaiser, N., Leibowitz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neuronal+nitric+oxide+synthase+protects+the+pancreatic+beta+cell+from+glucolipotoxicity-induced+endoplasmic+reticulum+stress+and+apoptosis%5BTitle%5D)+AND+%22Diabetologia%22%5BJournal%5D)">Neuronal nitric oxide synthase protects the pancreatic beta cell from glucolipotoxicity-induced endoplasmic reticulum stress and apoptosis.</a> Diabetologia. 53, 2177-2187 (2010).</li>
<li>Peyot, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Beta-cell+failure+in+diet-induced+obese+mice+stratified+according+to+body+weight+gain%3A+secretory+dysfunction+and+altered+islet+lipid+metabolism+without+steatosis+or+reduced+beta-cell+mass%5BTitle%5D)+AND+%22Diabetes%22%5BJournal%5D)">Beta-cell failure in diet-induced obese mice stratified according to body weight gain: secretory dysfunction and altered islet lipid metabolism without steatosis or reduced beta-cell mass.</a> Diabetes. 59, 2178-2187 (2010).</li>
<li>Hagman, D. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cyclical+and+alternating+infusions+of+glucose+and+intralipid+in+rats+inhibit+insulin+gene+expression+and+Pdx-1+binding+in+islets%5BTitle%5D)+AND+%22Diabetes%22%5BJournal%5D)">Cyclical and alternating infusions of glucose and intralipid in rats inhibit insulin gene expression and Pdx-1 binding in islets.</a> Diabetes. 57, 424-431 (2008).</li>
<li>Fontes, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Glucolipotoxicity+age-dependently+impairs+beta+cell+function+in+rats+despite+a+marked+increase+in+beta+cell+mass%5BTitle%5D)+AND+%22Diabetologia%22%5BJournal%5D)">Glucolipotoxicity age-dependently impairs beta cell function in rats despite a marked increase in beta cell mass.</a> Diabetologia. 53, 2369-2379 (2010).</li>
<li>Stein, D. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Essentiality+of+circulating+fatty+acids+for+glucose-stimulated+insulin+secretion+in+the+fasted+rat%5BTitle%5D)+AND+%22J.+Clin.+Invest%22%5BJournal%5D)">Essentiality of circulating fatty acids for glucose-stimulated insulin secretion in the fasted rat.</a> J. Clin. Invest. 97, 2728-2735 (1996).</li>
<li>Leahy, J. L., Cooper, H. E., Weir, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Impaired+insulin+secretion+associated+with+near+normoglycemia.+Study+in+normal+rats+with+96-h+in+vivo+glucose+infusions%5BTitle%5D)+AND+%22Diabetes%22%5BJournal%5D)">Impaired insulin secretion associated with near normoglycemia. Study in normal rats with 96-h in vivo glucose infusions.</a> Diabetes. 36, 459-464 (1987).</li>
<li>Hager, S. R., Jochen, A. L., Kalkhoff, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Insulin+resistance+in+normal+rats+infused+with+glucose+for+72+h%5BTitle%5D)+AND+%22The+American+Journal+of+Physiology%22%5BJournal%5D)">Insulin resistance in normal rats infused with glucose for 72 h.</a> The American Journal of Physiology. 260, 353-362 (1991).</li>
<li>Laybutt, D. R., Chisholm, D. J., Kraegen, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Specific+adaptations+in+muscle+and+adipose+tissue+in+response+to+chronic+systemic+glucose+oversupply+in+rats%5BTitle%5D)+AND+%22The+American+Journal+of+Physiology%22%5BJournal%5D)">Specific adaptations in muscle and adipose tissue in response to chronic systemic glucose oversupply in rats.</a> The American Journal of Physiology. 273, E1-E9 (1997).</li>
<li>Jonas, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High+glucose+stimulates+early+response+gene+c-Myc+expression+in+rat+pancreatic+beta+cells%5BTitle%5D)+AND+%22The+Journal+of+Biological+Chemistry%22%5BJournal%5D)">High glucose stimulates early response gene c-Myc expression in rat pancreatic beta cells.</a> The Journal of Biological Chemistry. 276, 35375-35381 (2001).</li>
<li>Tang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Glucose-induced+beta+cell+dysfunction+in+vivo+in+rats%3A+link+between+oxidative+stress+and+endoplasmic+reticulum+stress%5BTitle%5D)+AND+%22Diabetologia%22%5BJournal%5D)">Glucose-induced beta cell dysfunction in vivo in rats: link between oxidative stress and endoplasmic reticulum stress.</a> Diabetologia. 55, 1366-1379 (2012).</li>
<li>Alonso, L. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Glucose+infusion+in+mice%3A+a+new+model+to+induce+beta-cell+replication%5BTitle%5D)+AND+%22Diabetes%22%5BJournal%5D)">Glucose infusion in mice: a new model to induce beta-cell replication.</a> Diabetes. 56, 1792-1801 (2007).</li>
<li>Magnan, C., Gilbert, M., Kahn, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chronic+free+fatty+acid+infusion+in+rats+results+in+insulin+resistance+but+no+alteration+in+insulin-responsive+glucose+transporter+levels+in+skeletal+muscle%5BTitle%5D)+AND+%22Lipids%22%5BJournal%5D)">Chronic free fatty acid infusion in rats results in insulin resistance but no alteration in insulin-responsive glucose transporter levels in skeletal muscle.</a> Lipids. 31, 1141-1149 (1996).</li>
<li>Goh, T. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lipid-induced+beta-cell+dysfunction+in+vivo+in+models+of+progressive+beta-cell+failure%5BTitle%5D)+AND+%22Am.+J.+Physiol.+Endocrinol.+Metab%22%5BJournal%5D)">Lipid-induced beta-cell dysfunction in vivo in models of progressive beta-cell failure.</a> Am. J. Physiol. Endocrinol. Metab. 292, 549-560 (2007).</li>
<li>Pascoe, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Free+fatty+acids+block+glucose-induced+beta-cell+proliferation+in+mice+by+inducing+cell+cycle+inhibitors+p16+and+p18%5BTitle%5D)+AND+%22Diabetes%22%5BJournal%5D)">Free fatty acids block glucose-induced beta-cell proliferation in mice by inducing cell cycle inhibitors p16 and p18.</a> Diabetes. 61, 632-641 (2012).</li>
<li>Bell, C. G., Walley, A. J., Froguel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+genetics+of+human+obesity%5BTitle%5D)+AND+%22Nature+Reviews.+Genetics%22%5BJournal%5D)">The genetics of human obesity.</a> Nature Reviews. Genetics. 6, 221-234 (2005).</li>
<li>Fontes, G., Hagman, D. K., Latour, M. G., Semache, M., Poitout, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lack+of+preservation+of+insulin+gene+expression+by+a+glucagon-like+peptide+1+agonist+or+a+dipeptidyl+peptidase+4+inhibitor+in+an+in+vivo+model+of+glucolipotoxicity%5BTitle%5D)+AND+%22Diabetes+Res.+Clin.+Pract%22%5BJournal%5D)">Lack of preservation of insulin gene expression by a glucagon-like peptide 1 agonist or a dipeptidyl peptidase 4 inhibitor in an in vivo model of glucolipotoxicity.</a> Diabetes Res. Clin. Pract. 87, 322-328 (2010).</li>
<li>Crawford, P. A., Schaffer, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Metabolic+stress+in+the+myocardium%3A+Adaptations+of+gene+expression%5BTitle%5D)+AND+%22Journal+of+Molecular+and+Cellular+Cardiology%22%5BJournal%5D)">Metabolic stress in the myocardium: Adaptations of gene expression.</a> Journal of Molecular and Cellular Cardiology. , (2012).</li>
<li>Kewalramani, G., Bilan, P. J., Klip, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Muscle+insulin+resistance%3A+assault+by+lipids%2C+cytokines+and+local+macrophages%5BTitle%5D)+AND+%22Curr.+Opin.+Clin.+Nutr.+Metab+Care%22%5BJournal%5D)">Muscle insulin resistance: assault by lipids, cytokines and local macrophages.</a> Curr. Opin. Clin. Nutr. Metab Care. 13, 382-390 (2010).</li>
<li>Cusi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Role+of+obesity+and+lipotoxicity+in+the+development+of+nonalcoholic+steatohepatitis%3A+pathophysiology+and+clinical+implications%5BTitle%5D)+AND+%22Gastroenterology%22%5BJournal%5D)">Role of obesity and lipotoxicity in the development of nonalcoholic steatohepatitis: pathophysiology and clinical implications.</a> Gastroenterology. 142, 711-725 (2012).</li>
</ol>

Vincent Poitout is co-founder of and received research contracts from B&ecirc;tagenex Inc., a contract research organization which offers the infusion model presented in this article as a commercial service.

Although a number of previous studies have employed chronic infusions of glucose (e.g. 10-15) or lipids (e.g. 16,17) in rodents, to our knowledge the combined infusion of both fuels has only been reported in mice 18. The chronic infusion model presented here offers several advantages to study the effects on nutrient excess on a variety of biological functions in rats. First, it does not involve genetically obese rodents, and since common obesity in humans is polyge...

Chronically elevated levels of glucose and lipids in the circulation have been proposed to contribute to the pathogenesis of type 2 diabetes by altering the function of several organs implicated in the maintenance of glucose homeostasis including, but not limited to, the pancreatic beta-cell (reviewed in 1). The glucotoxicity hypothesis posits that chronic hyperglycemia aggravates the beta-cell defect which gave rise to hyperglycemia in the first place, thus creating a vicious cycle and contributing to the deterioration of glucose control in type 2 diabetes patients. Likewise, the glucolipotoxicity hypothesis proposes that concomitant elevations of glucose and lipid levels, as often observed in type 2 diabetes, are detrimental to the beta cell.
	Deciphering the cellular and molecular mechanisms of the deleterious effects of chronically elevated nutrients on pancreatic beta-cell function is key to the understanding of the pathogenesis of type 2 diabetes 1. To that end, a large number of studies have examined the mechanisms of chronic nutrient excess ex vivo in isolated islets of Langerhans or in vitro in clonal, insulin-secreting cell lines. However, translation of the findings obtained in these model systems to the whole organism is complex, in particular because the concentrations of fatty acids used in cultured cells or islets rarely match the circulating levels in the vicinity of the beta cells in vivo 2. On the other hand, the mechanisms of beta-cell failure in response to nutrient excess have been examined in rodent models of diabetes, as exemplified by the Zucker Diabetic Fatty rat 3,4, the gerbil Psammomys obesus 5 and the high-fat diet-fed mouse 6. These models, however, are characterized by intrinsic metabolic abnormalities and are not easily amenable to manipulations of blood glucose and/or lipid levels in a more controlled and less chronic setting. To be able to alter circulating nutrient levels in a timeframe of days in otherwise normal animals, we have developed a chronic infusion model in normal rats which enables us to examine the effects of lipids and glucose, alone or in combination, on physiological parameters and function 7,8.

<table><tbody><tr><td>Saline 0.9%</td><td>BD</td><td>JB1324</td><td></td></tr><tr><td>Dextrose 70%</td><td>McKesson</td><td></td><td></td></tr><tr><td>Intralipid 20%</td><td>Fresenius Kabi</td><td>JB6023</td><td></td></tr><tr><td>Metricide (Glutaraldehyde 2.6%)</td><td>Metrex</td><td>11-1401</td><td></td></tr><tr><td>Heparin Sodium 10,000 USP u/ml</td><td>PPC</td><td></td><td></td></tr><tr><td>Carprofen</td><td>Metacam</td><td></td><td></td></tr><tr><td>Glycopyrrolate</td><td>Sandoz</td><td></td><td></td></tr><tr><td>Isoflurane</td><td>Abbott</td><td></td><td></td></tr><tr><td>Chlohexidine 2%</td><td></td><td></td><td></td></tr><tr><td>Alcohol 70%</td><td></td><td></td><td></td></tr><tr><td>Iodine</td><td></td><td></td><td></td></tr><tr><td>PE-50</td><td>BD</td><td>427411</td><td></td></tr><tr><td>CO-EX T22</td><td>Instech Solomon</td><td>BCOEX-T22</td><td></td></tr><tr><td>Connector 22G</td><td>Instech Solomon</td><td>SC22/15</td><td></td></tr><tr><td>Swivel 22G</td><td>Instech Solomon</td><td>375/22PS</td><td></td></tr><tr><td>Y-Connector 22G</td><td>Instech Solomon</td><td></td><td></td></tr><tr><td>Counterbalance and arm</td><td>Instech Solomon</td><td>CM375BP</td><td></td></tr><tr><td>23 G blunted needles</td><td>Instech Solomon</td><td>LS23</td><td></td></tr><tr><td>23 G canulation pins</td><td>Instech Solomon</td><td>SP23/12</td><td></td></tr><tr><td>Tethers (12 inch)</td><td>Lomir</td><td>RT12D</td><td></td></tr><tr><td>Infusion jackets </td><td>Lomir</td><td>RJ01, RJ02, RJ03, RJ04</td><td></td></tr><tr><td>(SM-XL)</td><td></td><td></td><td></td></tr><tr><td>Tether attachment piece</td><td>Lomir</td><td>RS T1</td><td></td></tr><tr><td>60 ml syringe</td><td>BD</td><td>309653</td><td></td></tr><tr><td>1 ml syringe</td><td>BD</td><td>309602</td><td></td></tr></tbody></table>

a model of chronic nutrient infusion in the rat

A Model of Chronic Nutrient Infusion in the Rat

Chronic exposure to excessive levels of nutrients is postulated to affect the function of several organs and tissues and to contribute to the development of the many complications associated with obesity and the metabolic syndrome, including type 2 diabetes. To study the mechanisms by which excessive levels of glucose and fatty acids affect the pancreatic beta-cell and the secretion of insulin, we have established a chronic nutrient infusion model in the rat. The procedure consists of catheterizing the right jugular vein and left carotid artery under general anesthesia; allowing a 7-day recuperation period; connecting the catheters to the pumps using a swivel and counterweight system that enables the animal to move freely in the cage; and infusing glucose and/or Intralipid (a soybean oil emulsion which generates a mixture of approximately 80% unsaturated/20% saturated fatty acids when infused with heparin) for 72 hr. This model offers several advantages, including the possibility to finely modulate the target levels of circulating glucose and fatty acids; the option to co-infuse pharmacological compounds; and the relatively short time frame as opposed to dietary models. It can be used to examine the mechanisms of nutrient-induced dysfunction in a variety of organs and to test the effectiveness of drugs in this context.

Out of a series of 42 rats which underwent surgery, 5 rats were lost during the post-operative period and 1 rat was lost during the infusion, representing an overall success rate of 86%. The average body weight of the 37 rats that were eventually infused was 608±5 g before surgery and 588±6 g at the initiation of the infusion (mean±SE; n=37; P&lt;0.0001 by paired t-test). The following representative results were obtained in 2 infusion groups: Saline (SAL), and Glucose + Intralipid (GLU+IL). Figures 2A a...

A protocol for chronic infusions of glucose and Intralipid in rats is described. This model can be used to study the impact of nutrient excess on organ function and physiological parameters.

Chronic exposure to excessive levels of nutrients is postulated to  affect the function of several organs and tissues and to contribute to the  ...

The overall goal of this procedure is to catheterize the jugular veins and carotid arteries of rats to infuse glucose and lipids as a model for chronic nutrient excess. This is accomplished by first catheterizing, the jugular vein and carotid artery under general anesthesia. After the procedure, the rat is allowed to recover for six days.Then the catheters are connected to infusion pumps through a tether and swivel mounted on the cage top. Finally, the rat is infused with glucose and lipid solutions. Ultimately, results can be obtained that show the effects of chronic nutrient excess on glucose homeostasis.The main advantage of this technique over other models, such as genetic models, for instance, is that one can examine the early changes in beta cell function in response to nutrient excess. The other advantage is that one can specifically modulate the levels of glucose and intralipid or lipids in the circulation. Now, we use this technique to examine changes in beta cell function, but of course, it can be used to look at other physiological parameters such as insulin resistance.For example, demonstrating the procedure will be Grace Ferguson, an animal health technician in my laboratory, Prior to beginning, sterilize all surgical instruments by autoclave at least 16 to 24 hours before the procedure. Place the cannulation tubing into 2.6%glute aldehyde for sterilization. Using four rats per infusion condition, weigh each rat and then calculate dosages for the analgesia and anticholinergic drugs.After anesthetizing the rat with two to 3%iso, fluorine and oxygen, check the level of sedation by toe. Pinch and place the rat on its stomach. Shave the area behind the ears to the base of the shoulders, then turn the animal on its back, and shave the region under the neck to the front paws.Next, disinfect the surgical site three times with chlorhexidine alcohol and iodine, and then administer carprofen and glycopyrrolate. Transfer the rat to the surgical area, and then with aseptic technique. Using a PE 50 cannula attached to a one milliliter syringe filled with five units of heparinized saline, cannulate the right jugular vein and left carotid artery To avoid clotting during the recovery period, flush the cannulas with 50 units of heparinized saline through a blunted 23 gauge needle, and then use another 23 gauge pin to close each cannula after the surgery, trim about 2.5 millimeters off the bottom of the incisors, and place the rat in an infusion jacket to prevent the cannulas from being eaten.To help evacuate the ISO fluorine, administer oxygen at one liter per minute. Place the rat in a heated cage until it is fully awake. On days one and two, post-surgery.Weigh the rats, administer glycopyrrolate, subcutaneously twice on day one and once on day two, and provide additional treatments when necessary according to the text protocol on day seven post-surgery. After weighing each rat to calculate the flow rates for infusion, connect the cannula of the jugular vein by a tubing extension inside of the spring to the blue swivel. Flush the cannulas with five units of heparinized saline and connect them to the tether.Then connect the rat to the infusion system using the tether and swivel. Now flush the cannulas once more with five units of heparinized saline to prevent clotting. Allow the rats to acclimate to the tether and swivel for at least 24 hours before starting the infusion to carry out the infusion.Begin by drawing 0.15 milliliters of blood from the carotid of each rat and measure glycemia. Flush the jugular cannulas and use 50 units of heparinized saline to prevent clotting of both cannulas. Transfer the blood sample to a 0.5 milliliter collection tube containing 2%EDTA, then centrifuge at 10, 000 RBM for two minutes, and freeze the plasma at minus 20 degrees Celsius.Fill two 60 milliliter syringes for each of the infusion conditions listed here. Use y connectors and sterilized coex T 22 tubing to join each pair of solutions together. Then place the syringes on a Harbor 33 to syringe pump placing syringe one on the front position of the pump and syringe two on the back position.Next, change the cage bottom and remove all food from the cage grill top before replacing it with 150 grams of standard chow. Then connect the syringes to the swivel on the cage grill and flush the syringes properly. To remove trapped air from the lines using the most recently determined body weight, calculate the infusion flow rates using an Excel file that converts the glucose infusion rate or GIR into milliliters per hour based on predetermined glucose levels desired to maintain throughout the experiment.Set the pump to administer flow rates for 60 syringes according to the manufacturer's settings, and enter the rate for syringe one and syringe two. Then start the pump. After starting the pump, verify that there is no leaking from the swivel or from the cannulas and that the infusion tubing is not twisted.Also, verify that there are no air bubbles in the tubing. After three hours of infusion, take a small blood sample and use a portable glucose monitor to measure glycemia drawing. Small volumes will prevent altering blood volume and or hematocrit.Take additional blood samples using this timetable. Based on the measured glycemia values, modify the rate of syringe one to maintain blood glucose at 220 to 250 milligrams per deciliter. The rate of syringe two remains constant to maintain free fatty acids at one milli mole per liter.After 24 hours of infusion, change the cage bottom and weigh the food remaining in the cage grill. Return the UNE portion to the cage, grill and refill syringes as needed throughout the 72 hours. Observe the rat for any signs of inflammation or discomfort and administer appropriate care as needed.At the end of the infusion, rats can be subjected to hyperglycemic or u glycemic hyperinsulinemic clamp studies, and subsequently euthanized or euthanized to collect the pancreas for histology or eyelet isolation. This figure shows blood, glucose and fatty acid levels during the 72 hour infusion period. As shown here by design, the glucose levels are maintained around 220 to 250 milligrams per deciliter throughout the infusion.Rodents have a strong capacity to compensate for the glucose infusion by increasing endogenous insulin secretion. Therefore, the GIR must be increased during the course of the infusion for blood glucose levels to be maintained at a relative steady state. Nevertheless, blood glucose levels tend to decrease towards the end of the infusion.In addition, based on weight measurements of chow, since the glucose and intralipid infused animals are receiving caloric nutrients, they spontaneously decrease their food intake. Unlike the saline infused control rats once mastered, this technique can be performed in 30 to 40 minutes per rat if it is done properly. So following this procedure, we usually perform hyperglycemic or U glycemic hyper insulin anemic clamps to measure insulin secretion or insulin sensitivity respectively.

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Research

JoVE Journal

Medicine

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Intraduodenal Cannulation in a Rat Model for Immunopathology Studies

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Learn a chronic nutrient infusion protocol in rats to study glucose homeostasis and metabolic health effects. This method uses jugular vein and carotid artery catheterization with glucose and Intralipid solutions.

A Model of Chronic Nutrient Infusion in the Rat

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