Dr. Carvalho's work was supported by a research grant from the Office of Research on Women's Health and National Institute of Child Health and Human Development of the National Institutes of Health (5K12 HD043452). Dr Angst received supplies (On-Q PainBuster Post-Op Pain Relief System) and funds (for the biochemical assays) from I-Flow (Lake Forest, California).

1. Study Protocol
<ul>
	<li>Obtain Institutional Review Board approval and written informed consent.</li>
	<li>Select an appropriate surgical population for study conduct. This methodology has only been applied to cesarean delivery wounds but most surgical wounds would be appropriate.</li>
	<li>Consider inclusion and exclusion criteria for your study participation. Patients and procedures with high rates of surgical wound infection may not be appropriate.</li>
	<li>Standardize anesthesia and postoperative management for all patients to avoid potential confounders.</li>
</ul>
2. Nociceptive and Inflammatory Biochemical Mediator Collection
<ul>
	<li>Insert the ON-Q PainBuster with ON-Q SilverSoaker (I-Flow, Lake Forest, CA) into the subcutaneous layer just prior to wound closure (other tissue layers can be considered). The ON-Q PainBuster Pain Relief System continuously delivers normal saline at a rate of 2 ml/h. The carrier can be combined with or compared to drugs of interest such as a local anesthetic and non-steroidal anti-inflammatories.</li>
	<li>Incorporate a standard three-way stopcock into this system to allow aspiration of wound exudate at specified time points. The continuous infusion prevents the catheter from clotting and improves the reliability of the system to produce exudate samples. If aspiration of exudate is difficult (approximately 5% of cases), consider pushing gently above the wound, changing the subject's position (e.g., sitting the patient up or lying them flat), using a 0.5-1 ml normal saline flush or withdrawing the catheter 1-2 cm.</li>
	<li>At time points specified by the protocol (e.g., 1, 4, 6, 24, and 48 hr after cesarean delivery), aspirate and withdraw 1 ml of wound exudate. Transfer the exudate into a polyethylene cup containing 30 &mu;l of proteinase inhibitor. We have used Complete Proteinase Inhibitor (Roche Bioscience, Palo Alto, CA) for our purposes.</li>
	<li>At the same time intervals specified by the study protocol, collect 10 ml of blood. We have used a blood collection tube containing lithium heparin; at our institution this is a green-topped glass tube. Add an additional 300 &mu;l of proteinase inhibitor into the blood samples.</li>
	<li>Within 1 hr of collection, put samples on ice and centrifuge at 3,000 rpm for 10 min.</li>
	<li>Remove serum and wound supernate and place in a standard microcentrifuge tube and store at -20 &deg;C.</li>
</ul>
3. Assay Analysis
<ul>
	<li>Once all the study exudate and serum samples are collected and stored, thaw and analyze the samples at the same time.</li>
	<li>We have measured the cytokines and chemokines using a multiplex immunoassay. The multiplex immuno-assay technique allows us to assay numerous analytes from small (50 &mu;l) volume samples. The multiplex produce results comparable with those obtained with ELISA. We have used the 17-plex Bio-PlexTM system (BioRad, Hercules, CA) for our analysis. This assay is capable of measuring tumor necrosis factor &alpha; (TNF&alpha;), interferon &alpha; (INF&alpha;), interleukin 1&beta; (IL-1&beta;), interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6 (IL-6), interleukin 7 (IL-7), interleukin 8 (IL-8), interleukin (IL-10), interleukin 12 (IL-12), interleukin 13 (IL-13), interleukin 17 (IL-17), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), monocyte chemoattractant protein 1 (MCP-1) and macrophage inflammatory protein 1 (MIP-1&beta;). Nerve growth factor was measured with NGF DY256 antibody by adding it to the 17-plex plate with the Bio-Plex amine coupling kit.</li>
	<li>To ensure good quality analysis, each measurement should be made in duplicate. Standard curves for each analyte are generated by using reference analytes supplied by the manufacturer (at concentrations of 0.20, 0.78, 3.13, 12.5, 50, 200, 800, 3,200 pg/ml plus a zero standard for the normal saline). Standard curves are included in each run and sample concentrations are calculated with Bio-Plex Manager software.</li>
	<li>Substance P and prostaglandin E2 were measured using highly sensitivity ELISA Kits. We have used Assay Designs, Inc. (Ann Arbor, Michigan) for this analysis. Perform the analysis in duplicate according to the manufacturer's specification.</li>
</ul>

<ol>
	<li>Carvalho, B., Clark, D. J., Angst, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+and+Systemic+Release+of+Cytokines,+Nerve+Growth+Factor,+Prostaglandin+E2,+and+Substance+P+in+Incisional+Wounds+and+Serum+Following+Cesarean+Delivery.">Local and Systemic Release of Cytokines, Nerve Growth Factor, Prostaglandin E2, and Substance P in Incisional Wounds and Serum Following Cesarean Delivery.</a> J. Pain. 9, 650-657 (2008).</li><li>Carvalho, B., Clark, D. J., Yeomans, D. C., Angst, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+subcutaneous+instillation+of+bupivacaine+compared+to+saline+reduces+interleukin+10+and+increases+substance+P+in+surgical+wounds+after+cesarean+delivery.">Continuous subcutaneous instillation of bupivacaine compared to saline reduces interleukin 10 and increases substance P in surgical wounds after cesarean delivery.</a> Anesth. Analg. 111, 1452-1459 (2010).</li><li>Verri, W. A., Cunha, T. M., Parada, C. A., Poole, S., Cunha, F. Q., Ferreira, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypernociceptive+role+of+cytokines+and+chemokines:+targets+for+analgesic+drug+development.">Hypernociceptive role of cytokines and chemokines: targets for analgesic drug development.</a> Pharmacol. Ther. 112, 116-138 (2006).</li><li>Watkins, L. R., Maier, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+regulation+of+central+nervous+system+functions:+from+sickness+responses+to+pathological+pain.">Immune regulation of central nervous system functions: from sickness responses to pathological pain.</a> J. Intern. Med. 257, 139-155 (2005).</li><li>Beilin, B., Bessler, H., Mayburd, E., Smirnov, G., Dekel, A., Yardeni, I., Shavit, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+preemptive+analgesia+on+pain+and+cytokine+production+in+the+postoperative+period.">Effects of preemptive analgesia on pain and cytokine production in the postoperative period.</a> Anesthesiology. 98, 151-155 (2003).</li><li>Wu, C. T., Jao, S. W., Borel, C. O., Yeh, C. C., Li, C. Y., Lu, C. H., Wong, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+epidural+clonidine+on+perioperative+cytokine+response,+postoperative+pain,+and+bowel+function+in+patients+undergoing+colorectal+surgery.">The effect of epidural clonidine on perioperative cytokine response, postoperative pain, and bowel function in patients undergoing colorectal surgery.</a> Anesth. Analg. 99, 502-509 (2004).</li><li>Buvanendran, A., Kroin, J. S., Berger, R. A., Hallab, N. J., Saha, C., Negrescu, C., Moric, M., Caicedo, M. S., Tuman, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Upregulation+of+prostaglandin+E2+and+interleukins+in+the+central+nervous+system+and+peripheral+tissue+during+and+after+surgery+in+humans.">Upregulation of prostaglandin E2 and interleukins in the central nervous system and peripheral tissue during and after surgery in humans.</a> Anesthesiology. 104, 403-410 (2006).</li><li>Kristiansson, M., Soop, M., Sundqvist, K. G., Soop, A., Suontaka, A. M., Blomback, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+vs.+systemic+immune+and+haemostatic+response+to+hip+arthroplasty.">Local vs. systemic immune and haemostatic response to hip arthroplasty.</a> Eur. J. Anaesthesiol. 15, 260-270 (1998).</li><li>Liu, S. S., Richman, J. M., Thirlby, R. C., Wu, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+continuous+wound+catheters+delivering+local+anesthetic+for+postoperative+analgesia:+a+quantitative+and+qualitative+systematic+review+of+randomized+controlled+trials.">Efficacy of continuous wound catheters delivering local anesthetic for postoperative analgesia: a quantitative and qualitative systematic review of randomized controlled trials.</a> J. Am. Coll. Surg. 203, 914-932 (2006).</li><li>Eriksson, A. S., Sinclair, R., Cassuto, J., Thomsen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+lidocaine+on+leukocyte+function+in+the+surgical+wound.">Influence of lidocaine on leukocyte function in the surgical wound.</a> Anesthesiology. 77, 74-78 (1992).</li><li>Clark, J. D., Shi, X., Li, X., Qiao, Y., Liang, D., Angst, M. S., Yeomans, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphine+reduces+local+cytokine+expression+and+neutrophil+infiltration+after+incision.">Morphine reduces local cytokine expression and neutrophil infiltration after incision.</a> Mol. Pain. 3, 28 (2007).</li><li>Webb, S. T., Ghosh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intra-articular+bupivacaine:+potentially+chondrotoxic.">Intra-articular bupivacaine: potentially chondrotoxic.</a> Br. J. Anaesth. 102, 439-441 (2009).</li><li>Scott, N. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+infiltration+for+surgery.">Wound infiltration for surgery.</a> Anaesthesia. 65, 67 (2010).</li></ol>

No conflicts of interest declared.

This methodology outlined above has been found to be sensitive enough to detect biochemical inflammatory and nociceptive mediators in wound exudate. Sampling at various time-points has also allowed us to observe changes of these mediators over time.1,2 We are not aware of any other human surgical wound assay that can serially measure the release profile of inflammatory and nociceptive mediators in surgical wound exudate. Results from our methodology of wound exudate collection from the surgical incision site is different from studies utilizing drains from the hip joint,7,8 which suggests that subcutaneous wound mediator release may be different from intraarticular or other body cavities biochemical mediator release. Additionally, results obtained in drains may be confounded by blood loss into the wound, and therefore they may not accurately reflect local biochemical mediator release.
The poor correlation between wound and serum levels demonstrates the importance of determining site-specific release when studying surgical wounds. Local changes may not be reflected systemically due to many factors including differential systemic and local production in response to surgical trauma and stress. Although we have chosen to measure a particular selection of pro- and anti-inflammatory cytokines and chemokines as well as NGF, SP and PG-E2 from cesarean delivery wound exudate,1,2 this methodology can be applied to other surgical models and many other biochemical mediators.
Surgical wound local anesthetic instillation is a commonly used technique for improving postoperative pain.9 Applying the methodology, we have been able to demonstrate that local anesthetic instillation into surgical wounds does affect wound biochemistry.2 A decrease in IL-10 and increase in substance P in wound exudates with bupivacaine instillation suggests a disruption of anti-inflammatory modulation. Peripherally administered local anesthetics may thereby affect the inflammatory processes,10,11 which may partially account for local anesthetic-induced chondrolysis in joints after surgery.12
ON-Q PainBuster Pain Relief System is a US Food and Drug Administration (FDA) approved device since 1998 that is used for the delivery of local anesthetics to the surgical wound site to reduce pain following surgery. Continuous wound instillation utilizing this and other catheters has not been associated with increased wound infection.9,13 ON-Q PainBuster with ON-Q SilverSoaker is designed to infuse drugs and not to repeatedly sample wound exudate. Although we have had no wound infections with this technique, we stress an aseptic technique while sampling wound exudate from this system. Our proposed modification to facilitate sampling should be reserved for research purposes and not for routine clinical use. We also recommend that the duration the catheter remains in-situ be limited to 48-72 hours.
This surgical site bioassay technique provides a good model to understand the relationship between inflammatory and nociceptive biochemical mediators in postoperative pain. We will use the methodology in future studies to investigate effects of locally administered non-steroidal anti-inflammatory drugs and opioids. The technique may also lend itself to investigations examining wound healing, excessive surgical scarring and wound infection.

After a surgical incision, resident and migrating inflammatory cells release pro- and anti-inflammatory cytokines, prostanoids, neuropeptides and chemokines that are important for initiating and maintaining pain.3,4 The ability to collect site-specific biochemical markers is critical to determine the relationship between levels in serum and surgical wound, understand the complex relationship between mediator release and surgical pain, and to be able to evaluate the effect of both systemic and peripheral drug administration on surgical wound biochemistry. Studies have focused on systemic measurements of biochemical mediators and have not considered site-specific release.5,6 Serum measurements of cytokines and chemokines may not be reflective of local surgical wound biochemical events and only indicate a global systemic response to surgical injury. This article provides a detailed and unique visual description of this methodology that was first referenced in 2008 in the Journal of Pain.1 The aim of this JoVE article is to provide a unique and visually enhanced description of the methods.

collecting and measuring wound exudate biochemical mediators in surgical wounds

We describe a methodology by which we are able to collect and measure biochemical inflammatory and nociceptive mediators at the surgical wound site. Collecting site-specific biochemical markers is important to understand the relationship between levels in serum and surgical wound, determine any associations between mediator release, pain, analgesic use and other outcomes of interest, and evaluate the effect of systemic and peripheral drug administration on surgical wound biochemistry. This methodology has been applied to healthy women undergoing elective cesarean delivery with spinal anesthesia. We have measured wound exudate and serum mediators at the same time intervals as patient's pain scores and analgesics consumption for up to 48 hours post-cesarean delivery. Using this methodology we have been able to detect various biochemical mediators including nerve growth factor (NGF), prostaglandin E2 (PG-E2) substance P, IL-1&#x03B2;, IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, TNF&alpha;, INF&#x03B3;, G-CSF, GM-CSF, MCP-1 and MIP-1&#x03B2;. Studies applying this human surgical wound bioassay have found no correlations between wound and serum cytokine concentrations or their time-release profile (J Pain. 2008; 9(7):650-7).1 We also documented the utility of the technique to identify drug-mediated changes in wound cytokine content (Anesth Analg 2010; 111:1452-9).2

Using this methodology we have been able to detect all biochemical mediators outlined above with the exception of IL-5 in wound exudate and plasma.1,2 Some additional cytokines were not detected in plasma (IL-4, IL-17, TNF&alpha;, INF&gamma;, GM-CSF).1 The time course of measured cytokines and chemokines in reported studies were consistent, reaching a plateau within the first few hours that was sustained for the 24 hour observation period..1,2 However, NGF increased steadily over the 24-hours collection period, whereas PGE2 peaked within 1 hour and then steadily decreased.1 Particularly noteworthy results are markedly decreased levels of IL-10 and increased levels of substance P in wounds instilled with the local anesthetic bupivacaine, compatible with an overall pro-inflammatory effect of the drug (Figure 1).2 In a different study we demonstrated that analgesic consumption after cesarean delivery surgery was negatively correlated with wound exudate IL-6, IL-1 &beta;, and G-CSF concentrations (Figure 2).1
<img alt="Figure 1" fo:content-width="3in" fo:src="/files/ftp_upload/50133/50133fig1highres.jpg" src="/files/ftp_upload/50133/50133fig1.jpg" /> 
Figure 1. Interleukin-10 (IL-10) and substance P wound exudate levels levels (pg/ml) measured at 1, 3, 5, 7, and 24 hours post-cesarean delivery. The normal saline group is indicated in gray diamonds, and the bupivacaine group is shown with black squares. *=Significantly different between groups (P &lt; 0.005).
<img alt="Figure 2" src="/files/ftp_upload/50133/50133fig2.jpg" /> 
Figure 2. Correlation between interleukin-6 (pg/ml) and morphine consumption in the first 24 hr after cesarean delivery.

Watch this Scientific Journal Video about Collecting And Measuring Wound Exudate Biochemical Mediators In Surgical Wounds at JoVE.com

Collecting And Measuring Wound Exudate Biochemical Mediators In Surgical Wounds

This article provides a detailed and visual description of a methodology for collecting and measuring biochemical inflammatory and nociceptive mediators at the surgical wound site following cesarean delivery. This human bioassay has been used to determine correlations between wound and serum cytokine concentrations and drug-mediated changes in wound cytokines, chemokines and neuropetides.

We describe a methodology by which we are able to collect and measure biochemical inflammatory and nociceptive mediators at the surgical wound site. ...

collecting-measuring-wound-exudate-biochemical-mediators-surgical

Nanyang Technological University

Research

JoVE Journal

Medicine

11.9K Views.  Stanford University School of Medicine. This article provides a detailed and visual description of a methodology for collecting and measuring biochemical inflammatory and nociceptive mediators at the surgical wound site following cesarean delivery. This human bioassay has been used to determine correlations between wound and serum cytokine concentrations and drug-mediated changes in wound cytokines, chemokines and neuropetides.

Video: Collecting And Measuring Wound Exudate Biochemical Mediators In Surgical Wounds

Biochemical Measurement of Neonatal Hypoxia

Neonatal hypoxia ischemia is characterized by inadequate blood perfusion of a tissue or a systemic lack of oxygen. This condition is thought to cause/exacerbate well documented neonatal disorders including neurological impairment 1-3. Decreased adenosine triphosphate production occurs due to a lack of oxidative phosphorylation. To compensate for this energy deprived state molecules containing high energy phosphate bonds are degraded 2. This leads to increased levels of adenosine which is subsequently degraded to inosine, hypoxanthine, xanthine, and finally to uric acid. The final two steps in this degradation process are performed by xanthine oxidoreductase. This enzyme exists in the form of xanthine dehydrogenase under normoxic conditions but is converted to xanthine oxidase (XO) under hypoxia-reperfusion circumstances 4, 5. Unlike xanthine dehydrogenase, XO generates hydrogen peroxide as a byproduct of purine degradation 4, 6. This hydrogen peroxide in combination with other reactive oxygen species (ROS) produced during hypoxia, oxidizes uric acid to form allantoin and reacts with lipid membranes to generate malondialdehyde (MDA) 7-9. Most mammals, humans exempted, possess the enzyme uricase, which converts uric acid to allantoin. In humans, however, allantoin can only be formed by ROS-mediated oxidation of uric acid. Because of this, allantoin is considered to be a marker of oxidative stress in humans, but not in the mammals that have uricase.
We describe methods employing high pressure liquid chromatography (HPLC) and gas chromatography mass spectrometry (GCMS) to measure biochemical markers of neonatal hypoxia ischemia. Human blood is used for most tests. Animal blood may also be used while recognizing the potential for uricase-generated allantoin. Purine metabolites were linked to hypoxia as early as 1963 and the reliability of hypoxanthine, xanthine, and uric acid as biochemical indicators of neonatal hypoxia was validated by several investigators 10-13. The HPLC method used for the quantification of purine compounds is fast, reliable, and reproducible. The GC/MS method used for the quantification of allantoin, a relatively new marker of oxidative stress, was adapted from Gruber et al 7. This method avoids certain artifacts and requires low volumes of sample. Methods used for synthesis of MMDA were described elsewhere 14, 15. GC/MS based quantification of MDA was adapted from Paroni et al. and Cighetti et al. 16, 17. Xanthine oxidase activity was measured by HPLC by quantifying the conversion of pterin to isoxanthopterin 18. This approach proved to be sufficiently sensitive and reproducible.

A method is described to measure biochemical markers of neonatal hypoxia-ischemia. The approach utilizes high pressure liquid chromatography (HPLC) and Gas Chromatography Mass Spectrometry (GC/MS).

Neonatal hypoxia ischemia is characterized by inadequate blood perfusion of a tissue or a systemic lack of oxygen. This condition is thought to ...

Mouse Model of Intraluminal MCAO: Cerebral Infarct Evaluation by Cresyl Violet Staining

Stroke is the third cause of mortality and the leading cause of disability in the World. Ischemic stroke accounts for approximately 80% of all strokes. However, the thrombolytic tissue plasminogen activator (tPA) is the only treatment of acute ischemic stroke that exists. This led researchers to develop several ischemic stroke models in a variety of species. Two major types of rodent models have been developed: models of global cerebral ischemia or focal cerebral ischemia. To mimic ischemic stroke in patients, in whom approximately 80% thrombotic or embolic strokes occur in the territory of the middle cerebral artery (MCA), the intraluminal middle cerebral artery occlusion (MCAO) model is quite relevant for stroke studies. This model was first developed in rats by Koizumi et al. in 1986 1. Because of the ease of genetic manipulation in mice, these models have also been developed in this species 2-3.
	Herein, we present the transient MCA occlusion procedure in C57/Bl6 mice. Previous studies have reported that physical properties of the occluder such as tip diameter, length, shape, and flexibility are critical for the reproducibility of the infarct volume 4. Herein, a commercial silicon coated monofilaments (Doccol Corporation) have been used. Another great advantage is that this monofilament reduces the risk to induce subarachnoid hemorrhages. Using the Zeiss stereo-microscope Stemi 2000, the silicon coated monofilament was introduced into the internal carotid artery (ICA) via a cut in the external carotid artery (ECA) until the monofilament occludes the base of the MCA. Blood flow was restored 1 hour later by removal of the monofilament to mimic the restoration of blood flow after lysis of a thromboembolic clot in humans. The extent of cerebral infarct may be evaluated first by a neurologic score and by the measurement of the infarct volume. Ischemic mice were thus analyzed for their neurologic score at different post-reperfusion times. To evaluate the infarct volume, staining with 2,3,5-triphenyltetrazolium chloride (TTC) was usually performed. Herein, we used cresyl violet staining since it offers the opportunity to test many critical markers by immunohistochemistry. In this video, we report the MCAO procedure; neurological scores and the evaluation of the infarct volume by cresyl violet staining.

The intraluminal middle cerebral occlusion model in mice is herein presented. The extent of cerebral infarct is evaluated by a neurologic score and cresyl violet staining, an alternative staining to TTC, offering the great advantage to test in parallel many interest markers.

Stroke is the third  cause of mortality and the leading cause of disability in the World. Ischemic  stroke accounts for approximately 80% of all strokes. ...

Collecting Saliva and Measuring Salivary Cortisol and Alpha-amylase in Frail Community Residing Older Adults via Family Caregivers

Salivary measures have emerged in bio-behavioral research that are easy-to-collect, minimally invasive, and relatively inexpensive biologic markers of stress. This article we present the steps for collection and analysis of two salivary assays in research with frail, community residing older adults-salivary cortisol and salivary alpha amylase. The field of salivary bioscience is rapidly advancing and the purpose of this presentation is to provide an update on the developments for investigators interested in integrating these measures into research on aging. Strategies are presented for instructing family caregivers in collecting saliva in the home, and for conducting laboratory analyses of salivary analytes that have demonstrated feasibility, high compliance, and yield quality specimens. The protocol for sample collection includes: (1) consistent use of collection materials; (2) standardized methods that promote adherence and minimize subject burden; and (3) procedures for controlling certain confounding agents. We also provide strategies for laboratory analyses include: (1) saliva handling and processing; (2) salivary cortisol and salivary alpha amylase assay procedures; and (3) analytic considerations.

We demonstrate: (1) procedures for collection of salivary samples in cognitive impaired older adults by family caregivers in the home setting, (2) procedures for measuring stress activity via salivary cortisol and alpha amylase, and (3) representative profiles. Protocols that allow researchers to study stress-linked processes advance our understanding of biological sensitivity and susceptibility.

Salivary measures have emerged in bio-behavioral research  that are easy-to-collect, minimally invasive, and relatively inexpensive  biologic markers of ...

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

Congenital heart disease (CHD) is the most frequent noninfectious cause of death at birth. The incidence of CHD ranges from 4 to 50/1,000 births (Disease and injury regional estimates, World Health Organization, 2004). Surgeries that often compromise the quality of life are required to correct heart defects, reminding us of the importance of finding the causes of CHD. Mutant mouse models and live imaging technology have become essential tools to study the etiology of this disease. Although advanced methods allow live imaging of abnormal hearts in embryos, the physiological and hemodynamic states of the latter are often compromised due to surgical and/or lengthy procedures. Noninvasive ultrasound imaging, however, can be used without surgically exposing the embryos, thereby maintaining their physiology. Herein, we use simple M-mode ultrasound to assess heart rates of embryos at E18.5 in utero. The detection of abnormal heart rates is indeed a good indicator of dysfunction of the heart and thus constitutes a first step in the identification of developmental defects that may lead to heart failure.

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

Ultra-high frequency ultrasound is a powerful live imaging tool to examine cardiac abnormalities in small animals. Its noninvasiveness allows maintaining the physiologic state of embryos. Herein, we show the use of M-mode ultrasound to measure the heart rates of embryos at E18.5 in utero.

Congenital heart disease (CHD) is the most frequent noninfectious cause of death at birth. The incidence of CHD ranges from 4 to 50/1,000 births (Disease ...

Intravital Microscopy of Tumor-associated Vasculature Using Advanced Dorsal Skinfold Window Chambers on Transgenic Fluorescent Mice

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static information and is often not sufficient for a correct interpretation. Intravital evaluation, in which a process is followed in time, provides extra and often unexpected information. With the creation of transgenic animals expressing cell-specific markers and live cell tracers, improvements to imaging equipment, and the development of several imaging chambers, intravital microscopy has become an important tool to better understand biological processes. This paper describes an experimental design for the investigation of tumor vessel development and of therapeutic effects in a spatial and temporal manner. Using this setup, the stage of vessel development, tip cell and lumen formation, blood flow, extravasation, an established vascular bed, and vascular destruction can be visualized and followed. Furthermore, therapeutic effects, intratumoral fate, and the localization of chemotherapeutic compounds can also be followed.

This protocol describes the intravital imaging of transgenic mice expressing cell-specific fluorescent markers. Intravital imaging provides a non-invasive method for high-resolution observations in living animals using implantable windows through which the microscopic visualization of processes is possible. This method is especially useful to study longitudinal processes.

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static ...

Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles

Conventional imaging techniques can provide detailed information about cellular processes. However, this information is based on static images in an otherwise dynamic system, and successive phases are easily overlooked or misinterpreted. Live-cell imaging and time-lapse microscopy, in which living cells can be followed for hours or even days in a more or less continuous fashion, are therefore very informative. The protocol described here allows for the investigation of the fate of chemotherapeutic nanoparticles after the delivery of doxorubicin (dox) in living cells. Dox is an intercalating agent that must be released from its nanocarrier to become biologically active. In spite of its clinical registration for more than two decades, its uptake, breakdown, and drug release are still not fully understood. This article explores the hypothesis that lipid-based nanoparticles are taken up by the tumor cells and are slowly degraded. Released dox is then translocated to the nucleus. To prevent fixation artifacts, live-cell imaging and time-lapse microscopy, described in this experimental procedure, can be applied.

Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles

Live-cell imaging is a powerful tool to visualize dynamic processes. The examination of fixed cells provides only static pictures, which can lead to misinterpretation and confusion about the process. This work presents a method to study uptake, drug release, and intracellular localization of liposomal nanoparticles in living cells.

Conventional imaging techniques can provide detailed information about cellular processes. However, this information is based on static images in an ...

Collecting Hair Samples for Hair Cortisol Analysis in African Americans

The hormone cortisol is typically assessed in saliva, serum, or urine samples. More recently, cortisol has been successfully extracted from hair, including humans. The advantage of hair cortisol concentration is that it reflects a retrospective representation of hypothalamic-pituitary-adrenal (HPA) axis function over time, much like hemoglobin A1C represents glycemic control. However, obtaining hair samples can be challenging, due to the cultural beliefs and hair care practices of minority participants. For example, African Americans may be reluctant to provide samples. Additionally, few researchers are trained to collect hair samples from African Americans. The purpose of this paper is to present a culturally informed protocol to help researchers obtain hair samples from African Americans. To illustrate the representative results of this protocol implementation, de-identified data from African Americans that participated in a community-based study on chronic stress are provided. Hair practice preferences are assessed. The participants are made comfortable by showing pictures of hair samples prior to cutting their hair. The single strain twist and gently pull method is used to collect approximately 30 - 50 strands of hair from the posterior vertex region of the scalp. This protocol will significantly improve collection of hair samples from African Americans.

Hair cortisol concentration analysis provides an alternative to traditional measures of cortisol; however, to collect hair samples from African Americans, scientists need to be culturally informed and competent. The purpose of this protocol is to demonstrate a culturally informed technique to collect hair samples for cortisol analysis from African Americans.

The hormone cortisol is typically assessed in saliva, serum, or urine samples. More recently, cortisol has been successfully extracted from hair, ...

Protocol to Create Chronic Wounds in Diabetic Mice

Chronic wounds develop as a result of defective regulation in one or more complex cellular and molecular processes involved in proper healing. They impact ~6.5M people and cost ~$40B/year in the US alone. Although a significant effort has been invested in understanding how chronic wounds develop in humans, fundamental questions remain unanswered. Recently, we developed a novel mouse model for diabetic chronic wounds that have many characteristics of human chronic wounds. Using db/db-/- mice, we can generate chronic wounds by inducing high levels of oxidative stress (OS) in the wound tissue immediately after wounding, using a one-time treatment with inhibitors specific to the antioxidant enzymes catalase and glutathione peroxidase. These wounds have high levels of OS, develop biofilm naturally, become fully chronic within 20 days after treatment and can remain open more for more than 60 days. This novel model has many features of diabetic chronic wounds in humans and therefore can contribute significantly to advancing fundamental understanding of how wounds become chronic. This is a major breakthrough because chronic wounds in humans cause significant pain and distress to patients and result in amputation if unresolved. Moreover, these wounds are very expensive and time-consuming to treat, and lead to significant loss of personal income to patients. Advancements in this field of study through the use of our chronic wound model can significantly improve health care for millions who suffer under this debilitating condition. In this protocol, we describe in great detail the procedure to cause acute wounds to become chronic, which has not been done before.

Chronic wounds are developed from acute wounds on a diabetic mouse model by inducing high levels of oxidative stress after a full-thickness cutaneous wound. The wound is treated with inhibitors for catalase and glutathione peroxidase, resulting in impaired healing and biofilm development by bacteria present in the skin microbiome.

Chronic wounds develop as a result of defective regulation in one or more complex cellular and molecular processes involved in proper healing. They impact ...

Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to injury. In fact, the most common type of eye injury found in clinic is a corneal abrasion. Here, we utilize an ocular burr to induce an abrasion resulting in removal of the corneal epithelium in vivo on anesthetized mice. This method allows for targeted and reproducible epithelial disruption, leaving other areas intact. In addition, we describe the visualization of the abraded epithelium with fluorescein staining and provide concrete advice on how to visualize the abraded cornea. Then, we follow the timeline of wound healing 0, 18, and 72 h after abrasion, until the wound is re-epithelialized. The epithelial abrasion model of corneal injury is ideal for studies on epithelial cell proliferation, migration and re-epithelialization of the corneal layers. However, this method is not optimal to study stromal activation during wound healing, because the ocular burr does not penetrate to the stromal cell layers. This method is also suitable for clinical applications, for example, pre-clinical test of drug effectiveness.

This protocol describes a method to inflict an abrasion to the ocular surface of the mouse, and to follow the wound healing process thereafter. The protocol takes advantage of an ocular burr to partially remove the surface epithelium of the eye in anaesthetized mice.

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to ...

Targeting Gray Rami Communicantes in Selective Chemical Lumbar Sympathectomy

Chemical lumbar sympathectomy (CLS) is a commonly used, minimally invasive procedure for the treatment of conditions including ischemic diseases of the lower extremities, hyperhidrosis, etc. It is commonly practiced to position the puncture needle tip in front of the anterior fascia of the psoas major muscle and inject the inactivating agent around the sympathetic trunk, which is defined as conventional CLS. Although relatively rare, ureteropelvic damage is the most frequently reported complication of conventional CLS and can cause serious harm to patients. We found that injecting the inactivating agent behind the anterior fascia, which only targets gray rami communicantes, helped achieve therapeutic efficacy in&#160;vasodilation, sweat reduction, and pain relief comparable to conventional CLS, and serious complications were largely reduced. We define this procedure as selective CLS. Here, we present a protocol of selective CLS. The precise needle tract and accurate evaluation of the spreading of the contrast agent are critical to ensure that the drug is injected behind the anterior fascia of the psoas major muscle. The needle tip is at approximately one-third the dividing line of the vertebral body in the lateral view of a lumbar X-ray. The contrast is mainly confined around the needle tip and spreads outward and downward along the psoas muscle fibers. In this way, the anterior fascia provides a natural barrier for the ureteropelvic area, and the psoas major muscle provides a natural barrier for the lumbar nerve root. There are several highlights of this article, including 1) a detailed description of the selective CLS procedures, 2) an explanation of the anatomical basis for the implementation of selective CLS, and 3) an explanation of the differences between selective and conventional CLS.

We present a protocol for selective chemical lumbar sympathectomy (CLS), which inactivates only gray rami communicantes and not the sympathetic trunk. Selective CLS can help achieve therapeutic efficacy in vasodilation, sweat reduction, and pain relief that are comparable to conventional CLS, and serious complications, particularly ureteropelvic damages, can be reduced.

Chemical lumbar sympathectomy (CLS) is a commonly used, minimally invasive procedure for the treatment of conditions including ischemic diseases of the ...