This work was supported by departmental grants from the Multidisciplinary Pain Center (PCT), Anesthesiology and Critical Care Medicine departments of the Antwerp University Hospital (UZA), Belgium. In addition, an educational grant (Dehousse mandaat) was received from the University of Antwerp (UA). The authors want to thank Dr. Tom Schepens for his expert help during revision of this article.

This single-center cohort study was performed in accordance with the ethical standards of ICH-GCP and the Declaration of Helsinki after it was approved by the institutional review board and ethics committee of the Antwerp University Hospital, Belgium (study identifier: 16/33/334). The study was registered at Clinicaltrials.gov (NCT02916004) before its initiation.
All included patients were sedated in accordance with the standard hospital sedation protocol before study enrollment. The patients were titrated to a Richmond Agitation-Sedation Scale (RASS) set by the ICU physician. Patients were sedated to a RASS - 4 prior to study inclusion. All patients were routinely titrated to a Behavior Pain Scale (BPS) of 3 by the ICU analgosedation protocol.
Note: Determining therapeutic measures solely on the basis of the excitability of the recorded pain reflexes is not recommended. When interpreting the measurements, possible effects on the efferent branch of the reflex arc must be considered. Patients who are sedated or anesthetized have a higher pain reflex threshold than non-sedated patients. For reflex assessment, higher currents may be required. Monitoring of physiological parameters (heart rate, blood pressure, breathing rate) is recommended.
1. Safety Precautions
<ol>
	<li>Verify potential confounders for noise control (other devices, alternating mattress).</li>
	<li>Verify if the ambient temperature is in the normal range.</li>
</ol>
2. Positioning of the Subject
<ol>
	<li>Position the patient in the bed to maintain angles of 120&#176; of flexion of the hip and 130 - 160&#176; at the knee.</li>
	<li>Place the palmar side of the wrist upwards.</li>
	<li>Ensure that the non-measured eye is closed during reflex recording.</li>
</ol>
3. Preparation of the Skin for Electrode Application
Note: This will reduce electrode impedance.
<ol>
	<li>Clip or shave hair at the application sites.</li>
	<li>Check the application sites, they must be clean and dry. If necessary, remove any body lotion by cleaning the skin with soap and water and rub the skin gently with a dry wash cloth or gauze.</li>
	<li>Abrade the application sites with available abrasive material. Use the skin preparation paper over a large area rather than only a single swipe.</li>
	<li>Apply each electrode immediately after skin preparation.</li>
</ol>
4. Placement of the Electrodes for Pupil Dilation Reflex (PDR) Assessment
Note: Please see the Figures for an overview of electrodes application. Magnetic and electrical fields may appear as background noise or other artefacts in the measurement trace. Maximal acceptable noise level using the followings protocol is set from values higher than 10 &#181;V. High noise level is defined when the maximum amplitude in the area before the stimulation (&#8216;noise area&#8217;, i.e., -130 ms up to -10 ms before stimulation) exceeds this adjustable threshold (&#8216;maximum acceptable noise level&#8217;). Noise values are not used to calculate the threshold and the stimulation is repeated with the current intensity until an EMG signal with no noise is determined. To limit the occurrence of artefacts, verify the device has been updated to the latest version. Artefacts can be reduced by optimal electrode placement and skin preparation.
<ol>
	<li>Use Ag-AgCl electrodes with highly conductive wet gel to ensure an optimal signal during reflex recording.</li>
	<li>Maintain an inter-electrode distance of 30 mm (center-to-center).</li>
	<li>Place two stimulation electrodes for PDR recording, at the wrist on the skin area innervated by the median nerve, keeping the palmar side of the wrist facing upwards.</li>
</ol>
5. Placement of the Electrodes for Nociceptive Flexion Reflex (NFR) Assessment
Note: Please see the Figures for an overview of electrodes application.
<ol>
	<li>Use two stimulation electrodes at the ankle and place the electrodes distal to the lateral malleolus, stimulating the sural nerve area.</li>
	<li>Use two registration electrodes for EMG recording at the biceps femoris muscle. Place the electrodes four finger breadths above the popliteal fossa, posterior to the iliotibial band on the ipsilateral leg.</li>
	<li>Use one reference electrode, placed at the quadriceps tendon.</li>
</ol>
6. Safety Check
<ol>
	<li>Identify the materials: battery status (PDR tool), accessibility of a plug connection nearby (NFR evaluation monitor), lead wires, and connections to the labeled device sockets.</li>
	<li>Identify the patient: patient number, medical history, current medications, behavior pain scale, and sedation depth.</li>
</ol>
7. Pupillary Dilation Reflex Assessment: Getting Started
<ol>
	<li>Attach the lead wire to the stimulation electrodes at the wrist. Verify that the black-labeled part is attached to the most distal electrode.&#160;</li>
	<li>Turn the infrared camera on.</li>
	<li>Select the measurement protocol: &#8216;pupillary pain index&#8217; (PPI) through menu selection on the touch screen display.&#160;Perform an impedance control indicated by the colored symbols, if necessary repeat the preparation procedure.</li>
	<li>Clean the camera and eye cab with water and disinfect them.</li>
</ol>
8. Pupillary Dilation Reflex Assessment: Installation
<ol>
	<li>Open the eyelid and place the camera in an optimal position.
	<ol>
		<li>Let the rubber eyecup rest on the orbit, enclosing the whole eye.</li>
		<li>Verify whether pupil detection has been set correctly and adjust the camera if necessary. The operator may have to raise the eyelid more.</li>
		<li>Center the pupil in the middle of the screen and verify the position by pursuing a pupil completely colored green.</li>
	</ol>
	</li>
	<li>Close the contralateral eye, decreasing the consensual light response.</li>
	<li>Wait for a least 5 seconds to start the measurement, ensuring a stabilization period necessary for pupil accommodation (dark measurement environment).</li>
</ol>
9. Pupillary Dilation Reflex Assessment: Measurement
<ol>
	<li>Start the test by pushing the trigger button. Hold the button until the pupil assessment is complete (a few seconds). Ensure that the entire measurement cycle is executed by 2 audible signals (first at the start, second when the test is finished)
	<ol>
		<li>Do not move the camera during measurement; a countdown is shown on the screen when stimulation intensity is increasing automatically from 10 mA up to maximum 60 mA.</li>
	</ol>
	</li>
	<li>Identify the results automatically displayed after 15 seconds on the screen 
	Baseline pupil size (mm) before noxious stimulation (yellow horizontal line). 
	Maximal pupil size (mm) after noxious stimulation (white horizontal line). 
	Different levels of noxious stimulations by colored bands and values. 
	Maximal pupil variation (% and mm). 
	PPI score</li>
	<li>Save the measurement results by pressing the icon after pupil assessment.</li>
</ol>
10. Nociception Flexion Reflex Assessment: Getting Started
<ol>
	<li>Attach the lead wires for stimulation, recording and reference. Verify whether the black-labeled parts are attached to the most distal electrodes; white is for reference value recording at the knee.</li>
	<li>Turn the device on when connected to a power supply. Identify the USB flash drive if data storage is desired.</li>
</ol>
11. Nociceptive Flexion Reflex Assessment: Installation
<ol>
	<li>Press the settings button to go to the configuration menu to verify the stimulation settings and the threshold determination procedure for reflex measurement in unconscious sedated patients.
	<ol>
		<li>Verify Measurement technique is on threshold tracking.</li>
		<li>Verify the Stimulus type is determined as RIII reflex.</li>
		<li>Select off when asked for NRS input.</li>
		<li>Choose Peak Z Score as Evaluation criterion.</li>
		<li>Use &#62;100 number of stimuli.</li>
		<li>Initiate stimulation at 1 mA intensity, with minimum and maximum step size of 0.5 mA.</li>
		<li>Verify the Interstimulus interval is defined as 8 s with a reflex range of 90 &#8211; 180 ms.</li>
	</ol>
	</li>
</ol>
12. Nociceptive Flexion Reflex Assessment: Measurement
<ol>
	<li>Start the measurement, i.e., automatic reflex threshold tracking.</li>
	<li>Reduce impedances when &#8216;High noise level&#8217; appears by repeating the skin preparation protocol.</li>
	<li>Identify reflex features.
	<ol>
		<li>Identify the currents applied to the patient and number of stimulations.</li>
		<li>Identify the raw EMG displayed 200 ms before to 300 ms after stimulation via the EMG electrode on the thigh.</li>
		<li>Identify the reflex range and the reflex threshold value. The parameter is shown numerically (value in mA).</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

This paper describes the application of two nociceptive reflex devices for objective (patient-independent) pain assessment in adult ICU patients. Moreover, the evaluation of the PDR and the NFR characteristics are described.
Pain and delirium are common in hospitalized patients, often in combination, and may adversely affect outcome parameters. In the ICU, opioids are frequently administered, sometimes in combination with other sedative agents, to protect patients against stressful stimuli such as nursing care or various diagnostic or therapeutic procedures and to improve mechanical ventilation therapy, or they may be necessary due to critical illness. However, extensive evidence indicates that (unnecessary) prolonged administration of analgosedation to ICU patients negatively affects morbidity and mortality. Furthermore, the implementation of reliable evidence-based analgosedation protocols could further improve patient outcomes36,37,38.
The described reflex evaluation techniques can be considered quality indicators in healthcare and are closely associated with the use of opioids; further implementation could result in shorter ICU stays and improved short- and long-term outcomes. Furthermore, measuring nociceptive reflex thresholds through nociceptive assessments could result in targeted and patient-specific opioid administration. Therefore, evaluation and validation of the available objective pain assessment tools in critically-ill patients are urgently needed. Infrared pupillometry for PDR assessment has shown promising results39,40. Consistent with previous studies, this study demonstrated that pupillometry in unconscious patients in a very technological environment is feasible, fast, and straightforward41,42. Moreover, using the derived PPI score, the clinician is provided with an indication of the level of analgesia. Our study has clearly demonstrated that NFR can be routinely evaluated in ICU patients. However, it raises some significant points. First, NFR assessment may not be measurable due to persistent high electrode impedance despite maximal skin preparation. Secondly, we identified patients in whom NFR was not present, even with the maximal stimulation intensity. Despite the fact that NFR measurement is more challenging to perform, NFR threshold evaluation has shown promising results in patients under propofol-remifentanil sedation43.
Improving reflex assessment skills, the authors advise the performer to take some key steps into account. It is imperative to pursue a low electrode impedance for generating high quality output. As such, cleaning the skin with isopropyl alcohol should be limited to patients in whom electrode adhesion may be problematic (lotion-covered skin) since it may dehydrate the skin and therefore increase impedance. Abrading the skin at the electrode application site with intended material will optimize measurement variables. However, care should be taken not to injure the skin of the patient. Before reflex assessment, the user can easily perform an impedance control in a similar way for both devices, looking to the colored electrode symbol on the main screen. A green symbol indicates an optimal electrode impedance, a yellow symbol implies a &#39;good&#39; impedance. When the symbol is red colored, the impedance is too high for measurement and the skin preparation procedure should be repeated. In addition, the use of (very) small stimulation electrodes is recommended (i.e., 45 mm &#215; 30 mm) to avoid electrode overlap which may lead to incorrect reflex recording. Finally, explore the device settings before starting reflex measurement as default settings or stimulation characteristics can change between different patient populations. The issue of obvious concern is that of high unnecessary currents application in mainly awake, conscious patients.
Despite the growing interest in physiological pain assessment in unconscious patients2,16,17,18, there are some limitations that need to be acknowledged for both devices. Most notably, the pupillometer uses an inbuilt measurement model called &#39;pupillary pain index&#39; containing stepwise increasing tetanic stimulations. The measurement protocol is stopped when the pupil dilates more than 13% from its baseline size, a fixed cut-off criteria. By using this inbuilt limit, the occurrence of tachycardia and hypertension in response to nociceptive stimulations is assumed. Although pupillometry stimulation models are more frequently used, data confirming this hypothesis is lacking. Furthermore, the true challenge of this model lies in the practical implementation of these tests in routine clinical practice. Although more objective and patient-independent nociceptive reflex measurements may offer new perspectives for analgesic management, preparation and measurements require approximately 15 minutes (especially for NFR assessment), which remains challenging in a fast-paced work environment. Moreover, no normative data are currently available for &#39;normal reflex ranges&#39; in critically ill patients. Optimizing the skills and expertise of health care workers with respect to the use of these highly innovative tools may generate extraordinary results that can further classify analgesia levels, improve pain detection, prevent chronic pain disorders and enable (re)evaluation of pain management. Moreover, opportunities for economic valorization may arise, and the use of objective pain assessment tools may offer a unique translational platform for the testing of new pharmacological, analgesic compounds.
Measurements of more objective nociceptive reflexes, such as the PDR and NFR, may help clinicians evaluate patients&#39; specific analgesic needs, especially in those who are not able to report pain levels themselves. Whether these two assessment tools can be applied on a wide scale in daily practice remains to be determined. The ability of both innovative devices to predict nociceptive status and their ability to guide clinicians in optimizing analgesic treatment in non-communicative critically ill patients warrants further investigation.

Many critically ill patients in the Intensive Care Unit (ICU) are prone to experience pain during daily care or during diagnostic or therapeutic procedures. Substandard nociceptive evaluation and consequent suboptimal pain management may increase stress and anxiety1. Persistent pain not only increases circulating catecholamines, compromises tissue perfusion and reduces oxygen delivery2 but also activates catabolic hypermetabolism, thus contributing to hyperglycemia, lipolysis and muscle loss. All of these elements impair the healing process and increase the risk of infections3,4,5,6.
As stated by the International Association for the Study of Pain (IASP), clinicians must use pain assessment tools that are valid for all patients, and self-reports remain the golden standard for pain evaluation. However, there are many situations in which patients are unable to communicate, especially because of critical illness or when they are mechanically ventilated (MV). The increased interest in ICU patient-related outcome measures has amplified the need for structured and reliable techniques for nociceptive assessment when a patient is unable to report pain and discomfort. Attempts to address this need have been hampered by the lack of specific, reproducible and feasible monitoring tools. In recent years, considerable effort has been directed toward providing physicians with more objective nociceptive parameters. However, many studies executed in the ICU have focused on the use of vital signs as possible surrogates for pain assessment and underlie not to use blood pressure or heart rate as a specific parameter for pain7,8.
As reported in previous research, untreated pain significantly compromises patient outcomes and should therefore always be assessed independently of vital signs, and assessments should not be influenced by a patient&#39;s inability to communicate7,8,9,10,11,12. This approach of objective nociceptive assessment has gained considerable support due to the known negative consequences of pain. Especially in ICU patients, physiological and psychological effects can be substantial and long-lasting and may significantly decrease health-related quality of life13,14.
Currently, no objective pain monitoring protocol exists that can readily be applied to a large group of critically ill patients. The implementation of objective assessment tools in ICU patients could optimize pain management and thus prevent the development of central sensitization syndromes. Moreover, opioid-induced hyperalgesia (OIH), chronification of pain, and long-lasting pain-related morbidity may decrease. Finally, the application of nociceptive reflex evaluation tools may provide a unique translational platform on which new pharmacological analgesic compounds can be tested.
The aim of the proposed methodology is to provide an overview of the technical requirements and provide a precise description of the protocols used to assess nociceptive reflexes in non-communicative ICU patients. Overall, we aim to provide a comprehensive guide for the use of objective pain measurement tools in the ICU and in other circumstances in which sedated or unconscious patients need to be assessed.
Critically ill unconscious adults admitted to the ICU were screened for study inclusion from October 2016 until December 2017. All were mechanically ventilated and received a strict analgosedation protocol containing propofol/remifentanil or propofol/sufentanil, which are the two most commonly used schemes in our hospital. A history of ophthalmologic surgery, known pupil reflex disorders, Horner or Adie&#39;s syndrome, previous eye trauma, cranial nerve lesions or acute intracranial hypertension caused by traumatic brain injury, tumor compression or bleeding, fulminant stroke, known (poly)neuropathy related to diabetes or other neurological conditions known to influence reflex activity, intra- or extracorporeal treatment (pacemaker, intra-aortic balloon pump, extracorporeal life support), chronic opioid use (&#62;3 months), age &#60;18 years, and the use of topical interfering eye drops (atropine, phenylephrine), &#945;2 adrenergic agonists15, the use of other analgosedation protocols than described by the inclusion criteria or neuromuscular blocking agents were defined as exclusion criteria.
The demographic variables and medical data of the enrolled subjects, including the Simplified Acute Physiology Score II (SAPS II),16 were extracted from the digital patient data management system (e.g., Metavision).
Pain Assessment
ICU patients were screened for study inclusion, which required a medical history and admission diagnosis to assess the inclusion and exclusion criteria mentioned above. Physiological reflexes were assessed in the ICU environment under real-life conditions: no specific modifications were made regarding temperature or noise control. Reflex assessment was executed during daytime working hours at the individual patient room of approximately 20 &#176;C. All generated data (reflex characteristics) can be stored by each of the two devices when this function is enabled on the touch screen display.
Measurement of the Pupil Dilation Reflex
A pupillometry device was used for pupil dilation reflex (PDR) assessment using infrared video recording for quantitative pupil size evaluation. For the application of standardized nociceptive stimulation, two low-impedance Ag-AgCl electrodes were placed on the skin area innervated by the median nerve on the left arm after skin preparation (Figure 1). The current was fixed at 60 milliamp&#232;res (mA) with a maximum acceptable resistance of 5 kOhms, defining a voltage limitation of 300 volts (V).
PDR assessment was performed using an inbuilt pupillary pain index (PPI) measurement protocol that generates an automatic electric stimulation pattern for dynamic pupil reflex evaluation. Standardized noxious stimulation was applied with increasing intensity (from 10 mA to 60 mA with incremental steps of 10 mA, a duration of 1 s, and a pulse width of 200 &#181;s) until pupillary dilation greater than 13% ([maximal diameter - minimal diameter]/maximal diameter * 100) or maximal stimulation at 60 mA was achieved. When the defined criteria were reached, stimulation was automatically interrupted, and a PPI score was displayed (Table 1). Baseline pupil size (before standardized noxious stimulation), pupil reflex amplitude (PRA), stimulation intensity and the PPI score were recorded. The duration of PDR measurement was between 2 and 16 seconds depending on the number of required stimulations.
Several studies have suggested the use of pupillometry in non-communicative ICU adults. Paulus et al. demonstrated that PDR evaluation may predict analgesia requirements during endotracheal aspiration17. Moreover, this method may be able to reveal different levels of analgesia and could have discriminatory properties regarding different types of noxious procedures18,19. Recently, scientific interest has been directed toward the use of specific protocols for PDR assessment because of their low stimulation currents. The PPI protocol suggested in our approach has been previously investigated in anesthetized adults, revealing a significant correlation between PDR and opioid administration20. Furthermore, Sabourdin et al.21 demonstrated that PDR can be used to guide individual intraoperative remifentanil administration and therefore reduce intraoperative opioid consumption and postoperative rescue analgesia requirements.
Measurement of the Nociceptive Flexion Reflex
To assess the role of primary afferent fibers in the transmission of nociceptive signals from peripheral nociceptors to the sympathetic chain, the nociceptive flexion reflex (NFR) was evaluated. Reflex elicitation is mediated after A-delta fibers are activated by a complex interaction between neurons located in the dorsal horn of the spinal cord22. Rhudy and colleagues described the RIII reflex, a late response of the NFR with high-threshold nociceptive characteristics measured electromyographically (EMG) over the biceps femoris muscle after nociceptor activation.23
Increasing electrical stimulations are performed via cutaneous Ag-AgCl electrodes at the lateral malleolus, triggering the solely sensory sural nerve. The reflex response is evaluated in time and amplitude through EMG recording (Figure 2; Reprinted with permission of PH Dr med. Jan Baars, Managing Director, Dolosys GmbH.).
Following Willer et al., using the described reflex registration setup, the required stimulation intensity to elicit the NFR (threshold tracking) can be used as an objective nociceptive assessment correlating with subjective pain scores24,25,26,27,28. Subsequently, numerous studies have been conducted to identify reflex characteristics (mainly reflex threshold and amplitude) and their correlation with pain intensity sensation in conscious adults. These studies revealed that the reflex threshold and response amplitude is closely related to pain intensity27,29,30.Furthermore, standardized NFR scoring criteria, such as the reflex peak and the mean reflex EMG activity, can be used as reliable criteria for defining this NFR23,31,32. According to recent research, the defined reflex characteristics contributing to the NFR, despite their empirically derived origin, showed good test-retest reliabilities33,34. The duration of NFR recording, taking into account the (variable) step size range (0.5 mA - 2 mA), interstimulus interval of 8 seconds with an interval randomization of 20% to avoid possible habituation and reflex range between 90 - 180 ms after stimulation35, was between 5 and 15 minutes depending on the necessary stimulation intensity to elicit the NFR and therefore the number of required stimulations (maximum of 100 mA).

<table border="0" cellpadding="0" cellspacing="0" width="1021">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Neurolight Algiscan</td>
			<td style="width:215px;">ID Med, Marseille, France&#160;</td>
			<td style="width:221px;">Pupillometre 13235</td>
			<td style="width:369px;">Infra red camera for pupil dilation reflex measurement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Paintracker</td>
			<td style="width:215px;">Dolosys GmbH, Belin, Germany</td>
			<td style="width:221px;">Paintracker V1 2497</td>
			<td>Nociception flexion reflex assessment tool</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Red DotTrace Prep</td>
			<td style="width:215px;">3M, Ontario, Canada</td>
			<td style="width:221px;">CV-0001-7353-0</td>
			<td>Skin surface preparation tape</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Electrodes - BlueSensor N</td>
			<td style="width:215px;">Ambu, Ballerup, Denmark</td>
			<td style="width:221px;">BlueSensor N N-00-S/25&#160;</td>
			<td>Low-impedance Ag-AgCl skin electrodes</td>
		</tr>
	</tbody>
</table>

objective nociceptive assessment in ventilated icu patients: a feasibility study using pupillometry and the nociceptive flexion reflex

The concept of objective nociceptive assessment and optimal pain management have gained increasing attention. Despite the known negative short- and long-term consequences of unresolved pain or excessive analgosedation, adequate nociceptive monitoring remains challenging in non-communicative, critically ill adults. In the intensive care unit (ICU), routine nociceptive evaluation is carried out by the attending nurse using the Behavior Pain Scale (BPS) in mechanically ventilated patients. This assessment is limited by medication use (e.g., neuromuscular blocking agents) and the inherent subjective character of nociceptive evaluation by third parties.
Here, we describe the use of two nociceptive reflex testing devices as tools for objective pain evaluation: the pupillary dilation reflex (PDR) and nociception flexion reflex (NFR). These measurement tools are non-invasive and well tolerated, providing clinicians and researchers with objective information regarding two different nociceptive processing pathways: (1)&#160;the pain-related autonomic reactivity&#160;and (2) the ascending component of the somatosensory system. The use of PDR and NFR measurements are currently limited to specialized pain clinics and research institutions because of impressions that these are technically demanding or time-consuming procedures, or even because of a lack of knowledge regarding their existence.
By focusing on the two abovementioned nociceptive reflex assessments, this study evaluated their feasibility as a physiological pain measurement method in daily practice. Pursuing novel technologies for evaluating the analgesia level in unconscious patients may further improve individual pharmacological treatment and patient related outcome measures. Therefore, future research must include large well-designed clinical trials in a real-life environment.

We used both reflex assessments in a total of 40 critically ill ventilated subjects (38% females) at the ICU department using the previously described protocol. Patients with various indications for analgo-sedation were included: 58% for primary respiratory insufficiency, 23% due to multiple organ failure, 10% of the patients had a septic shock, and 9% were defined as being sedated for other reasons (e.g., cardiogenic reasons). All measurements were performed by the same investigator. Sedative agent dosing was never adjusted during the assessment. The pupil characteristics and EMG responses are shown in Table 2.
Vital signs remained unchanged during measurements, even with high (&#62;60 mA) nociceptive stimulation. Therefore, no nociceptive reflex assessment had to be terminated early due to an increase in blood pressure, heart rate or change in ventilatory parameters. Identification of the PDR was possible in all subjects using the described protocol. Nevertheless, the NFR was identified in only 72% of the patients. Moreover, NFR threshold tracking was not possible in 13% of the patients despite optimal measurement conditions, suggesting a deep analgosedation level. However, excessive nociceptive stimulation (i.e., stimulation currents above 100 mA) was not used.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57972/57972fig1.jpg" /> 
Figure 1: Schematic presentation of electrode application for standardized nociceptive stimulation used to elicit the PDR. Application of two stimulation electrodes at the skin area innervated by the median nerve.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57972/57972fig2.jpg" /> 
Figure 2: Schematic presentation of electrode application for NFR assessment. Application of two stimulation electrodes at the skin area innervated by the sural nerve, two recording electrodes placed at the ipsilateral biceps femoris, and 1 reference electrode. Please note that the black lead wire is attached to the most distally placed electrode, and the red lead wire is attached to the proximal electrode (courtesy of Dolosys GmbH, PD Dr med. Jan Baars, Managing Director). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57972/57972fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Maximum stimulation intensity (mA)</td>
			<td>Pupil reactivity</td>
			<td>Generated PPI score</td>
		</tr>
		<tr>
			<td>10</td>
			<td>Pupil dilation is greater than 13% during 10-mA stimulation</td>
			<td>9</td>
		</tr>
		<tr>
			<td>20</td>
			<td>Pupil dilation is greater than 13% during 20-mA stimulation</td>
			<td>8</td>
		</tr>
		<tr>
			<td>30</td>
			<td>Pupil dilation is greater than 13% during 30-mA stimulation</td>
			<td>7</td>
		</tr>
		<tr>
			<td>40</td>
			<td>Pupil dilation is greater than 13% during 40-mA stimulation</td>
			<td>6</td>
		</tr>
		<tr>
			<td>50</td>
			<td>Pupil dilation is greater than 13% during 50-mA stimulation</td>
			<td>5</td>
		</tr>
		<tr>
			<td>60</td>
			<td>Pupil dilation is greater than 13% during 60-mA stimulation</td>
			<td>4</td>
		</tr>
		<tr>
			<td>60</td>
			<td>Pupil dilation is greater than 13% during the second 60-mA stimulation</td>
			<td>3</td>
		</tr>
		<tr>
			<td>60 (5% &#60; dilation &#60; 13%)</td>
			<td>Pupil dilation is greater than 13% during the third 60-mA stimulation</td>
			<td>2</td>
		</tr>
		<tr>
			<td>60 (dilation &#8804; 5%)</td>
			<td>Pupil dilation is greater than 13% during the last 60-mA stimulation</td>
			<td>1</td>
		</tr>
		<tr>
			<td colspan="2">Note: if the pupil dilation is over 20% during stimulation, the PPI score is increased with one point</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1: PPI scoring algorithm.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Analgesia Protocol</td>
			<td>Overall</td>
			<td>Remifentanil</td>
			<td>Sufentanil</td>
			<td>No opioid</td>
		</tr>
		<tr>
			<td>Number of subjects&#160;</td>
			<td>40</td>
			<td>32</td>
			<td>5</td>
			<td>3</td>
		</tr>
		<tr>
			<td>PDR elicitable</td>
			<td>100%</td>
			<td>100%</td>
			<td>100%</td>
			<td>100%</td>
		</tr>
		<tr>
			<td>PDR stimulation intensity (mean &#177; SD, mA)&#160;</td>
			<td>49.75 &#177; 12.91</td>
			<td>49.69 &#177; 2.31</td>
			<td>54.00 &#177; 6.00</td>
			<td>43.33 &#177; 6.67</td>
		</tr>
		<tr>
			<td>PDR PPI score (mean &#177; SD)</td>
			<td>4.55 &#177; 0.39</td>
			<td>5.09 &#177; 0.50</td>
			<td>4.00 &#177; 1.73</td>
			<td>6.33 &#177; 0.88&#160;&#160;</td>
		</tr>
		<tr>
			<td>NFR elicitable</td>
			<td>72%</td>
			<td>69%</td>
			<td>60%</td>
			<td>0%</td>
		</tr>
		<tr>
			<td>NFR measurement error (no reflex assessed)</td>
			<td>15%</td>
			<td>19%</td>
			<td>20%</td>
			<td>-</td>
		</tr>
		<tr>
			<td>NFR threshold (mean &#177; SD)</td>
			<td>44.93 &#177; 4.93</td>
			<td>39.93 &#177; 4.65</td>
			<td>48.22 &#177; 16.84</td>
			<td>53.33 &#177; 8.37</td>
		</tr>
	</tbody>
</table>
Table 2: Pupil characteristics and EMG responses after nociceptive reflex assessment. &#39;Error&#39; measurements are defined as high impedance or noise problems during measurements. This can be explained by skin moisturing problems or skin pathology resulting in sub-optimal measurements or wall-outlet dysfunction.

Watch this Scientific Journal Video about Objective Nociceptive Assessment in Ventilated ICU Patients: A Feasibility Study Using Pupillometry and the Nociceptive Flexion Reflex at JoVE.com

Objective Nociceptive Assessment in Ventilated ICU Patients: A Feasibility Study Using Pupillometry and the Nociceptive Flexion Reflex

Pain assessment in anesthetized patients who cannot communicate with the outside world in any way remains challenging despite the development of innovative objective pain evaluation tools. In this project, the pupillary dilation reflex and the nociception flexion reflex are assessed in critically ill, mechanically ventilated adult patients.

The concept of objective nociceptive assessment and optimal pain management have gained increasing attention. Despite the known negative short- and ...

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8.7K Views.  Antwerp University Hospital (UZA), University of Antwerp (UA). Pain assessment in anesthetized patients who cannot communicate with the outside world in any way remains challenging despite the development of innovative objective pain evaluation tools. In this project, the pupillary dilation reflex and the nociception flexion reflex are assessed in critically ill, mechanically ventilated adult patients.

Video: Objective Nociceptive Assessment in Ventilated ICU Patients: A Feasibility Study Using Pupillometry and the Nociceptive Flexion Reflex

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Three-dimensional Imaging of Nociceptive Intraepidermal Nerve Fibers in Human Skin Biopsies

A punch biopsy of the skin is commonly used to quantify intraepidermal nerve fiber densities (IENFD) for the diagnosis of peripheral polyneuropathy 1,2. At present, it is common practice to collect 3 mm skin biopsies from the distal leg (DL) and the proximal thigh (PT) for the evaluation of length-dependent polyneuropathies 3. However, due to the multidirectional nature of IENFs, it is challenging to examine overlapping nerve structures through the analysis of two-dimensional (2D) imaging. Alternatively, three-dimensional (3D) imaging could provide a better solution for this dilemma.
In the current report, we present methods for applying 3D imaging to study painful neuropathy (PN). In order to identify IENFs, skin samples are processed for immunofluorescent analysis of protein gene product 9.5 (PGP), a pan neuronal marker. At present, it is standard practice to diagnose small fiber neuropathies using IENFD determined by PGP immunohistochemistry using brightfield microscopy 4. In the current study, we applied double immunofluorescent analysis to identify total IENFD, using PGP, and nociceptive IENF, through the use of antibodies that recognize tropomyosin-receptor-kinase A (Trk A), the high affinity receptor for nerve growth factor 5. The advantages of co-staining IENF with PGP and Trk A antibodies benefits the study of PN by clearly staining PGP-positive, nociceptive fibers. These fluorescent signals can be quantified to determine nociceptive IENFD and morphological changes of IENF associated with PN. The fluorescent images are acquired by confocal microscopy and processed for 3D analysis. 3D-imaging provides rotational abilities to further analyze morphological changes associated with PN. Taken together, fluorescent co-staining, confocal imaging, and 3D analysis clearly benefit the study of PN.

In order to study the changes of nociceptive intraepidermal nerve fibers (IENFs) in painful neuropathies (PN), we developed protocols that could directly examine three-dimensional morphological changes observed in nociceptive IENFs. Three-dimensional analysis of IENFs has the potential to evaluate the morphological changes of IENF in PN.

A punch biopsy of the skin is commonly used to quantify intraepidermal nerve fiber densities (IENFD) for the diagnosis of peripheral polyneuropathy 1,2. ...

Computerized Dynamic Posturography for Postural Control Assessment in Patients with Intermittent Claudication

Computerized dynamic posturography with the EquiTest is an objective technique for measuring postural strategies under challenging static and dynamic conditions. As part of a diagnostic assessment, the early detection of postural deficits is important so that appropriate and targeted interventions can be prescribed. The Sensory Organization Test (SOT) on the EquiTest determines an individual&#39;s use of the sensory systems (somatosensory, visual, and vestibular) that are responsible for postural control. Somatosensory and visual input are altered by the calibrated sway-referenced support surface and visual surround, which move in the anterior-posterior direction in response to the individual&#39;s postural sway. This creates a conflicting sensory experience. The Motor Control Test (MCT) challenges postural control by creating unexpected postural disturbances in the form of backwards and forwards translations. The translations are graded in magnitude and the time to recover from the perturbation is computed.
Intermittent claudication, the most common symptom of peripheral arterial disease, is characterized by a cramping pain in the lower limbs and caused by muscle ischemia secondary to reduced blood flow to working muscles during physical exertion. Claudicants often display poor balance, making them susceptible to falls and activity avoidance. The Ankle Brachial Pressure Index (ABPI) is a noninvasive method for indicating the presence of peripheral arterial disease and intermittent claudication, a common symptom in the lower extremities. ABPI is measured as the highest systolic pressure from either the dorsalis pedis or posterior tibial artery divided by the highest brachial artery systolic pressure from either arm. This paper will focus on the use of computerized dynamic posturography in the assessment of balance in claudicants.

Individuals with intermittent claudication exhibit poor balance compared to healthy controls. Computerized dynamic posturography is an objective method for measuring an individual's postural responses to balance disturbances. This provides an objective reflection of the person&#x2019;s ability to respond to situations under which sensory stimuli are altered and unexpected perturbations occur.

Computerized dynamic posturography with the EquiTest is an objective technique for measuring postural strategies under challenging static and dynamic ...

Myocardial Infarction and Functional Outcome Assessment in Pigs

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One important prerequisite is a good understanding of all safety and efficacy aspects obtained in a large animal model that validly reflect the human scenario of myocardial infarction (MI). Pigs are widely used in this regard since their cardiac size, hemodynamics, and coronary anatomy are close to that of humans. Here, we present an effective protocol for using the porcine MI model using a closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. This approach is based on 90 min of myocardial ischemia, inducing large left ventricle infarction of the anterior, septal and inferoseptal walls. Furthermore, we present protocols for various measures of outcome that provide a wide range of information on the heart, such as cardiac systolic and diastolic function, hemodynamics, coronary flow velocity, microvascular resistance, and infarct size. This protocol can be easily tailored to meet study specific requirements for the validation of novel cardioregenerative biologics at different stages (i.e. directly after the acute ischemic insult, in the subacute setting or even in the chronic MI once scar formation has been completed). This model therefore provides a useful translational tool to study MI, subsequent adverse remodeling, and the potential of novel cardioregenerative agents.

This protocol describes the porcine myocardial infarction (MI) model using a 90 min closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. Furthermore, the protocol for several outcome parameters, such as cardiac function, hemodynamics, microvascular resistance, and infarct size, are also presented.

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One ...

Assessment of Vascular Function in Patients With Chronic Kidney Disease

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this is only partially explained by traditional CVD risk factors. Vascular dysfunction is an important non-traditional risk factor, characterized by vascular endothelial dysfunction (most commonly assessed as impaired endothelium-dependent dilation [EDD]) and stiffening of the large elastic arteries. While various techniques exist to assess EDD and large elastic artery stiffness, the most commonly used are brachial artery flow-mediated dilation (FMDBA) and aortic pulse-wave velocity (aPWV), respectively. Both of these noninvasive measures of vascular dysfunction are independent predictors of future cardiovascular events in patients with and without kidney disease. Patients with CKD demonstrate both impaired FMDBA, and increased aPWV. While the exact mechanisms by which vascular dysfunction develops in CKD are incompletely understood, increased oxidative stress and a subsequent reduction in nitric oxide (NO) bioavailability are important contributors. Cellular changes in oxidative stress can be assessed by collecting vascular endothelial cells from the antecubital vein and measuring protein expression of markers of oxidative stress using immunofluorescence. We provide here a discussion of these methods to measure FMDBA, aPWV, and vascular endothelial cell protein expression.

The degree of vascular dysfunction and contributing physiological mechanisms can be assessed in patients with chronic kidney disease by measuring brachial artery flow-mediated dilation, aortic pulse-wave velocity, and vascular endothelial cell protein expression.

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this ...

Clinical Assessment of Spatiotemporal Gait Parameters in Patients and Older Adults

Spatial and temporal characteristics of human walking are frequently evaluated to identify possible gait impairments, mainly in orthopedic and neurological patients1-4, but also in healthy older adults5,6. The quantitative gait analysis described in this protocol is performed with a recently-introduced photoelectric system (see&#160;Materials table) which has the potential to be used in the clinic because it is portable, easy to set up (no subject preparation is required before a test), and does not require maintenance and sensor calibration. The photoelectric system consists of series of high-density floor-based photoelectric cells with light-emitting and light-receiving diodes that are placed parallel to each other to create a corridor, and are oriented perpendicular to the line of progression7. The system simply detects interruptions in light signal, for instance due to the presence of feet within the recording area. Temporal gait parameters and 1D spatial coordinates of consecutive steps are subsequently calculated to provide common gait parameters such as step length, single limb support and walking velocity8, whose validity against a criterion instrument has recently been demonstrated7,9. The measurement procedures are very straightforward; a single patient can be tested in less than 5 min&#160;and a comprehensive report can be generated in less than 1&#160;min.

This protocol is used to evaluate spatial and temporal gait variables of neurological/orthopedic patients and older persons by means of a recently-introduced floor-based photocell system.

Spatial and temporal characteristics of human walking are frequently evaluated to identify possible gait impairments, mainly in orthopedic and ...

Using Saccadometry with Deep Brain Stimulation to Study Normal and Pathological Brain Function

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including Parkinson's and Huntington's can disrupt it. People with Parkinson's disease, for example, tend to have increased saccadic latencies. Consequently, the quantitative measurement of saccadic eye movements has received considerable attention as a potential biomarker for neurodegenerative conditions. A lot more can be learned about the brain in both health and disease by observing what happens to eye movements when the function of specific brain areas is perturbed. Deep brain stimulation is a surgical intervention used for the management of a range of neurological conditions including Parkinson's disease, in which stimulating electrodes are placed in specific brain areas including several sites in the basal ganglia. Eye movement measurements can then be made with the stimulator systems both off and on and the results compared. With suitable experimental design, this approach can be used to study the pathophysiology of the disease being treated, the mechanism by which DBS exerts it beneficial effects, and even aspects of normal neurophysiology.

This paper describes the use of quantitative measurement of eye movements in conjunction with stimulation of focal areas of the deep brain in order to study physiology, pathophysiology, and the mechanisms of deep brain stimulation.

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including ...

Assessment of Child Anthropometry in a Large Epidemiologic Study

A high proportion of children have overweight and obesity in the United States and other countries. Accurate assessment of anthropometry is essential to understand health effects of child growth and adiposity. Gold standard methods of measuring adiposity, such as dual X-ray absorptiometry (DXA), may not be feasible in large field studies. Research staff can, however, complete anthropometric measurements, such as body circumferences and skinfold measurements, using inexpensive portable equipment. In this protocol we detail how to obtain manual anthropometric measurements from children, including standing and sitting height, weight, waist circumference, hip circumference, mid-upper arm circumference, triceps skinfold thickness, and subscapular skinfold thickness, and procedures to assess the quality of these measurements. To demonstrate accuracy of these measurements, among 1,110 school-aged children in the pre-birth cohort Project Viva we calculated Spearman correlation coefficients comparing manual anthropometric measurements with a gold standard measure of body fat, DXA fat mass1. To address reliability, we evaluate intra-rater technical error of measurement at a quality control session conducted on adult female volunteers.

In epidemiologic studies of children, well-trained research staff can accurately and precisely assess weight, height, sitting height, skinfold thicknesses, and body circumferences.

A high proportion of children have overweight and obesity in the United States and other countries. Accurate assessment of anthropometry is essential to ...

Using a Knee Arthrometer to Evaluate Tissue-specific Contributions to Knee Flexion Contracture in the Rat

Normal knee range of motion (ROM) is critical to well-being and allows one to perform basic activities such as walking, climbing stairs and sitting. Lost ROM is called a joint contracture and results in increased morbidity. Due to the difficulty of reversing established knee contractures, early detection is important, and hence, knowing risk factors for their development is essential. The rat represents a good model with which the effect of an intervention can be studied due to the similarity of rat knee anatomy to that of humans, the rat's ability to tolerate long durations of knee immobilization in flexion, and because mechanical data can be correlated with histologic and biochemical analysis of knee tissue.
Using an automated arthrometer, we demonstrate a validated, precise, reproducible, user-independent method of measuring the extension ROM of the rat knee joint at specific torques. This arthrometer can be used to determine the effects of interventions on knee joint ROM in the rat.

The goal of the protocol is to measure the extension range of motion of the rat knee. The effects of various diseases that increase the stiffness of the knee joint and the effectiveness of treatments can be quantified.

Normal knee range of motion (ROM) is critical to well-being and allows one to perform basic activities such as walking, climbing stairs and sitting. Lost ...

Semi-quantitative Assessment Using [18F]FDG Tracer in Patients with Severe Brain Injury

Patients with severe traumatic brain injury (sTBI) have difficulty knowing whether they are accurately expressing their thoughts and emotions because of disorders of consciousness, disrupted higher brain function, and verbal disturbances. As a consequence of an insufficient ability to communicate, objective evaluations are needed from family members, medical staff, and caregivers. One such evaluation is the assessment of functioning brain areas. Recently, multimodal brain imaging has been used to explore the function of damaged brain areas. [18F]-fluorodeoxyglucose positron emission tomography-computed tomography ([18F]FDG-PET/CT) is a successful tool for examining brain function. However, the assessment of brain glucose metabolism based on [18F]FDG-PET/CT is not standardized and depends on several varying parameters, as well as the patient&#39;s condition. Here, we describe a series of semiquantitative assessment protocols for a region-of-interest (ROI) image analysis using self-produced [18F]FDG tracers in patients with sTBI. The protocol focuses on screening the participants, preparing the [18F]FDG tracer in the hot lab, scheduling the acquisition of [18F]FDG-PET/CT brain images, and measuring glucose metabolism using the ROI analysis from a targeted brain area.

Semi-quantitative Assessment Using [18F]FDG Tracer in Patients with Severe Brain Injury

[18F]-fluorodeoxyglucose (FDG) positron emission tomography-computed tomography is useful for studying glucose metabolism related to brain function. Here, we present a protocol for an [18F]FDG tracer set-up and semiquantitative assessment of the region-of-interest analysis for targeted brain areas associated with clinical manifestations in patients with severe traumatic brain injury.

Patients with severe traumatic brain injury (sTBI) have difficulty knowing whether they are accurately expressing their thoughts and emotions because of ...