The catheter of a telemetry device is implanted into the abdominal aorta in order to continuously collect beat-by-beat hemodynamic data from animals pre and post-high thoracic spinal cord transection. A novel JAVA software was employed to analyze hemodynamic parameters as well as frequency and intensity of spontaneous episodes of autonomic dysreflexia.
Spinal cord injury (SCI) is a debilitating neurological condition characterized by somatic and autonomic dysfunctions. In particular, SCI above the mid-thoracic level can lead to a potentially life-threatening hypertensive condition called autonomic dysreflexia (AD) that is often triggered by noxious or non-noxious somatic or visceral stimuli below the level of injury. One of the most common triggers of AD is the distension of pelvic viscera, such as during bladder and bowel distension or evacuation. This protocol presents a novel pattern recognition algorithm developed for a JAVA platform software to study the fluctuations of cardiovascular parameters as well as the number, severity and duration of spontaneously occurring AD events. The software is able to apply a pattern recognition algorithm on hemodynamic data such as systolic blood pressure (SBP) and heart rate (HR) extracted from telemetry recordings of conscious and unrestrained animals before and after thoracic (T3) complete transection. With this software, hemodynamic parameters and episodes of AD are able to be detected and analyzed with minimal experimenter bias.
Autonomic dysreflexia (AD) is a life-threatening emergency in individuals after acute or chronic spinal cord injury (SCI) at cervical or high-thoracic segments and is usually characterized by episodes of persistent hypertension and bradycardia1. AD is principally caused by disruption of descending spinal pathways that usually provide input from supraspinal centers to the spinal sympathetic preganglionic neurons that control sympathetic activity and vascular tone1-4. AD episodes are characterized by a spike in systolic blood pressure (SBP) up to 300 mmHg and if left untreated may lead to seizures, intracranial hemorrhage, myocardial infarction, and even death5-8. A variety of noxious and non-noxious stimuli act as a trigger of AD, including bowel and bladder distension, spasms, pressure sores, urinary bladder catheterization or iatrogenic procedures9-12.
The temporal development of AD in response to SCI has been investigated in both human9 and animal models13,14. Typically these studies have used an ‘induced AD’ method (i.e., urodynamics, penile vibrostimulations in humans or colorectal distension in animals) to determine the temporal development of AD. Such an approach is limited by the need for repeated assessments at isolated time-points that may preclude an accurate determination of the temporal development of AD. The use of 24-hr blood pressure monitoring in humans allows serial blood pressure measurements to be made at pre-determined intervals. This technique has recently been employed to monitor spontaneously occurring AD in patients with chronic SCI. In animal models, solid-state pressure transducers are being increasingly used to chronically monitor beat-by-beat arterial blood pressure. Recently, Rabchesvky et al. (2012), developed an algorithm that extracted one second averages of mean arterial pressure (MAP) and compared against a moving average threshold15. Spontaneous AD events were characterized based on MAP peaks that are 10 mmHg or greater above threshold concurrently with a HR drop of 10 bpm or greater.
Here a novel JAVA software that has a built in AD Detection Algorithm is presented. This algorithm works by detecting pre-determined patterns in arterial blood pressure (ABP) and heart rate (HR) that are indicative of a spontaneously occurring AD event. The user is able to manually adjust all input variables to the software such that the ‘detection algorithm’ can be easily customized to the specific parameters of interest. The software is also able to dichotomize ABP and HR into a given epoch such that diurnal rhythmicity of hemodynamic parameters can be analyzed16. In the present manuscript, a detailed explanation is given of the surgical technique that is used to implant the telemetry devices and conduct the SCI surgery. Examples are also provided with respect to the post-processing capabilities of the AD Detection software and how cardiovascular function is altered post-SCI. For comparative purposes, the methodology and results obtained from a method of induced AD known as colorectal distension (CRD) is also illustrated.
Male Wistar (Hsd: WI Wistar) rats at 7 weeks of age and weighing 300-350 g were used in this experiment. All rats were maintained on a 12 hr light/dark cycle and received standard laboratory rat chow and water ad libitum. All experimental procedures conformed with the guide to the Care and Use of Experimental Animals established by the Canadian Council on Animal Care and granted ethics approval by the University of British Columbia. Surgery and animal care were conducted according to standard procedures in our laboratory (Ramsey et al. 2010)17.
1. Preparation of the Animals: Surgical Procedures
2. Telemetry Monitoring of Hemodynamic Parameters
3. Assessment of Spontaneous Incidences of Autonomic Dysreflexia (AD)
Note: The frequency, severity, and duration of spontaneous AD events were assessed using an algorithm developed for our own novel AD Detection JAVA platform software (Figure 4). A novel algorithm has been developed to automatically detect spontaneous AD events based on 24 hr SBP and HR telemetry recordings before and after SCI utilizing parameters specified in Figure 2.
4. Colorectal Distension to Intentionally Elicit AD
Note: The severity of induced AD can be determined through colorectal distension (CRD), a clinically relevant stimulus that mimics the bowel routine3,18,19.
Using telemetry, arterial blood pressure is sampled at a frequency of 1,000 Hz continuously for 24 hr. An illustrative recording of arterial blood pressure (ABP) using LabChart is shown in Figure 1B. The sample ABP was monitored by a solid state pressure sensor inserted into the descending aorta. The novel JAVA platform AD Detection software is able to extract relevant SBP (mmHg) peaks (Figure 1C). We may also extract the HR (bpm) from the time interval between adjacent SBP peaks (Figure 1D).
SBP and HR extracted from beat by beat ABP sampled at 1,000 Hz are chosen as the input variables for the AD Detection software. The software characterizes spontaneous AD events by an increase in SBP greater than 20 mmHg accompanied by a decrease in HR of 40 bpm or greater, as presented in Figure 2. These events are initially identified utilizing a 240 sec SBP moving average baseline transposed vertically 20 mmHg. The AD Detection software ensures the detected AD event is ‘real’ by checking for a HR drop of 40 bpm or greater with respect to the onset of the AD event. The software detects and characterizes spontaneous AD events from continuous beat by beat, 24 hr SBP and HR telemetry recordings. Along with the time of day and frequency of these AD events, the AD Detection software provides the following tabular information: the max SBP, pressor response, duration of the AD event, min HR and HR drop upon onset of the spontaneous AD event.
The representative graphs of SBP and HR of Figure 3 present the severity and duration of an induced AD episode during CRD. As the balloon of the catheter is inflated, there is a rapid and persistent increase in SBP, accompanied by pronounced bradycardia. There is a gradual stabilization of the cardiovascular parameters as distension progresses, and normalization of hemodynamic parameters as the pediatric Foley catheter is deflated. After the insertion of the Foley catheter, SBP and HR indices are averaged for 60 sec before inflation to generate a baseline. From the baseline, the severity or pressor response associated with the spike in SBP (mmHg) and drop in HR (bpm) can be determined.
Figure 1. Schematic of Telemetry Device Implantation and Representative Trace of Arterial Blood Pressure (ABP). Schematic illustration of the telemetry transducer implantation with the tip of the catheter directed to the occluded descending aorta and the body of the telemetry device secured to the abdominal wall16. Example data of ABP sampled at 1,000 Hz from the telemetry device for a 24 hr period. From this recording, it is possible to extract cardiovascular indices, such as systolic blood pressure (SBP; mmHg; Figure 1B) and heart rate (HR; bpm; Figure 1C). Please click here to view a larger version of this figure.
Figure 2. A Spontaneous Episode of Autonomic Dysreflexia (AD) in Spinal cord Injured Animals. The novel JAVA platform software detected spontaneous AD events from 24 hr telemetry recordings of systolic blood pressure (SBP; Figure 2A) and heart rate (HR; Figure 2B). An increase of 20 mmHg or greater in SBP together with a drop in HR of 40 bpm or greater upon onset of the spike in SBP is considered an AD event. The "upper HR threshold" consists of the mean 10% of the HR values upon onset of the potential event. The "lower HR threshold" consists of the mean 75% of the heart rate values from the end of the potential event. Subtract the "lower HR threshold" from the "higher HR threshold" in order to ensure a corresponding drop of 40 bpm or greater.
Figure 3. Representative Trace of Induced Autonomic Dysreflexia (AD) by Colorectal Distension (CRD) in Spinal Cord Injured Animals. Upon induction of CRD, there is a sudden and persistent increase in SBP (Figure 3A) accompanied by a marked drop in HR (Figure 3B).
Figure 4. JAVA Platform Spontaneous Autonomic Dysreflexia (AD) Detection Software. A CSV format continuous arterial blood pressure excel file with interbeat interval (IBI), systolic blood pressure (SBP), mean arterial pressure (MAP) and time (Figure 4A) is uploaded to the program and the AD detection parameters are specified (Figure 4B) as outlined in the protocol. The program presents the temporal phenotype of these spontaneously occurring AD events (Figure 4C-D). Please click here to view a larger version of this figure.
The protocol describes a detailed implementation of a novel JAVA platform AD Detection software which would be combined with a telemetry device, for a long-term thorough analysis of ABP in SCI-animals (Figure 1B). This is the first software that allows for the characterization of ABP patterns to detect spontaneous AD events as they occur sporadically throughout the duration of the day. A well-characterized T3 SCI animal model can illustrate the functional capacity of the software to detect the frequency, pressor response and duration of spontaneous AD. Along with the detection of spontaneous AD events, the software can analyze oscillations in ABP and discern diurnal variations of hemodynamic parameters.
The novel AD Detection software efficiently, reliably and accurately detected and characterized spontaneous AD events in acute and chronic SCI animals. An AD event is considered to occur upon an increase in SBP of 20 mmHg or greater (Figure 2A) and is often associated with pronounced bradycardia9. In this particular study, SBP fluctuations were considered a primary indicator for the appearance of a dysreflexic episode, although the software has the ability to also use MAP should the investigator wish. The software has the ability to discern the pronounced bradycardia by detecting the HR drop of a user determined amount upon onset of the AD event (Figure 2B). Rabchevsky et al. utilized a characteristic drop of 10 bpm as the threshold upon which a hypertensive event is classified as a spontaneous AD event. This must be questioned, as rodents have a very high resting heart rate; hence, 10 bpm is likely insufficient to define bradycardia. HR drop restrictions may be altered by the user to not only determine drops in HR but also increases in HR depending on the level and completeness of the SCI.
Prior to this software, CRD-induced AD was found to be a robust method of induced AD that is both experimentally feasible and well characterized (Figure 3A, 3B)15,20. Repetitive induction of AD in rat animal models with high SCI was initially found to mimic the spontaneous and frequent episodes of AD associated with high thoracic SCI4. CRD is a potent, noninvasive stimulus for AD in rats with high thoracic SCI14 which emulates some of the most common causes of AD clinically such as constipation and fecal impaction3. CRD-induced AD does not account for the wide variety of afferent stimuli that may also evoke these reflex mediated hypertensive events. Thus CRD-induced AD in conjunction with detection and characterization of spontaneous AD provide the ideal scope in which we can study the temporal phenotype of AD.
Telemetry is a state-of-the-art method for monitoring physiological functions in awake and freely moving animals while minimizing stress-associated artifacts, such as distress, handling and anesthesia21. In this case, a solid-state pressure transducer made of a lightweight biosilicone material is used. As opposed to the low pressure response of fluid based catheters, a solid state pressure sensor can monitor subtle changes in cardiovascular parameters. These transducers can sample continuous, beat by beat blood pressure and core body temperature at frequencies up to 2 kHz. Solid state sensors also have the benefit of preventing motion artifacts commonly encountered in fluid based catheters. Though telemetry devices are invasive and costly, it accurately monitors the diurnal rhythm of hemodynamic parameters15,16,21,22. The recovery time after implantation is critically important for the survival of the animal, since mechanical obstruction to the blood flow in the abdominal aorta by the implant itself may result in insufficient blood supply to the hind body15.
Telemetric detection of spontaneous AD, in addition to CRD induced AD, account for the wide spectrum of stimuli associated with the onset of these life threatening episodes. Therefore, detecting and characterizing AD events is critical for considering treatments for SCI patients. There are currently no feasible noninvasive techniques available for humans that allow for chronic, beat by beat, monitoring of hemodynamic parameters. Ambulatory blood pressure monitoring is insufficient due to the low temporal resolution. Animals models are necessary in order to accurately detect and characterize the onset of these spontaneous AD events utilizing the novel AD Detection software. Upon advent of new continuous hemodynamic monitoring technologies, the software may be applied as a vital tool clinically to monitor the onset of these events. The use of telemetry devices in conjunction with the AD Detection software might be a useful future strategy for clinical ambulatory monitoring of acute AD.
The authors have nothing to disclose.
This research if funded by the Canadian Institute of Health Research and the Heart and Stroke Foundation of BC and Yukon. We would like to acknowledge Mr. Rayshad Gopaul and Dr. Shelly McErlane for technical support and expertise in animal care.
11 Male Wistar Rats -Hsd-WI (250-300g) | Envigo (formerly Harlan Laboratories) | 141 | |
Lab Chart (PowerLab® Data Acquisition System) | AD Instruments | ||
Pressure Telemeter | Millar Inc. | RP-TRM54P | |
Configurator | Millar Inc. | TR190 | |
SmartPad | Millar Inc. | TR180 | |
Isoflurane (Aerrane) | Baxter Corp. | DIN: 02225875 | |
Enrofloxacin (Baytril) | Bayer Healthcare | DIN: 02169428 | |
5-0 Silk Sutures | Ethicon | S182 | |
4-0 Vicryl Subcuticular | Ethicon | J496G | |
Buprenorphine (Temgesic) | Reckitt Benckiser | DIN: 0281250 | |
Bupivicaine Hydrochloride (Marcaine 0.5%) | Hospira Healthcare Corp. | DIN: 02305909 | |
Ketoprofen (Anafen) | Merial | DIN: 02150999 | |
Ketamine Hydrochloride (Vetalar) | Bioniche | DIN: 01989529 | |
Dexmedetomidine Hydrochloride (Domitor) | Pfizer | DIN: 02333929 | |
Lactated Ringer's Solution | Braun Medical Inc. | DIN: 01931636 | |
Gelfoam #12 | Pharmacia & Upjohn Company | 03603-14-1 | |
Microscissors | Fine Science Tools | 15003-008 | |
Iris Spatulae | Fine Science Tools | 10094-13 | |
10 French 35cm Foley Catheter | Coloplast | AA6110 | |
Dietgel® | Clear H2O, Westbrook, ME | 76A | |
LabDiet Rodent Diet 5001 | Purina Mills (PMI®) | 5001 | |
Chlorhexadine (Hibitane) | Wyeth Animal Health, Guelph, Ontario | DIN 00245097 | |
Atipamezole Hydrochloride(Antisedan) | Orion Pharma | DIN: 02237744 |