The authors would like to acknowledge the strong support of the DHMRI CLAS animal care staff, Facility Manager Mr. Daniel Peralta and attending veterinarian, Dr. Glicerio Ignacio, DVM MRCVS.

All animal procedures were approved by the David H. Murdock Research Institute IACUC located on the North Carolina Research Campus (NCRC), under protocol 14-017, Characterization of a nonhuman primate model of diabetes and prediabetes/insulin resistance and efficacy of therapeutics to improve insulin sensitivity and metabolic function.
1. Animal Selection and Study Preparation
<ol>
	<li>Select diet and weight stable animals based on monthly food intake and body weight records. 
	NOTE: Animals that have exhibited a recent decline in appetite should not be characterized until their food intake has stabilized.
	<ol>
		<li>For mature animals (&#62; 5 - 6 yr), do not select animals whose body weights between consecutive months differ by more than 10% without first examining the monkey and ruling out causes other than changing metabolic status for the dramatic change in weight.</li>
	</ol>
	</li>
	<li>For glucose, insulin and c-peptide analytes, prepare K2EDTA sample collection tubes with protease inhibitor (Aprotinin and DPP4I). 
	NOTE: The DPP4I + Aprotinin cocktail provides a broad spectrum of protease inhibition should this be required for additional analytes (glucagon, GLP-1). In the event that additional analytes are not collected, the blood tubes should be prepared in the same way to maintain consistency in sample collection. Samples for assays not validated for this method should be collected separately, according to the recommendations of the manufacturer.
	<ol>
		<li>Prepare the protease inhibitor cocktail by mixing 100 mg lyophilized Aprotinin with 10 ml of DPP4I. Add 10 &#181;l of the Aprotinin + DPP4I mixture to each blood tube for every milliliter of blood collected.</li>
		<li>Add an additional 10 &#181;l of the protease inhibitor cocktail to each tube for possible collection overage. Store the treated-blood tubes at -20 &#176;C until use. Keep the tubes on wet ice during the procedure and spin at 4 &#176;C.</li>
		<li>Use a serum separator tube to collect a blood sample at baseline for standard serum chemistry analysis. For the Complete Blood Cell count (CBC), use a standard K2EDTA without protease inhibitors. Use cryovials to aliquot plasma and serum after the blood samples have been spun down.</li>
		<li>Label the blood tubes and the cryovials appropriately with the animal identification, date, procedure, timepoint, and sample volume. Label the cryovials with the analyte(s) in plasma for appropriate assay.</li>
	</ol>
	</li>
	<li>Prepare the heparinized saline flush by injecting 0.15 ml of 1,000 USP units per ml heparin into a 250 ml bag of normal saline. Obtain a solution of 0.06 mg heparin/ml. Draw 40 - 60 ml of this solution into a saline lock for flushing between samples. Draw an additional 1 ml and 5 ml into separate syringes for flushing the dextrose infusion port before and after the infusion, respectively.</li>
</ol>
2. Animal Sedation and Preparation
<ol>
	<li>Remove food from the animal&#39;s cage no less than 14 hr before the procedure, and no more than 18 hr. 
	NOTE: It is important that animals be fasted for the procedure to avoid any post-prandial variation in glycemic values. It is also a precaution to avoid regurgitation and aspiration of stomach contents while anesthetized.</li>
	<li>Sedate animals for the duration of the IVGTT procedure with ketamine given intramuscularly as a general anesthetic, at 10 mg/kg. Administer additional ketamine (5 - 10 mg/kg) at intervals of 20 - 30 min, or as needed, during the procedure.
	<ol>
		<li>Weigh the sedated animal. Place the animal in a laterally recumbent position on a heated procedure table.</li>
		<li>Monitor clinical parameters every 15 to 20 min to ensure the animal is in a stable plane of anesthesia. Measure heart rate (100 - 200 bpm) and SPO2 (&#62; 92%) with a pulse oximeter. Measure respiratory rate (20 - 50 breaths/min) with a stopwatch, counting respirations visually or by hand over fifteen seconds and multiplying by four. Measure temperature (&#62; 97 &#176;F) rectally. Monitor the color of the mucous membrane around the gum and lips (moist, pink).</li>
	</ol>
	</li>
	<li>Prepare two cannula sites. Use hair clippers to trim the hair from the area of interest where the catheter will be inserted, and sterilize the entire region with alternating scrubs of chlorhexidine and 70% alcohol.
	<ol>
		<li>Place one catheter in the region of the left or right cephalic or saphenous veins and attach it to a heparin saline flush (0.06 mg heparin/ml) with a three-way stopcock. This is the sampling blood draw site.</li>
		<li>Place the second catheter in another leg or arm in the region of the cephalic or saphenous veins and attach a port. Use this site for the dextrose infusion. Use a small, 1 ml flush of heparinized saline to keep the cannula patent prior to dextrose infusion.</li>
	</ol>
	</li>
</ol>
3. IVGTT Procedure
NOTE: The IVGTT procedure consists of 8 blood draw sampling time-points (Table 1).
<ol>
	<li>Take the baseline sample and use a hand held glucometer to measure the fasting blood glucose level. Obtain a serum sample for standard chemistry analysis, as well as a whole blood sample for a CBC in order to assess the overall health of the animal. Collect plasma samples to assay glucose and insulin levels from the baseline sample using a kit validated for use with macaques as per the manufacturer&#39;s instructions 8, 9. 
	NOTE: It is important to have a pre-draw of 0.5 ml taken from the cannula prior to taking any sample for blood collection to remove residual blood or heparin in the dead-space of the cannula.</li>
	<li>After obtaining the baseline sample, infuse the dose of 50% dextrose (250 mg/kg) over 30 sec into the dextrose infusion port. 
	NOTE: Higher dose models (500 mg/kg) can be used, though the dose should be fixed across procedures in order to make longitudinal comparisons.
	<ol>
		<li>Flush the infusion port with 5 ml of heparinized saline to make sure that there is no dextrose left in the port. The end of the infusion is the T0. Have the technician replace the gloves, as residual dextrose from the infusion may contaminate subsequent blood samples.</li>
	</ol>
	</li>
	<li>The first post-infusion sample time point is at T3 min, from the end of dextrose infusion, followed by T5 min, T7 min, T10 min, T15 min, T20 min, and the last sample time point is at T30 min. Collect plasma from each timepoint to assay glucose and insulin levels with the baseline sample (see step 3.1).
	<ol>
		<li>At the T3 min time point, use the hand held glucometer to once again check the blood glucose level. 
		NOTE: The glucometer readings at baseline and T3 are only to confirm the infusion of the dextrose. The blood glucose level at the T3 min time point should be ~100 mg/dl higher compared to the fasting baseline plasma glucose level.</li>
	</ol>
	</li>
</ol>
4. Animal Recovery and Sample Processing
<ol>
	<li>Remove the cannulas and apply pressure to the catheterized sites for hemostasis after the T30 min time-point. Monitor the animal until it has regained consciousness and is sitting up. Offer feed once the animal is fully recovered.</li>
	<li>Immediately place each whole blood sample into K2EDTA tubes on ice. Centrifuge at 3,000 rpm at a temperature of 4 &#176;C within 10 min of collection. Aliquot plasma samples into cryovials, freeze and store at -80 &#176;C until analysis.
	<ol>
		<li>Allow the blood into the serum tube for standard serum chemistry analysis to sit at room temperature for no less than 20 min and no more than 30 min before centrifugation at 3,000 rpm at RT. Freeze the serum samples until assayed within 48 hr of collection. 
		NOTE: Refrigerate the whole blood samples collected for CBC analysis until assayed within 24 hr of collection.</li>
	</ol>
	</li>
</ol>
5. Data Treatment
<ol>
	<li>After establishing the plasma insulin and glucose curves, determine the glucose clearance rate K from the slope of the natural log of glucose values above baseline 16, 17. 
	NOTE: A healthy NHP may be expected to have a glucose clearance rate K well above 1, often greater than 2 or more, as a healthy animal will often return to their baseline glucose values within 30 min. As insulin production drops off, the glucose clearance rate K will drop more dramatically, falling below 1.</li>
	<li>Calculate the AUC as a whole, as the sum of totals of the area of the trapezoids representing the area under the curve of each line segment between time-points, through T30 16, 17. 
	NOTE: Traditionally, the AUC of the first ten minutes of the procedure is considered the acute insulin response to glucose (AIR), while the AUC from the last 20 min of the procedure is considered the late insulin response (LIR). As the animal becomes more dysmetabolic, the insulin AUC will increase, reflecting compensation for increasing insulin insensitivity. As the animal transitions to overt diabetes, however, the AUC will decrease, often initially in the acute insulin phase, captured by the AIR.</li>
</ol>

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Invest. 79, 790-800 (1987).</li><li>Hovorka, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partitioning+glucose+distribution/transport,+disposal,+and+endogenous+production+during+IVGTT.">Partitioning glucose distribution/transport, disposal, and endogenous production during IVGTT.</a> Am. J. Physiol. Endocrinol. 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The authors are affiliated with a contract research organization (Crown Bioscience) active in the field of metabolic disease.

The IVGTT assesses the capacity of glucose-stimulated insulin release by a single dextrose infusion based on body weight 5, 12, 13. From the assay, the fasting blood glucose and insulin level is attained, and it allows an assessment of the animal&#39;s capacity to release insulin and return the elevated glucose level towards baseline. This provides the user with information to characterize the animal as a normal glucose and insulin level healthy control, a hyperinsulinemic dysmetabolic animal with normoglycemi...

The IVGTT is a convenient functional assay that is routinely used to determine the &#946;-cell function in humans at different metabolic states 5, 7. In animal models of T2D, it is well recognized as a tool to characterize animals that show metabolic disease progression from a healthy to a dysmetabolic hyperglycemic state 8, 9. The closest animal model of T2D is demonstrated in nonhuman primates (NHPs), of which rhesus and cynomolgus macaques are notable examples. These animals naturally develop T2D with the same risk factors of age and obesity contributing to its incidence as in humans 10. Furthermore, there is a similar disease progression and pancreatic pathology showing amyloid deposits as the dysmetabolic disease progresses 11.
Here we report on our standard method of performing an IVGTT in NHPs as part of our colony characterization of metabolic status in these animals. This method is easy to perform relative to other, more time consuming and costly techniques 2. The IVGTT is useful for characterizing a large colony of animals rapidly and frequently. When taken into consideration with the level of glycated hemoglobin (HbA1C), the animal&#39;s diet and food intake history, as well as their percent lean mass and body fat, the IVGTT is normally sufficient for characterizing an animal&#39;s metabolic status and progression toward overt diabetes 6, 8.
HbA1C represents the average glycemic level over the life of a red blood cell, providing a reliable measure of glucose levels over the previous six weeks to three months. When measured from the fasted baseline blood sample of the IVGTT, this value provides a window into glycemic control during the months between procedures. If the animal has transitioned from dysmetabolic to diabetic since their last IVGTT, an HbA1C value much higher than their previous value would indicate that the transition began soon after their last IVGTT, whereas, an HbA1C value closer to their previous value would indicate that they have only recently transitioned. In general, in rhesus macaques, HbA1C values greater than 6% are considered abnormal, and indicate poor glycemic control 10, 23.
Glycemic levels should be interpreted within the context of the behavior and general health of the animal as a whole. Diabetic macaques &#8212; like humans &#8212; exhibit hyperphagia, polydipsia, and polyuria. Group housing of animals provides significant challenges to measurement of these indicators and the individual care required for dysmetabolic and diabetic monkeys. We recommend singly housing the animals in order that more personalized care may be provided, and behavioral markers of the health of the monkey more easily be monitored 8. Additionally, diabetic macaques will exhibit weight loss, as well as an elevated lipid profile (increased cholesterol, hypertriglyceridemia) and disturbed mineral metabolism in serum chemistry. It is important to measure markers of liver and kidney function in serum chemistry, as damage to these organs are often complications of advancing metabolic disorder/diabetes, and may be co-determinants of glycemic, lipid and mineral imbalances 9, 11, 18, 24.
When using this method, the historic values generated from multiple, frequent characterizations over the life of a monkey are of particular value. If other procedures, such as a glucose clamp or graded glucose infusion (GGI), are needed to fully assess an animal&#39;s health, it is commonly upon initial characterization when their history is unavailable. However, once a baseline has been established, repeated IVGTTs of a frequency of every three months are normally sufficient to track an animal&#39;s progress. This is particularly important when the animals are enrolled into multiple studies throughout a calendar year based upon their metabolic status. While their health may remain relatively stable for years at a time, when the metabolic status of an animal worsens, a dramatic increase in insulin resistance and glucose intolerance can occur very rapidly. HbA1C values allow for some interpolation of the decline or improvement of the health status of the animal between procedures scheduled three months apart. For this reason, this method is ideal for characterizing animals used in multiple, longitudinal studies over the course of their natural lifespan.

<table border="0" cellpadding="0" cellspacing="0" width="670">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Allegra X-15R Centrifuge</td>
			<td style="width:147px;"></td>
			<td style="width:119px;"></td>
			<td style="width:188px;">plasma: 4C @3000 rpm for 10 min</td>
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		<tr height="42">
			<td height="42" style="height:42px;">Sorvall ST16R Centrifuge</td>
			<td></td>
			<td style="width:119px;"></td>
			<td style="width:188px;">serum: 22C @3000 rpm for 10 min</td>
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			<td height="65" style="height:65px;width:216px;">Thermo Scientific -86C Freezer, Forma 88000 Series</td>
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			<td>Model: 88500A</td>
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			<td height="21" style="height:21px;">Dextrose 50% (D50)</td>
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			<td style="width:188px;">I.V. glucose infusate</td>
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			<td height="42" style="height:42px;width:216px;">5ml Luer Lock Syringe</td>
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			<td style="width:188px;">delivery of I.V. D50</td>
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			<td height="42" style="height:42px;width:216px;">4 ml serum separator tubes&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">K2EDTA, 2mL</td>
			<td style="width:147px;">VWR</td>
			<td style="width:119px;">95057-239</td>
			<td style="width:188px;">blood collection tubes</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Aprotinin, 100mg</td>
			<td style="width:147px;">Sigma</td>
			<td style="width:119px;">A1153-100MG</td>
			<td style="width:188px;">blood collection tube protease additive</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">22g x 1&#34; Catheters</td>
			<td style="width:147px;">Midwest Veterinary Suppy</td>
			<td style="width:119px;">193.75250.2</td>
			<td style="width:188px;">I.V. catheter&#160;</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Injection Plug W/ Cap</td>
			<td style="width:147px;">Midwest Veterinary Suppy</td>
			<td style="width:119px;">001.11500.2</td>
			<td style="width:188px;">%50 dextrose infusion port</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Porus Tape, 1/2&#34; x 10yd&#160;</td>
			<td style="width:147px;">Midwest Veterinary Suppy</td>
			<td style="width:119px;">001.85000.2</td>
			<td style="width:188px;">maintain adherance of catheters and hep. Locks</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Chlorhexidine Solution 2%</td>
			<td style="width:147px;">Midwest Veterinary Suppy</td>
			<td style="width:119px;">193.08855.3</td>
			<td style="width:188px;">prep catheter site</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">70% Ethanol</td>
			<td style="width:147px;">VWR</td>
			<td style="width:119px;">71001-654</td>
			<td style="width:188px;">prep catheter site</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">tourniquet</td>
			<td style="width:147px;">Webster</td>
			<td style="width:119px;">07-8003432</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">3 way stopcock</td>
			<td style="width:147px;">Midwest Veterinary Supply</td>
			<td style="width:119px;">366.28510.4</td>
			<td style="width:188px;">hep. lock</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">37&#34; extension set</td>
			<td style="width:147px;">Webster</td>
			<td style="width:119px;">07-8454200</td>
			<td style="width:188px;">hep. lock</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Exel 50-60cc LL Syringes</td>
			<td style="width:147px;">Midwest Veterinary Suppy</td>
			<td style="width:119px;">001.12250.2</td>
			<td style="width:188px;">Heparinized saline flush</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">250 ml bag 0.9% saline</td>
			<td style="width:147px;">Webster</td>
			<td style="width:119px;">07-8365593</td>
			<td style="width:188px;">flush</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1,000 U Heparin, 10 ml</td>
			<td style="width:147px;">Webster</td>
			<td style="width:119px;">07-883-4916</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ketamine, (Ketaset) 100mg/mL</td>
			<td style="width:147px;">Fort Dodge</td>
			<td style="width:119px;">(AV ordered)</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Precision Xtra glucose test strips 50/bx</td>
			<td style="width:147px;">Abbott (American Diabetes Wholesale)</td>
			<td style="width:119px;">9381599728K7</td>
			<td style="width:188px;">test baseline/ T3 blood glucose levels</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Masimo Rad 57</td>
			<td style="width:147px;">DRE</td>
			<td style="width:119px;">6052057V</td>
			<td style="width:188px;">pulse-oximeter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pavia rectal thermometer</td>
			<td style="width:147px;">Patterson</td>
			<td style="width:119px;">07-8391335</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Precision Xtra Glucometer</td>
			<td style="width:147px;">Abbott</td>
			<td style="width:119px;">9381599728K7</td>
			<td style="width:188px;">Handheld glucometer</td>
		</tr>
	</tbody>
</table>

characterization of metabolic status in nonhuman primates with the intravenous glucose tolerance test

The intravenous glucose tolerance test (IVGTT) plays a key role in the characterization of glucose homeostasis. When taken together with serum biochemical profiles, inclusive of blood glucose levels in both the fed and fasted state, HbA1c, insulin levels, clinical history of diet, body composition, and body weight status, an assessment of normal and abnormal glycemic control can be made. Interpretation of an IVGTT is done through measurement of changes in glucose and insulin levels over time in relation to the dextrose challenge. Critical components to be considered are: peak glucose and insulin levels reached in relation to T0 (end of glucose infusion), the glucose clearance rate K derived from the slope of rapid glucose clearance in the first 20 min (T1 to T20), the time to return to glucose baseline, and the area under the curve (AUC). These IVGTT measures will show characteristic changes as glucose homeostasis moves from a healthy to a diseased metabolic state5. Herein we will describe the characterization of nonhuman primates (Rhesus and Cynomolgus macaques), which are the most relevant animal model of Type II diabetes (T2D) in humans and the IVGTT and clinical profiles of these animals from a lean healthy, to obese dysmetabolic, and T2D state 8, 10, 11.

The results shown in Figure 1 are demonstrative of typical glucose and insulin curves from mature, healthy and diabetic cynomolgus macaques over the course of a 30 min IVGTT. Data from healthy and advanced diabetic monkeys are shown in order to contrast the obvious differences between animals from both extreme ends of the range of metabolic characterization. This IVGTT protocol has been used successfully by the authors in rhesus macaques with similar results.
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Watch this Scientific Journal Video about Characterization of Metabolic Status in Nonhuman Primates with the Intravenous Glucose Tolerance Test at JoVE.com

Characterization of Metabolic Status in Nonhuman Primates with the Intravenous Glucose Tolerance Test

The goal of this protocol is to present a standard method to perform intravenous glucose tolerance tests (IVGTTs) to assess glycemic control in nonhuman primates and assess their metabolic status from healthy to dysmetabolic.

The intravenous glucose tolerance test (IVGTT) plays a key role in the characterization of glucose homeostasis. When taken together with serum biochemical ...

characterization-metabolic-status-nonhuman-primates-with-intravenous

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JoVE Journal

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10.8K Views.  Crown Bioscience. The goal of this protocol is to present a standard method to perform intravenous glucose tolerance tests (IVGTTs) to assess glycemic control in nonhuman primates and assess their metabolic status from healthy to dysmetabolic.

Video: Characterization of Metabolic Status in Nonhuman Primates with the Intravenous Glucose Tolerance Test

Making Sense of Listening: The IMAP Test Battery

The ability to hear is only the first step towards making sense of the range of information contained in an auditory signal. Of equal importance are the abilities to extract and use the information encoded in the auditory signal. We refer to these as listening skills (or auditory processing AP). Deficits in these skills are associated with delayed language and literacy development, though the nature of the relevant deficits and their causal connection with these delays is hotly debated.
When a child is referred to a health professional with normal hearing and unexplained difficulties in listening, or associated delays in language or literacy development, they should ideally be assessed with a combination of psychoacoustic (AP) tests, suitable for children and for use in a clinic, together with cognitive tests to measure attention, working memory, IQ, and language skills. Such a detailed examination needs to be relatively short and within the technical capability of any suitably qualified professional. Current tests for the presence of AP deficits tend to be poorly constructed and inadequately validated within the normal population. They have little or no reference to the presenting symptoms of the child, and typically include a linguistic component. Poor performance may thus reflect problems with language rather than with AP. To assist in the assessment of children with listening difficulties, pediatric audiologists need a single, standardized child-appropriate test battery based on the use of language-free stimuli.
We present the IMAP test battery which was developed at the MRC Institute of Hearing Research to supplement tests currently used to investigate cases of suspected AP deficits. IMAP assesses a range of relevant auditory and cognitive skills and takes about one hour to complete. It has been standardized in 1500 normally-hearing children from across the UK, aged 6-11 years. Since its development, it has been successfully used in a number of large scale studies both in the UK and the USA. IMAP provides measures for separating out sensory from cognitive contributions to hearing. It further limits confounds due to procedural effects by presenting tests in a child-friendly game-format. Stimulus-generation, management of test protocols and control of test presentation is mediated by the IHR-STAR software platform. This provides a standardized methodology for a range of applications and ensures replicable procedures across testers. IHR-STAR provides a flexible, user-programmable environment that currently has additional applications for hearing screening, mapping cochlear implant electrodes, and academic research or teaching.

A test battery (IMAP) for performing an in-depth assessment of auditory and cognitive abilities contributing to listening skills is described. It is quick to administer, child-friendly and free from linguistic confounds. Stimulus generation and protocol management are controlled via a software platform (IHR-STAR) to ensure replicable procedures.

The ability to hear is only the first step towards making sense of the range of information contained in an auditory signal. Of equal importance are the ...

Delivery of Therapeutic Agents Through Intracerebroventricular (ICV) and Intravenous (IV) Injection in Mice

Despite the protective role that blood brain barrier plays in shielding the brain, it limits the access to the central nervous system (CNS) which most often results in failure of potential therapeutics designed for neurodegenerative disorders 1,2. Neurodegenerative diseases such as Spinal Muscular Atrophy (SMA), in which the lower motor neurons are affected, can benefit greatly from introducing the therapeutic agents into the CNS. The purpose of this video is to demonstrate two different injection paradigms to deliver therapeutic materials into neonatal mice soon after birth. One of these methods is injecting directly into cerebral lateral ventricles (Intracerebroventricular) which results in delivery of materials into the CNS through the cerebrospinal fluid 3,4. The second method is a temporal vein injection (intravenous) that can introduce different therapeutics into the circulatory system, leading to systemic delivery including the CNS 5. Widespread transduction of the CNS is achievable if an appropriate viral vector and viral serotype is utilized. Visualization and utilization of the temporal vein for injection is feasible up to postnatal day 6. However, if the delivered material is intended to reach the CNS, these injections should take place while the blood brain barrier is more permeable due to its immature status, preferably prior to postnatal day 2. The fully developed blood brain barrier greatly limits the effectiveness of intravenous delivery. Both delivery systems are simple and effective once the surgical aptitude is achieved. They do not require any extensive surgical devices and can be performed by a single person. However, these techniques are not without challenges. The small size of postnatal day 2 pups and the subsequent small target areas can make the injections difficult to perform and initially challenging to replicate.

Delivery of Therapeutic Agents Through Intracerebroventricular ICV and Intravenous IV Injection in Mice

This article demonstrates two very different methods of injection: 1) into the brain (intracerebroventricular) and 2) systemic (intravenous) to introduce the therapeutic agents into the central nervous system of neonatal mice.

Despite the protective role that blood brain barrier plays in shielding the brain, it limits the access to the central nervous system (CNS) which most ...

Tilt Testing with Combined Lower Body Negative Pressure: a "Gold Standard" for Measuring Orthostatic Tolerance

Orthostatic tolerance (OT) refers to the ability to maintain cardiovascular stability when upright, against the hydrostatic effects of gravity, and hence to maintain cerebral perfusion and prevent syncope (fainting). Various techniques are available to assess OT and the effects of gravitational stress upon the circulation, typically by reproducing a presyncopal event (near-fainting episode) in a controlled laboratory environment. The time and/or degree of stress required to provoke this response provides the measure of OT. Any technique used to determine OT should: enable distinction between patients with orthostatic intolerance (of various causes) and asymptomatic control subjects; be highly reproducible, enabling evaluation of therapeutic interventions; avoid invasive procedures, which are known to impair OT1.
In the late 1980s head-upright tilt testing was first utilized for diagnosing syncope2. Since then it has been used to assess OT in patients with syncope of unknown cause, as well as in healthy subjects to study postural cardiovascular reflexes2-6. Tilting protocols comprise three categories: passive tilt; passive tilt accompanied by pharmacological provocation; and passive tilt with combined lower body negative pressure (LBNP). However, the effects of tilt testing (and other orthostatic stress testing modalities) are often poorly reproducible, with low sensitivity and specificity to diagnose orthostatic intolerance7.
Typically, a passive tilt includes 20-60 min of orthostatic stress continued until the onset of presyncope in patients2-6. However, the main drawback of this procedure is its inability to invoke presyncope in all individuals undergoing the test, and corresponding low sensitivity8,9. Thus, different methods were explored to increase the orthostatic stress and improve sensitivity.
Pharmacological provocation has been used to increase the orthostatic challenge, for example using isoprenaline4,7,10,11 or sublingual nitrate12,13. However, the main drawback of these approaches are increases in sensitivity at the cost of unacceptable decreases in specificity10,14, with a high positive response rate immediately after administration15. Furthermore, invasive procedures associated with some pharmacological provocations greatly increase the false positive rate1.
Another approach is to combine passive tilt testing with LBNP, providing a stronger orthostatic stress without invasive procedures or drug side-effects, using the technique pioneered by Professor Roger Hainsworth in the 1990s16-18. This approach provokes presyncope in almost all subjects (allowing for symptom recognition in patients with syncope), while discriminating between patients with syncope and healthy controls, with a specificity of 92%, sensitivity of 85%, and repeatability of 1.1&plusmn;0.6 min16,17. This allows not only diagnosis and pathophysiological assessment19-22, but also the evaluation of treatments for orthostatic intolerance due to its high repeatability23-30. For these reasons, we argue this should be the "gold standard" for orthostatic stress testing, and accordingly this will be the method described in this paper.

We describe a "gold standard" for evaluating orthostatic tolerance (OT) using tilt testing with combined lower body negative pressure (LBNP). This can be combined with non-invasive evaluations of cardiovascular reflex control. Normal and abnormal responses are defined.

Orthostatic tolerance (OT)  refers to the ability to maintain cardiovascular stability when upright,  against the hydrostatic effects of gravity, and ...

Improving Strength, Power, Muscle Aerobic Capacity, and Glucose Tolerance through Short-term Progressive Strength Training Among Elderly People

This protocol describes the simultaneous use of a broad span of methods to examine muscle aerobic capacity, glucose tolerance, strength, and power in elderly people performing short-term resistance training (RET). Supervised progressive resistance training for 1 h three times a week over 8 weeks was performed by RET participants (71&#177;1 years, range 65-80). Compared to a control group without training, the RET showed improvements on the measures used to indicate strength, power, glucose tolerance, and several parameters of muscle aerobic capacity. Strength training was performed in a gym with only robust fitness equipment. An isokinetic dynamometer for knee extensor strength permitted the measurement of concentric, eccentric, and static strength, which increased for the RET group (8-12% post- versus pre-test). The power (rate of force development, RFD) at the initial 0-30 ms also showed an increase for the RET group (52%). A glucose tolerance test with frequent blood glucose measurements showed improvements only for the RET group in terms of blood glucose values after 2 h (14%) and the area under the curve (21%). The blood lipid profile also improved (8%). From muscle biopsy samples prepared using histochemistry, the amount of fiber type IIa increased, and a trend towards a decrease in IIx in the RET group reflected a change to a more oxidative profile in terms of fiber composition. Western blot (to determine the protein content related to the signaling for muscle protein synthesis) showed a rise of 69% in both Akt and mTOR in the RET group; this also showed an increase in mitochondrial proteins for OXPHOS complex II and citrate synthase (both ~30%) and for complex IV (90%), in only the RET group. We demonstrate that this type of progressive resistance training offers various improvements (e.g., strength, power, aerobic capacity, glucose tolerance, and plasma lipid profile).

The effect of short-term resistance training on elderly people was investigated through the simultaneous use of several methods. Compared to a control group, many improvements were seen, including on muscle aerobic capacity, glucose tolerance, strength, power, and muscle quality (i.e., protein involved in cell signaling and muscle fiber type composition).

This protocol describes the simultaneous use of a broad span of methods to examine muscle aerobic capacity, glucose tolerance, strength, and power in ...

Dissection of the Mouse Pancreas for Histological Analysis and Metabolic Profiling

We have been investigating the pancreas specific transcription factor, 1a cre-recombinase; lox-stop-lox- Kristen rat sarcoma, glycine to aspartic acid at the 12 codon (Ptf1acre/+;LSL-KrasG12D/+) mouse strain as a model of human pancreatic cancer. The goal of our current studies is to identify novel metabolic biomarkers of pancreatic cancer progression. We have performed metabolic profiling of urine, feces, blood, and pancreas tissue extracts, as well as histological analyses of the pancreas to stage the cancer progression. The mouse pancreas is not a well-defined solid organ like in humans, but rather is a diffusely distributed soft tissue that is not easily identified by individuals unfamiliar with mouse internal anatomy or by individuals that have little or no experience performing mouse organ dissections. The purpose of this article is to provide a detailed step-wise visual demonstration to guide novices in the removal of the mouse pancreas by dissection. This article should be especially valuable to students and investigators new to research that requires harvesting of the mouse pancreas by dissection for metabolic profiling or histological analyses.

This video article provides a detailed demonstration of the procedures required to successfully remove the pancreas from a mouse by dissection for histological analysis and metabolic profiling.

We have been investigating the pancreas specific transcription factor, 1a cre-recombinase; lox-stop-lox- Kristen rat sarcoma, glycine to aspartic acid at ...

Studying the Hypothalamic Insulin Signal to Peripheral Glucose Intolerance with a Continuous Drug Infusion System into the Mouse Brain

Insulin regulates systematic metabolism in the hypothalamus and the peripheral insulin response. An inflammatory reaction in peripheral adipose tissues contributes to type 2 diabetes mellitus (T2DM) development and appetite regulation in the hypothalamus. Chemokine CCL5 and C-C chemokine receptor type 5 (CCR5) levels have been suggested to mediate arteriosclerosis and glucose intolerance in type 2 diabetes mellitus (T2DM). In addition, CCL5 plays a neuroendocrine role in the hypothalamus by regulating food intake and body temperature, thus, prompting us to investigate its function in hypothalamic insulin signaling and the regulation of peripheral glucose metabolism.
The micro-osmotic pump brain infusion system is a quick and precise way to manipulate CCL5 function and study its effect in the brain. It also provides a convenient alternative approach to generating a transgenic knockout animal. In this system, CCL5 signaling was blocked by intracerebroventricular (ICV) infusion of its antagonist, MetCCL5, using a micro-osmotic pump. The peripheral glucose metabolism and insulin responsiveness was detected by the Oral Glucose Tolerance Test (OGTT) and Insulin Tolerance Test (ITT). Insulin signaling activity was then analyzed by protein blot from tissue samples derived from the animals.
After 7-14 days of MetCCL5 infusion, the glucose metabolism and insulin responsiveness was impaired in mice, as seen in the results of the OGTT and ITT. The IRS-1 serine302 phosphorylation was increased and the Akt activity was reduced in mice hypothalamic neurons following CCL5 inhibition. Altogether, our data suggest that blocking CCL5 in the mouse brain increases the phosphorylation of IRS-1 S302 and interrupts hypothalamic insulin signaling, leading to a decrease in insulin function in peripheral tissues as well as the impairment of glucose metabolism.

This protocol studies the role of chemokine (C-C motif) ligand 5 (CCL5) in the hypothalamus by delivering an antagonist, MetCCL5, into the mouse brain using a micro-osmotic pump brain infusion system. This transient inhibition of CCL5 activity interrupted hypothalamic insulin signaling, leading to glucose intolerance and peripheral systemic insulin sensitivity.

Insulin regulates systematic metabolism in the hypothalamus and the peripheral insulin response. An inflammatory reaction in peripheral adipose tissues ...

Study of In Vivo Glucose Metabolism in High-fat Diet-fed Mice Using Oral Glucose Tolerance Test (OGTT) and Insulin Tolerance Test (ITT)

Obesity represents the most important single risk factor in the pathogenesis of type 2 diabetes, a disease which is characterized by a resistance to insulin-stimulated glucose uptake and a gross decompensation of systemic glucose metabolism. Despite considerable progress in the understanding of glucose metabolism, the molecular mechanisms of its regulation in health and disease remain under-investigated, while novel approaches to prevent and treat diabetes are urgently needed. Diet derived glucose stimulates the pancreatic secretion of insulin, which serves as the principal regulator of cellular anabolic processes during the fed-state and thus balances blood glucose levels to maintain systemic energy status. Chronic overfeeding triggers meta-inflammation, which leads to alterations in peripheral insulin receptor-associated signaling and thus reduces the sensitivity to insulin-mediated glucose disposal. These events ultimately result in elevated fasting glucose and insulin levels as well as a reduction in glucose tolerance, which in turn serve as important indicators of insulin resistance. Here, we present a protocol for the generation and metabolic characterization of high-fat diet (HFD)-fed mice as a frequently used model of diet-induced insulin resistance. We illustrate in detail the oral glucose tolerance test (OGTT), which monitors the peripheral disposal of an orally administered glucose load and insulin secretion over time. Additionally, we present a protocol for the insulin tolerance test (ITT) to monitor whole-body insulin action. Together, these methods and their downstream applications represent powerful tools to characterize the general metabolic phenotype of mice as well as to specifically assess alterations in glucose metabolism. They may be especially useful in the broad research field of insulin resistance, diabetes and obesity to provide a better understanding of pathogenesis as well as to test the effects of therapeutic interventions.

Study of In Vivo Glucose Metabolism in High-fat Diet-fed Mice Using Oral Glucose Tolerance Test OGTT and Insulin Tolerance Test ITT

The current article describes the generation and metabolic characterization of high-fat diet-fed mice as a model of diet-induced insulin resistance and obesity. It further features detailed protocols to perform the oral glucose tolerance test and the insulin tolerance test, monitoring whole-body alterations of glucose metabolism in vivo.

Obesity represents the most important single risk factor in the pathogenesis of type 2 diabetes, a disease which is characterized by a resistance to ...

Induction of Nephrotic Syndrome in Mice by Retrobulbar Injection of Doxorubicin and Prevention of Volume Retention by Sustained Release Aprotinin

Nephrotic syndrome is the most extreme manifestation of proteinuric kidney disease and characterized by heavy proteinuria, hypoalbuminemia, and edema due to sodium retention and hyperlipidemia. To study the pathophysiology of this syndrome, rodent models have been developed based on the injection of toxic substances such as doxorubicin causing podocyte damage. In mice, only few strains are susceptible to this model. In wildtype 129S1/SvImJ mice, the administration of doxorubicin by rapid intravenous injection to the retrobulbar sinus induces experimental nephrotic syndrome that features all the symptoms of human disease including sodium retention and edema. After the onset of proteinuria, mice exhibit increased urinary serine protease activity that leads to the activation of the epithelial sodium channel (ENaC) and sodium retention. Pharmacological inhibition of urinary serine proteases by the treatment with sustained release aprotinin abrogates ENaC activation and prevents sodium retention. This model is ideal to study the pathophysiology of proteasuria, i.e., the excretion of active serine proteases that cause ENaC activation by the proteolysis of its &#947;-subunit. This can be regarded as the primary mechanism of ENaC activation and sodium retention in proteinuric kidney disease.

Here, we describe the induction of experimental nephrotic syndrome in 129S1/SvImJ mice by rapid retrobulbar injection of doxorubicin. We also treat nephrotic mice with sustained release pellets containing aprotinin to inhibit urinary serine protease activity and prevent sodium retention.

Nephrotic syndrome is the most extreme manifestation of proteinuric kidney disease and characterized by heavy proteinuria, hypoalbuminemia, and edema due ...

Mechanisms Underlying Gut Hormone Secretion Using the Isolated Perfused Rat Small Intestine

The gut is the largest endocrine organ of the body, producing more than 15 different peptide hormones that regulate appetite and food intake, digestion, nutrient absorption and distribution, and post-prandial glucose excursions. Understanding the molecular mechanisms that regulate gut hormone secretion is fundamental for understanding and translating gut hormone physiology. Traditionally, the mechanisms underlying gut hormone secretion are either studied in vivo (in experimental animals or humans) or using gut hormone-secreting primary mucosal cell cultures or cell lines. Here, we introduce an isolated perfused rat small intestine as an alternative method for studying gut hormone secretion. The virtues of this model are that it relies on the intact gut, meaning that it recapitulates most of the physiologically important parameters responsible for the secretion in in vivo studies, including mucosal polarization, paracrine relationships and routes of perfusion/stimulus exposure. In addition, and unlike in vivo studies, the isolated perfused rat small intestine allows for almost complete experimental control and direct assessment of secretion. In contrast to in vitro studies, it is possible to study both the magnitude and the dynamics of secretion and to address important questions, such as what stimuli cause secretion of different gut hormones, from which side of the gut (luminal or vascular) is secretion stimulated, and to analyze in detail molecular sensors underlying the secretory response. In addition, the preparation is a powerful model for the study of intestinal absorption and details regarding the dynamics of intestinal absorption including the responsible transporters.

Here, we present a powerful and physiological model to study the molecular mechanisms underlying gut hormone secretion and intestinal absorption &#8212; the isolated perfused rat small intestine.

The gut is the largest endocrine organ of the body, producing more than 15 different peptide hormones that regulate appetite and food intake, digestion, ...

Incorporation of a Survivable Liver Biopsy Procedure in Mice to Assess Non-alcoholic Steatohepatitis (NASH) Resolution

Clinical trials assessing therapies for the treatment of non-alcoholic steatohepatitis (NASH) involve a baseline and end of study liver biopsy, and assessment of improvement in disease endpoints, often reflected as a percent of each treatment arm that improved, worsened or remained unchanged. Traditional preclinical rodent studies for putative NASH therapies are often limited by not knowing the level of liver disease/NASH present at the start of therapeutic intervention, instead of randomizing treatment groups on easily measurable endpoints such as body weight, metabolic status or similar. Here, we describe a liver biopsy technique in a diet-induced NASH mouse model, for the assessment of baseline liver disease in order to exclude mice that do not exhibit fibrosis and to equally distribute animals with similar fibrosis between treatment groups. These levels can then be compared to the terminal, post-intervention levels for a truer understanding of in vivo pharmacological effects and thus more accurately reflect clinical trial design strategies. The mouse is properly anesthetized and prepared for the surgery using sterile conditions. A small incision is made in the upper abdomen and the left lateral lobe of the liver is exposed. A wedge of the liver is surgically removed, and a similar-sized piece of absorbable gelatin is put in its place to stop any bleeding. The mouse is surgically sutured and stapled closed and will recover back to normal within 1 day. The entire process takes 5-10 min per mouse. Here we exemplify the utility of this procedure by leveraging the pre-study biopsy to assess the impact of the glucagon-like peptide-1 (GLP-1) receptor agonist liraglutide on NASH endpoints in mice.

Incorporation of a Survivable Liver Biopsy Procedure in Mice to Assess Non-alcoholic Steatohepatitis NASH Resolution

In clinical settings, a liver biopsy is used to assess stages of non-alcoholic steatohepatitis (steatosis, inflammation, hepatocyte ballooning, and fibrosis). Here, we illustrate a survivable liver biopsy in mice which can be used for histological analyses to enable assessment of therapeutic agents in a manner more aligned with clinical trials.

Clinical trials assessing therapies for the treatment of non-alcoholic steatohepatitis (NASH) involve a baseline and end of study liver biopsy, and ...