The susceptibility of cardiac arrhythmias after spinal cord crush injury in rats

Abstract

High-level spinal cord injury (SCI) often interrupts supraspinal regulation of sympathetic input to the heart. Although it is known that dysregulated autonomic control increases the risk for cardiac disorders, the mechanisms mediating SCI-induced arrhythmias are poorly understood. Here, we employed a rat model of complete spinal cord crush injury at the 2nd/3rd thoracic (T2/3) level to investigate cardiac rhythm disorders resulting from SCI. Rats with T9 injury and naïve animals served as two controls. Four weeks after SCI, rats were implanted with a radio-telemetric device for electrocardiogram and blood pressure monitoring. During 24-h recordings, heart rate variability in rats with T2/3 but not T9 injury exhibited a significant reduction in the time domain, and a decrease in power at low frequency but increased power at high frequency in the frequency domain which indicates reduced sympathetic and increased parasympathetic outflow to the heart. Pharmacological blockade of the sympathetic or parasympathetic branches confirmed the imbalance of cardiac autonomic control. Activation of sympatho-vagal input during the induction of autonomic dysreflexia by colorectal distention triggered various severe arrhythmic events in T2/3 injured rats. Meanwhile, intravenous infusion of the β1-adrenergic receptor agonist, dobutamine, caused greater incidence of arrhythmias in rats with T2/3 injury than naïve and T9 injured controls. Together, the results indicate that high-level SCI increases the susceptibility to developing cardiac arrhythmias likely owing to compromised autonomic homeostasis. The T2/3 crush model is appropriate for studying abnormal cardiac electrophysiology resulting from SCI.

1. Introduction

Spinal cord injury (SCI) is a debilitating condition associated with high morbidity and mortality. According to a report from the national spinal cord statistical center, the incidence of SCI in the United States is approximately 40 cases per million people each year. The primary causes of SCI are motor vehicle accidents, falls, violence, and sports/miscellaneous accidents (Devivo, 2012). Depending on the level and type of injury, patients with SCI exhibit a myriad of symptoms ranging from sensorimotor loss to autonomic dysfunction. Impairment of the autonomic nervous system may cause abnormalities in respiratory, gastrointestinal, urogenital, cardiovascular, and thermoregulatory function. Despite current advances in medical treatment, mortality rates in autonomic dysfunction resulting from SCI remain high. Among them, cardiovascular disorders are one of the leading contributors (~40%) in SCI patients (Garshick et al., 2005).

Individuals with SCI at the cervical or high thoracic level often exhibit unstable heart rate and blood pressure. This is due at least partly to interruption of supraspinal control of sympathetic input to the heart or splanchnic vascular bed. In neural regulation of the heart, sympathetic preganglionic neurons (SPNs) are in the upper thoracic (T1-T4) spinal cord segments. After high thoracic SCI, cardiac sympathetic activity is reduced and is responsible for the loss of supraspinal regulation, whereas cranial parasympathetic cardiac pathways originating from the brainstem (vagal) are largely spared. Consequently, autonomic regulation of the heart becomes out of balance, resulting in cardiovascular disorders (Collins et al., 2006). The compromised neuronal control presumably alters cardiac electrophysiology and increases the risk of arrhythmias (Ravensbergen et al., 2012; Hector et al., 2013). Although cardiac arrythmias are infrequently observed at rest in the chronic phase of SCI, the heart becomes vulnerable to specific provocations when patients are exposed to increased heart stress or burden, such as urodynamic examinations (Liu et al., 2017), penile vibratory stimulation for ejaculation (Claydon et al., 2006), and tracheal intubation before surgery (Yoo et al., 2010). This is likely attributable to the occurrence of autonomic dysreflexia during these scenarios. Autonomic dysreflexia is a life-threatening cardiovascular disorder that develops at both the acute and chronic stages of high-level SCI. It is characterized by episodic bouts of hypertension due to exaggerated sympathetic activity and accompanying bradycardia caused by high vagal discharge in response to a somatic or visceral sensory stimulus, e.g., colorectal distention (CRD) (Maiorov et al., 1997; Maiorov et al., 1998). In this state, concurrent increases in sympathetic control of blood vessels and parasympathetic activity of the heart change cardiac electrical conduction. Further, previous studies in SCI individuals showed that repetitive and severe autonomic dysreflexia impairs left ventricular function because of high blood pressure (Squair et al., 2018a; Squair et al., 2018b), which may progress into myocardial infarction and eventually heart failure related to calcium overload (Rodenbaugh et al., 2003b; Yang et al., 2015). It was reported that paraplegic rats had a lower electrical stimulation threshold to induce ventricular arrhythmias than intact animals (Rodenbaugh et al., 2003a). Clinical studies have shown that patients with SCI are more susceptible to ventricular arrhythmias than able-bodied individuals (Phillips et al., 1998; Bartholdy et al., 2014).

The mechanisms underlying cardiac arrhythmias following SCI are not fully understood. For example, it is still unknown how reduced sympathetic or unopposed parasympathetic outflow affects rhythm after a SCI interrupts sympathetic regulation. The β₁ receptor agonist dobutamine, which recapitulates cardiac sympathetic response but keeps vagal response untouched to accentuate cardiac contractility, was recently utilized as a tool to study proarrhythmogenic risk following SCI (Lucci et al., 2021). Meanwhile, transplanting early-stage neurons is a promising approach to reconstituting damaged neuronal circuits and restore somatic or autonomic function following SCI (Lu et al., 2012; Hou et al., 2020). Yet, this approach is hindered by some negative factors, particularly severe fibrotic scarring and cavitation in the lesion site of spinal cord transection, thus limiting the extent of graft-host integration. Later, these issues were successfully addressed using a complete spinal cord crush injury in rats (Hou et al., 2018). However, it is unknown if the injury model tends to be arrhythmogenic, and if so, whether it can be used for neuronal machinery and experimental therapeutic research. In the present study, we employed a rat spinal cord crush at the T2/3 level to partially disrupt supraspinal regulation of sympathetic input to the heart. Cardiac arrhythmogenicity was examined during rest, autonomic dysreflexia, and pharmacologically triggered stress situations. The results help understand the underpinning of arrhythmias and establish a reliable rat model for cell-based therapeutics to improve cardiac disorders after SCI.

2. Materials and methods

2.1. Animals

Adult Female Fischer 344 rats (2–3 months old, weighing 150–200 g, Charles River) were used. Their inbred phenotype is an advantage for cell transplantation studies (Lu et al., 2012). The rats were housed in micro-isolator cages and maintained in a light and temperature-controlled room. All animal procedures were performed according to the Drexel University Institutional Animal Care and Use Committee and Society for Neuroscience guidelines.

2.2. SCI surgery and Basso, Beattie and Bresnahan (BBB) testing

Rats were anesthetized with 4% isoflurane before initiating surgery and later reduced to 2% during surgery. Since sympathetic innervation of the heart ranges from thoracic level T1 to T4, we initially attempted to completely crush the C8/T1 spinal segment to eliminate the entire supraspinal regulation of sympathetic innervation to the heart. However, rats receiving this injury could not survive long in practice, and often developed severe respiratory symptoms and forelimb paralysis most likely due to impaired control of intercostal muscles and the brachial plexus. In contrast, rats receiving a complete crush SCI at the T2/3 level, which partially interrupted the descending regulation of the heart, survived long after injury. Accordingly, a crush injury at the level of T2/3 spinal cord was selected for the experimental groups. Rats receiving T9 injury that sustains intact cardio-autonomic control and naïve animals served as controls. A skin incision was made on the back, and the connective muscles were separated until the T2 spinal process was exposed. A T2 laminectomy was performed, and the T2/3 spinal segment was exposed. Subsequently, the spinal cord was crushed using fine forceps. The forceps were positioned around the spinal cord while touching the ventral vertebrae and the forceps were pressed tightly for 10 s (Hou et al., 2018). For the injury control, the laminectomy was performed on the dorsal part of T8 vertebra and then the T9 spinal segment was crushed. The injury site was visually confirmed. Following the injury, the muscle layer was sutured closed, and the skin was stapled. Post-operative care was performed by administering subcutaneous injections of buprenex (0.035 mg/kg) and cefazolin (10 mg/kg). Bladder care was performed 3–4 times daily until sacrifice. The Basso, Beattie, Bresnahan (BBB) locomotor scoring was used to evaluate hindlimb locomotor function to verify the completeness of the injury in an open field prior to radio-transmitter implantation. Each rat was observed for 4 min by two trained experimenters to assess hindlimb locomotor function. Animals with a BBB score <3 were included for transmitter implantation as a low BBB score suggests no extensive tissue sparing (Basso et al., 1995). The final number of 37 rats was used: T2/3 injury, n = 12; T9 injury, n = 12; naïve, n = 13. Among them, those used for heart rate variability and arrhythmia tests included: T2/3 injury, n = 7; T9 injury, n = 6; naïve, n = 7, while another cohort of rats were used for pharmacological interventions of autonomic tone: T2/3 injury, n = 5; T9 injury, n = 6; Naïve, n = 6.

2.3. Transmitter implantation

Three weeks post-SCI, rats were anesthetized with isoflurane, as described above. A ventral midline skin incision was made to access the abdominal cavity. The organs were pushed aside, and the abdominal aorta was exposed and separated from the vena cava. Next, a radio-transmitter (HD-S11, Data Sciences International) was placed in the abdominal cavity with the tip of its sensory catheter being inserted into the aorta. The insertion site was sealed with tissue glue. The organs were moved to their original position. The transmitter body was sutured to the abdominal wall, followed by suturing of the skin. With the help of the electrode guide, the electrode leads were subcutaneously placed with the positive lead on the lower chest and the negative lead on the upper chest respectively. The biopotential electrode leads senses the voltage changes of the heart. The catheter and electrodes record the mean arterial pressure (MAP) and electrocardiogram (ECG), respectively. Post-operative care was performed by administering subcutaneous injections of buprenex and cefazolin.

2.4. ECG and blood pressure recordings and analysis

At least 4 days to one week after telemeter implantation surgery, ECG and MAP were recorded using the Ponemah software (V6.3, Data Sciences International). Prior to the recordings, each individual receiver was frequency-matched to a single radio-transmitter. Animals with specific radio-transmitters were placed on their individual receivers. The transmitters were switched on using a magnet. For the resting state, the pressure limit was set at a high of 200 mmHg and the ECG voltage at 2 mV. The logging (sampling) rate was set to 1 h for 24-h parameter recordings. The data from the receivers were transmitted using the radiofrequencies and further analyzed using Ponemah variability plugin and Data Insights software (DSI). It was previous reported that resting hemodynamics has diurnal rhythms in SCI rats (West et al., 2015), the parameters recorded were thus separated to day and night for analyses, in which the data were consistently extracted as a 3-h period at a certain time, 3:00–6:00 pm for day and 11:00 pm-2:00 am for night.

2.4.1. 24-h ECG recordings for heart rate variability

ECG recordings were performed for 24 h at rest. Acquired data was subjected for automated heart rate variability (HRV) analysis in time and frequency domains. The time domain exhibits information regarding the amount of variability, whereas the frequency domain subcategorizes the R-R interval into different frequencies. HRV parameters in the time domain included the standard deviation of R-R interval (SDNN), the root mean square of the R-R interval (RMSDD), the number of R-R intervals (NNx), and the proportion of R-R intervals lower than the highest R-R interval (pNNx). The time domain was interpolated by applying the default software values. For instance, the reporting period was set to 5 min, the pNNx was set to 9 ms and the maximum standard deviation for outlier removal was set to 3. The derived time domain parameters such as the SDNN and RMSSD correspond to the amount of variability in heart rate. Low SDNN and RMSDD values typically suggest low variability (Shaffer and Ginsberg, 2017). The frequency domain parameters included low frequency (lf), high frequency (hf) and low-to-high frequency (lf/hf) ratio. The lf spans 0.25 to 1 Hz, while the hf ranges from 1.00 to 3.00 Hz (Rubini et al., 1993). The frequency domain was calculated using the default parameters of the variability plugin of the Ponemah software, wherein the segment duration was set to 30 s and the frequency axis range was set from 0 to 5 Hz. Moreover, the normalized (n) lf or hf was calculated as “nlf or nhf = lf or hf / (Total Power - vlf)” to remove very low frequency (vlf) component. The lf corresponds to sympathetic activity, while the hf represents parasympathetic activity. Hence, the ratio of lf/hf provides a non-invasive method to gauge the autonomic state of an animal. If the lf/hf ratio is <1, it suggests lower sympathetic activity and therefore autonomic imbalance (Malik and Camm, 1990; Sassi et al., 2015; Li et al., 2019). The calculation of HRV parameters kept consistent for all experimental groups.

2.4.2. Pharmacological tests for cardiac sympathetic and parasympathetic tone

To validate the data collected from HRV analysis, pharmacological interventions of the heart rate (HR) were employed to determine cardiac autonomic tone (Lujan et al., 2018). Each individual rat was housed in a small cage and placed on the telemetric receiver. Basal hemodynamics was recorded for 30 min after at least 1-h equilibration. Two drugs were then sequentially administered intraperitoneally (i.p.). The recording was stopped when rats were kept in the cage for injections. The muscarinic-cholinergic receptor blockade, atropine (3 mg/kg, MCE) was delivered to block parasympathetic activity and the HR was recorded for 15 min. Because the HR response to the drug reached the peak in 10–15 min, only data collected within last 5 min were used for calculation (Lujan et al., 2018). Subsequently, β₁-adrenergic receptor antagonist metoprolol (10 mg/kg, APEX) was administered i.p. to block sympathetic tone and HR was recorded for 20 min. Likewise, only data collected within last 5 min were averaged for calculation. During this short period, animals were usually in the steady-state hemodynamics without obvious body movement. All doses of drugs were calculated relative to the body weight. Sympathetic tone was calculated as HR_m – HR_i, where HR_m is the HR after muscarinic-cholinergic receptor blockade and HR_i is the intrinsic HR after complete autonomic blockade. On the following day, animals were placed on the receiver for hemodynamic recordings again but the order of the two blockers was reversed for calculation of the parasympathetic tone, which was extrapolated as HR_β – HR_i, where HR_β is HR after blocking β₁-adrenergic receptor with metoprolol.

2.4.3. Colorectal distention (CRD) induced autonomic dysreflexia

Autonomic dysreflexia is accompanied by concurrently high sympathetic and parasympathetic activity after high-level SCI (Collins et al., 2006). To induce autonomic dysreflexia, a balloon-tipped catheter coated with lubricant was inserted into the rectum of each rat. The catheter, which was attached to a syringe, was secured to the tail with a tape. The rats were allowed to rest for at least 15 min while ECG and blood pressure recordings were continuously acquired. To initiate CRD, a volume of 1.4 ml of air was injected to inflate the rectal catheter within 10 s and held in for 1 min. ECG and blood pressure were recorded 1 min before, during and after CRD, respectively. The procedure was performed twice per rat. After the recording, the catheter was removed from the rectum and the rats were injected with buprenex, as previously described (Hou et al., 2013; Trueblood et al., 2019). The data acquired were analyzed to obtain changes in HR and MAP. ECG recordings were examined for arrhythmias. The number of arrhythmias during CRD was counted both manually and automatically using Data Insights.

2.4.4. Dobutamine test

A butterfly needle connected to a catheter was inserted into the tail vein, under isoflurane anesthesia, and anchored on the tail for intravenous (i.v.) drug delivery. The rats were removed from the anesthesia and then placed into a plastic restraint tube. Subsequently, the animals were kept on their respective receivers for ECG and blood pressure recordings 1 h post-anesthesia. To mimic exercise-induced cardiac sympathetic activity, the β₁-adrenergic receptor agonist, dobutamine, was administered by continuous-infusion with a syringe pump. Dobutamine was delivered in three different doses: 20, 50, and 100 μg/kg. For each dose, 100 μl of drug solution was administered continuously for 4 min during ECG and blood pressure recordings (DeVeau et al., 2018). Arrhythmias were detected using Data Insights, and the number of arrhythmias was counted during drug administration.

2.5. Histology

Animals were anesthetized with an i.p. injection of overdosed Euthasol and perfused transcardially with 0.1 M PBS, pH 7.4, followed by 4% paraformaldehyde. The spinal cord was dissected, post-fixed overnight and cryoprotected in 30% sucrose in 0.1 M Tris-buffered saline (TBS). Spinal cord segment was trimmed to 1 cm containing the injury site in the center and was embedded in M1 matrix (Epredia)for cryosectioning. The spinal cord was serially sectioned in a longitudinal, horizontal plane at 35 μm in three series of sections mounted on the slides. For immunohistochemistry, sections were blocked in TBS with 0.25% Triton X and 5% donkey serum for 1 h, followed by incubation with primary antibodies for glial fibrillary acidic protein (GFAP, 1:1000, abcam) to label astrocytes, serotonin (5-HT, 1:1000, Immunostar) to label serotonergic axons, and collagen III (Col-III, 1:1000, abcam) to label collagen scars. Spinal cord sections were incubated with primary antibody overnight in 4 °C followed by incubation with AlexaFluor-488-, 594-, or 647-conjugated donkey secondary antibodies (1:500, Invitrogen). After thorough washing, the sections were coverslipped with mounting media. The slides were imaged using the fluorescent microscope (DM5500B, Leica Microsystems), connected to a digital camera (C11440, Hamamatsu), and installed with a Slidebook 6 software (Intelligent Imaging Innovations).

2.6. Statistical analysis

Baseline 24 h recordings for systolic, diastolic, MAP and HR were analyzed using mixed-effect model of the two-way analysis of variance (ANOVA) followed by a Tukey's post hoc test. CRD-induced autonomic dysreflexia was analyzed using an unpaired t-test. MAP and HR data as well as total arrythmias during dobutamine stress test were assessed using a two-way repeated measures ANOVA with Tukey's post hoc test, whereas HRV and pharmacological data of sympathetic and parasympathetic tone were analyzed by a one-way ANOVA followed by Tukey's post hoc test. The number of arrhythmias during autonomic dysreflexia was analyzed and compared between groups using a non-parametric measure of Mann Whitney U test due to non-normal data distribution. For all statistical assessment, p < 0.05 was marked as significant. All data are expressed as mean ± standard error of the mean (SEM).

3. Results

3.1. Resting hemo-parameters change and HRV decreases after high-level SCI

Resting hemodynamics and ECG were recorded for 24-h to examine changes and analyze HRV four weeks post-injury. Hemodynamic parameters were extracted and analyzed with diurnal and nocturnal divisions, including systolic pressure, diastolic pressure, MAP and HR (Fig. 1A). Irrespective of injury level, telemetric recording showed significantly reduced resting systolic (T2/3 117.5 ± 2.6, p < 0.001; T9 126.4 ± 1.5, p < 0.01; naïve 141.4 ± 1.8 mmHg, two-way ANOVA followed by Tukey's), diastolic (T2/3 88 ± 2.6, p < 0.01; T9 88.2 ± 1.5, p < 0.01; naïve 105.2 ± 1.8 mmHg), and MAP (T2/3 102.3 ± 2.5, p < 0.001; T9 107.5 ± 1.4, p < 0.01; naïve 123.3 ± 1.6 mmHg,) in two SCI groups compared to the naïve during the day. Similarly, the T2/3 injured rats had a significant decrease in systolic pressure and MAP in comparison to naïve animals at night. A significant decrease in MAP was also observed in T9 injured rats during the night (systolic: T2/3 121.2 ± 3.81, p < 0.001; T9 129.4 ± 1.7, p > 0.05; naïve 138.1 ± 1.5 mmHg; MAP: T2/3 105.9 ± 3.7, p < 0.001; T9 111.2 ± 1.5, p < 0.01; naïve 120.3 ± 1.5 mmHg). On the contrary, there was no significant difference in diastolic pressure at night (T2/3 90.8 ± 3.7, T9 92.4 ± 1.5, naïve 102.2 ± 1.6 mmHg, all p > 0.05) between all three experimental groups. Moreover, no significant change was observed in HR between injured and naïve groups (all p > 0.05). All parameters had no meaningful difference between T9 and T2/3 SCI rats during both day and night (all p > 0.05). The results suggest a reduced MAP at both day and night following thoracic SCI, regardless of injury levels.

Fig. 1.

Changes in hemodynamics, heart rate variability, and autonomic tone after T2/3 crush SCI. A, Telemetric recording at daytime indicates significantly reduced resting systolic, diastolic, and mean arterial blood pressure (MAP) but not heart rate (HR) in both T2/3 and T9 injured compared to naïve rats. However, recordings at night shows a significantly reduced systolic and MAP but not diastolic blood pressure and HR in T2/3 injured rats in comparison to naive (**p < 0.01, ***p < 0.001, two-way ANOVA followed by Tukey's). B, Representative tracings show dynamics in standard deviation of R-R interval (R-R SD), an index of HRV, during 24- or 5-h ECG recordings at rest. The variability was reduced slightly to modestly in rats with T9 injury and remarkably in those with T2/3 injury compared to the naïve. C, Pharmacological interventions of autonomic activity reveal significantly reduced sympathetic and elevated parasympathetic tone in T4 injured rats when compared with T9 injured or naïve controls (*p < 0.05, **p < 0.01, one-way ANOVA followed by Tukey's).

An overall decrease in HRV, represented as a curve of the standard deviation of R-R intervals during 24-h, was observed in T2/3 injured rats compared to T9 injured and naïve animals ( Fig. 1B ). HRV was further analyzed using frequency andand time domains.

Frequency domain:

Compared to naïve animals, there was a significant decrease in normalized low frequency power (nlf) of HRV, denoting cardiac sympathetic activity, in rats with T2/3 injury (p < 0.05, one-way ANOVA followed by Tukey's), as well as a lower ratio of low/high frequency power (lf/hf) (p < 0.05). Meanwhile, normalized high frequency power (nhf) of HRV, which reflects cardiac parasympathetic activity, was greater in rats with T2/3 injury than in naïve animals (p < 0.05). Additionally, rats with T2/3 injury exhibited a significant decrease in nlf and nlf/nhf ratio, and a greater increase in nhf relative to T9 injured animals (all p < 0.05) (Table 1). In contrast, there was no difference in these parameters between T9 injured and naïve rats. The results indicate a reduced sympathetic activity and an overall imbalance in autonomic control of cardiac function in rats with T2/3 injury.

Table 1.

HRV analysis of 24-h basal ECG recording in frequency domains.

Group	nlf	nhf	lf/hf
T2/3	0.29 ± 0.02*#	0.71 ± 0.02*#	0.51 ± 0.03*#
T9	0.47 ± 0.01	0.55 ± 0.01	1.24 ± 0.10
Naïve	0.51 ± 0.02	0.49 ± 0.03	1.39 ± 0.03

lf, low frequency; hf, high frequency; nlf, normalized low frequency; nhf, normalized high frequency; lf/hf, ratio of low frequency to high frequency.

^*p < 0.05 to naïve

^#p < 0.05 to T9.

Time domain:

HRV analysis in the time domain showed a significant decrease in root mean square of standard deviation (RMSSD) in T2/3 injured rats compared to naïve animals (p < 0.05, one-way ANOVA followed by Tukey's). However, there was no significant difference in all parameters between naïve and T9 injured rats (Table 2); This suggests that rats with T2/3 injury display lower HRV.

Table 2.

HRV analysis of 24-h basal ECG recording in time domains.

Group	NN Interval	SDNN	RMSSD	NNx	pNNx	Cycles
T2/3	142.9 ± 4.0	5.7 ± 1.1	2.0 ± 0.4*	27.7 ± 15.8	1.50 ± 0.9	2068.9 ± 53.6
T9	146.7 ± 1.4	6.5 ± 0.7	2.6 ± 0.3	14.8 ± 5.2	0.78 ± 0.3	2009.9 ± 17.4
Naïve	153.5 ± 3.0	8.0 ± 0.7	4.1 ± 0.6	45.0 ± 14.6	2.66 ± 0.8	1882.2 ± 60.8

NN, average of R peak of the ECG wave; SDNN, standard deviation of R-R interval; RMSSD, root Mean Square of the R-R interval; NNx, Number of R-R intervals; PNNx, proportion of R-R intervals lower than the highest R-R interval; Cycles, number of all R-R intervals.

^*p < 0.05 to naïve.

3.2. Pharmacological interventions validate autonomic imbalance after T2/3 SCI

To verify the results obtained from HRV analysis, pharmacological interventions were used as the second approach to characterize autonomic tone. Sympathetic or parasympathetic tone was derived using pharmacological blockades in T2/3, T9 SCI or naïve rats. It was revealed a significant decrease in sympathetic tone in T2/3 injured rats (57.4 ± 21.1 bpm) compared to those with T9 SCI (131 ± 7.3 bpm, p < 0.01, one-way ANOVA followed by Tukey's) and naïve controls (124.3 ± 8.9 bpm, p < 0.05). In contrast, parasympathetic tone was significantly increased in T2/3 injured rats (17.9 ± 2.7 bpm) compared to rats with T9 injury (−7.4 ± 2.9 bpm, p < 0.05) and naïve controls (−24.3 ± 12.5 bpm, p < 0.01). The results suggest a reduced sympathetic tone and elevated parasympathetic tone to the heart in high-level SCI, thereby corroborating the results from HRV analysis (Fig. 1C)

3.3. CRD triggers arrhythmias during autonomic dysreflexia

To determine if excitation of cardiac parasympathetic arm triggered arrhythmias, noxious CRD was performed to induce autonomic dysreflexia in SCI rats. During CRD tests, the change in MAP and HR was greatly enhanced in rats with T2/3 injury (Δ MAP 47.7 ± 5.6 mmHg, ΔHR −108.9 ± 15.2 bpm) compared to T9 injured controls (Δ MAP 17.3 ± 3.2 mmHg, p < 0.0001; ΔHR −24.0 ± 9.6 bpm, p < 0.01, Unpaired t-test) (Fig. 2A-C). Simultaneous occurrence of hypertension and bradycardia manifested as a typical autonomic dysreflexia response (Krassioukov et al., 2007). Visual inspection of the ECG data showed that the incidence of arrhythmias was rarely seen during autonomic dysreflexia in animals with T9 injury (Fig. 2D, E), whereas noxious CRD elicited various types of arrhythmias in rats with T2/3 injury, such as sinus pause, left bundle branch block, and atrial fibrillation (Fig. 2F, G). Specifically, sinus pause represents failure of the sinus node to conduct electrical impulses and is depicted as an increased duration in R-R interval in the ECG. Left bundle branch block results from the inability of the left ventricle to contract before the right ventricle and is depicted by an inverted QRS complex. On the other hand, atrial fibrillation is depicted by the absence of P wave followed by irregular R waves in the ECG waveform, which suggests irregular contractions of the atria.

Fig. 2.

Colorectal distension (CRD)-induced autonomic dysreflexia triggers cardiac arrhythmias following high thoracic SCI. Four weeks after T2/3 or T9 injury, noxious CRD was performed during electrocardiogram (ECG) and blood pressure (BP) recordings in conscious rats. A, In rats with T9 SCI, CRD triggers only a slight increase in mean blood pressure (MAP) and decrease in HR. B, C, Typical autonomic dysreflexia is induced with CRD in rats with T2/3 SCI. AD is characterized by an episode of sudden hypertension accompanied by bradycardia (****p < 0.0001, **p < 0.01, Unpaired t-test). D-G, ECG recordings reveal very few arrhythmias in rats with T9 injury (D, E) whereas various arrhythmias occur in rats with T2/3 injury (F, G) during CRD, such as bradycardia with sinus arrest (g1), possible atrial fibrillation (g2), and sinus pause with aberrant conduction vs. ventricular escape beat (g3).

Subsequently, various arrhythmias during CRD-induced autonomic dysreflexia were automatically detected using Data Insights software (Fig. 3A & Fig. 4) (Table 3). The overall number of arrhythmias in rats with T2/3 injury was greater than T9 injury controls. Some common instances of arrhythmias obseived in T2/3 injured rats included atrioventricular (AV) block (11 ± 10.3), ventricular ectopic (55 ± 35.3), junctional arrhythmias (314 ± 100.5), premature atrial complex (19 ± 7.5), ventricular ectopic singles (42 ± 25.3) and premature ventricular complex (17 ± 11.1) (Fig. 3B). Certain types of arrhythmias such as sinus pause, ventricular bigeminy ventricular couplets, ventricular trigeminy and ventricular triplet occurred less frequently. However, the occurrence of arrhythmias including AV blocks (3.9 ± 3.6, ventricular ectopic (15.8 ± 5.1), and junctional arrhythmias (273.5 ± 88.8) were lower in T9 injury controls (Fig. 3B). Quantitative analysis showed that T2/3 injured rats had more severe arrhythmias without junctional complex that is benign and occurs in normal subjects (p < 0.05, Mann Whitney U test) compared to those with T9 injury (Fig. 3C). The results indicate that cardiac arrhythmias are highly prevalent during CRD-induced autonomic dysreflexia in rats with high-level SCI.

Fig. 3.

Detection of incidence and different types of arrhythmias during colorectal distension (CRD) in SCI rats. A, Using a Data Insights software, events of various arrhythmias are color-coded in rats with T2/3 or T9 SCI. B, A distribution pattern indicates high frequencies of most types of arrhythmia occurrence in rats with T2/3 but not T9 SCI. C, Statistical analysis of arrhythmias without benign junctional events (–) indicates significantly (*p < 0.05, Mann Whitney U test) greater number of arrhythmias in rats with T2/3 compared to rats with T9 injury. AV, atrioventricular; V, ventricular; Junctional, junctional complex; PAC, premature atrial complex; PVC, premature ventricular complex.

Fig. 4.

Typical arrhythmia cases occur in rats with T2/3 injury. These rhythmic disorders are observed not only in colorectal distension to stimulate cardiac parasympathetic pathways but also in dobutamine tests for sympathetic excitation.

Table 3.

Types of arrhythmias and the number of animals triggered by CRD during ECG recording.

Name	Identification	Implication	Num of animals
			T2/ 3 n = 7	T9 n = 6
Junctional Complex	No P wave or a P wave with a short PR interval	AV node is the origin of the electrical impulse	7	7
Second Degree AV Block	Elongated PR intervals	His-purkinje system cannot conduct impulses from atria to ventricles	4	2
Premature Atrial Complex	Slow decrease in R-R interval relative to the previous cycle	Premature heartbeat of the atria	7	2
Sinus Pause	Long R-R interval	Inability of the SA node to generate an electrical impulse	4	0
Premature Ventricular Complex	Premature ventricular ectopic bracketed by two sinuses	Heart-beat origin is in the ventricles	6	3
Bigeminy	Repeating pattern of two or more ventricular beats that are separated by one sinus	The PVC occurs in two beat pattern and has the same implication as PVC	2	0
Trigeminy	Repeating pattern of two or more ventricular beats that are separated by two sinuses	The PVC occurs in three beat pattern and has the same implication as PVC	2	2

Num: number of animals that exhibited the arrhythmic events.

3.4. Dobutamine administration induces a greater number of arrhythmias after T2/3 injury

To determine whether stimulating cardiac sympathetic arm provoked arrhythmias, serial doses of dobutamine were administered to both naïve and SCI rats. Blood pressure and ECG were recorded during continuous administration of various doses (20, 50, and 100 μg/kg) of dobutamine (Fig. 5A). In either naïve or T9 injured rats, unexpectedly, the infusion did not change HR while MAP gradually decreased (high dose vs. baseline: T9, p < 0.05; Naïve, p = 0.07, two-way repeated measures ANOVA with Tukey's). By contrast, high dose of dobutamine significantly (both p < 0.05) increased HR and decreased MAP in T2/3 injured rats (Fig. 5B). More importantly, dobutamine administration triggered various incidence of arrhythmias in rats (Fig. 5C). Quantitative analysis showed that, in all three doses of dobutamine delivery, the total number of arrhythmias in T2/3 SCI rats was higher compared to T9 SCI and naïve animals (low dose: naïve 18.9 ± 13.13, T2/3 178.67 ± 111.61, T9 77.6 ± 49.4; middle dose: naïve 6 ± 2.5, T2/3 141 ± 71.1, T9 87.4 ± 44.73), and was statistically significant at the high dose between naïve and T2/3 injured rats (naïve 7 ± 2.3, T2/3 297 ± 139.75, T9 81.8 ± 34.7; p < 0.05, two-way repeated measures ANOVA with Tukey's) (Fig. 5D). If junctional complex was excluded, the number of arrhythmias at the high dose was significantly higher (both p < 0.01) in T2/3 injured rats than two controls. Moreover, the highest incidence of arrhythmias observed in T2/3 injured rats was AV and junctional block. Certain arrhythmias had low rate of occurrence but were consistently higher in T2/3 injured animals in comparison to T9 injured and naïve rats (Fig. 5C, D). Overall, continuous administration of dobutamine elicited high rates of arrhythmias in T2/3 injured rats that had a cumulative response with increasing dosage. The results suggest that rats with T2/3 injury have reduced capacity to manage cardiac stress, thereby leading to increased susceptibility to cardio-electric disorders.

Fig. 5.

Administration of dobutamine induces cardiac arrhythmias in SCI rats. Four weeks after SCI the β₁-adrenergic receptor agonist dobutamine, was administered (20, 50, and 100 μg/kg) during blood pressure (BP) and electrocardiogram (ECG) recordings. A, Various arrhythmias are induced during continuous i.v. infusion of low, middle and high doses (4 min per dose) of dobutamine. All arrhythmia detections are color coded. High dose tracing is represented in right panel. B, In either naïve or T9 injured rats, continuous infusion of dobutamine gradually decreased MAP but did not change HR. In contrast, high dose dobutamine significantly decreased MAP and increased HR in T2/3 injured rats (*p < 0.05, **p < 0.01, two-way repeated measures ANOVA followed by Tukey's). C, The distribution of arrhythmias is graphically represented. D, Quantitative analysis reveals that at high doses the total number of arrhythmias without junctional (−) was significantly higher in T2/3 SCI rats compared to T9 injured and naïve animals, while that with junctional (+) was also higher in T2/3 injured rats vs. naïve. Notably, very few arrhythmias were triggered with every dose of dobutamine in naïve rats). AV, atrioventricular; ectopic, ectopic beat; Junctional, Junctional complex; PAC, premature atrial complex; PVC, premature ventricular complex.

3.5. Histological analysis reveals completeness of injury

To examine completeness of the lesion, triple immunostaining was performed in either T2/3 or T9 injured spinal cord sections. It was revealed that GFAP-labeled spinal cord tissue was disrupted in the lesion site which was filled with Col-III-labeled collagen, a fibrotic scar component. Importantly, 5-HT-labeled serotonergic fibers, as a representative of supraspinal pathways, were observed in the rostral spinal cord but not caudal to the lesion, indicating interruption of supraspinal connections and completeness of injury (Fig. 6). The result was consistent in all injured spinal cords regardless of T2/3 or T9 level.

Fig. 6.

Histological analysis demonstrates the completeness of the lesion in the spinal cord. In a serial horizontal longitudinal spinal cord sections spanning dorsal to ventral from T2/3 injured rats, triple immunostaining reveals that GFAP-labeled spinal tissue disrupts in the lesion site which is filled with Col-III-labeled collagen, a fibrotic scar component. Importantly, 5-HT-labeled descending serotonergic fibers, as a representative of supraspinal pathways, were observed in the rostral spinal cord but not in the segments caudal to the lesion, suggesting interruption of supraspinal connections and completeness of injury. This is consistent in either rats with T2/3 or T9 SCI. Scale bar: 1 mm.

4. Discussion

Sympathovagal imbalance occurred in neural regulation of the heart after high thoracic SCI. Disordered cardiac electrophysiology was detected using various methods, including HRV, CRD-induced autonomic dysreflexia, and dobutamine stress test. Under these conditions, stimulating either sympathetic or parasympathetic arm triggered high incidence of various cardiac arrhythmias which indicates the involvement of these two components in conduction problems. Thus, the high-thoracic crush SCI rat model is susceptible to cardiac abnormalities and is reliable to investigate the prevalence of SCI-induced cardiac rhythmic disorders.

HRV reflects cardiac autonomic control, which provides a representation of autonomic balance between sympathetic and parasympathetic components. A high value of HRV indicates healthy autonomic and cardiovascular response while a low one is suggestive of improper coordination between sympathetic and parasympathetic systems to provide an appropriate HR response. An analysis in time domain examines the amount of variability at any given time, wherein a low variability implies that overall autonomic response to external and internal cues is minimal and hence denotes decreased adaptability. We observed a significant decrease in RMSSD in rats with T2/3 SCI, which suggests a general decrease in HRV and an abnormal autonomic function (Quintana et al., 2016; Young and Benton, 2018). Using frequency domain analysis, previous studies have verified that nlf is associated with sympathetic activity while nhf is related to cardiovagal activity, and the ratio of lf/hf indicates autonomic balance (Sassi et al., 2015). We demonstrated that rats with T2/3 but not T9 injury displayed decreased nlf, increased nhf, and a decrease in lf/hf ratio. Together, these results indicate that T2/3 SCI causes decreased sympathetic but increased parasympathetic activity, an overall state of autonomic imbalance. Notably, pharmacological assessments showed reduced cardiac sympathetic tone and elevated parasympathetic tone in T2/3 SCI rats, validating the results obtained from HRV spectral analysis. These observations are similar to those reported previously in T3 spinal cord transected rats (Inskip et al., 2012) and SCI patients (Claydon and Krassioukov, 2008). Accordingly, our results suggest that the crushed SCI at T2/3 level successfully disrupts the harmony in autonomic regulation of the heart.

Although the mechanisms mediating cardiac arrhythmias following SCI are not well defined, the main contributor may involve impaired balance of sympathetic and parasympathetic input to the heart (Lujan and DiCarlo, 2007). Morphological evidence from recent studies demonstrated that the development of various arrhythmias following SCI may be related either to sympathetic denervation or hyper-innervation of the heart (Bai et al., 2008; Lujan et al., 2010; Lujan et al., 2012; Gardner et al., 2016). SCI may lead to neuronal death causing denervation of axon terminals associated with sympathetic control of the heart. On the other hand, elevation in the level of nerve growth factor (NGF) in both the spinal cord and peripheral organs, such as the heart, could trigger neuronal fiber sprouting (Lujan et al., 2010). Simultaneously, increased dendritic arborization and synapse number in stellate ganglia also enhances sympathetic tone, contributing to arrhythmia generation (Lujan et al., 2012). In spite of statement that the consequence of denervation or hyper-innervation is dependent on the level of SCI, this is still controversial and needs more experiments to be addressed. Furthermore, cardiac tissue remodeling occurs following SCI. The changes in myocardial structure following SCI includes a decrease in myocyte length and ventricular mass. It leads to the alteration of axon terminal distribution per unit of tissue in the organ that are also related to cardiac dysfunction (West et al., 2012; DeVeau et al., 2018). In addition, the alterations in the molecular and cellular properties like β-adrenergic receptor sensitivity or expression (Liggett et al., 2000) and Ca²⁺ signaling pathway (Bers, 2008) provide other basis for arrhythmogenesis (Kanazawa et al., 2010; Fallavollita et al., 2014; Gardner et al., 2015). For instance, activation of sprouted sympathetic fibers stimulates β₁-adrenergic receptors in the sinus node and AV node, which generates and conducts action potential in the heart by increasing AV conductance and contractile force (Pogwizd et al., 2001). However, loss of sympathetic fibers to the heart can cause β₁-adrenergic receptor super-sensitivity and increase susceptibility of focal arrhythmias around the denervated myocardium (Gardner et al., 2015). Thus, arrhythmia generation can be a cumulative effect of various events occurring in the heart, sympathetic ganglia, and spinal cord after SCI.

Regulation of cardiac function by the autonomic nervous system involves a reciprocal relationship between sympathetic and parasympathetic activity. Following high-level SCI, the interruption of supraspinal neuronal circuits causes dysregulated cardiovascular reflex. Instances of co-activation of these two components is seen during episodes of autonomic dysreflexia, in which noxious sensory stimulation evokes high sympathetic discharges to constrict blood vessels and result in hypertension, while baroreceptor reflex mediated increased vagal activity reduces HR (Bradycardia). This is known as “autonomic conflict” and is assumed to be highly proarrhythmogenic (Shattock and Tipton, 2012). Recurrent autonomic dysreflexia also weakens cardiac muscles, contributing to electric transduction abnormalities (Spinale et al., 1998; Ferreira and Santos, 2015; Gopinathannair et al., 2015). Indeed, previous studies showed the occurrence of arrhythmias during autonomic dysreflexia in a rat SCI model (Collins et al., 2006; Sachdeva et al., 2021). In the present study, CRD successfully induced typical autonomic dysreflexia in the crushed SCI model at the T2/3 level but not those with T9 injury, similar to studies with complete spinal cord transection models (Hou et al., 2013). During the episode of autonomic dysreflexia, various arrhythmia events were detected, including sinus pause, bradycardia, atrial fibrillation, AV block, junctional escape, and ventricular bigeminies and trigeminies. Although some arrhythmia incidences occurred during CRD in rats with T9 SCI, the number of these occurrences was much less than rats with T2/3 SCI. Notably, junctional arrhythmias consistently occurred in both T2/3 and T9 SCI rats. They are characterized by the generation of cardiac electrical impulse near or in the AV node. If not coupled with any other arrhythmias, junctional arrhythmias are benign and even occasionally emerges under normal conditions. Considering its much higher rate of incidence than others, this parameter was separated from total arrhythmia count and more focus was given to the prevalence of junctional arrhythmias accompanied by other serious arrhythmias, e.g., sinus pause, that were solely observed in T2/3 SCI rats. Sinus pause is visualized by a stopped impulse in the SA node and can be detrimental if occurrs repeatedly. Clinical studies have documented some abnormal ECG features from SCI subjects such as ST segment depression, T-wave inversion, ventricular conduction delays, premature ventricular contractions and low QRS amplitude (Blocker et al., 1983). Similarly, markers of susceptibility to cardiac arrhythmias, such as QT variability and QT interval mean, were prevalent in T2 contusive rats (Lucci et al., 2021). Here, disordered cardiac electrical conduction exhibiting as various arrhythmias was detected in T2/3 injured rats in different stressor conditions, which is complementary to previous reports.

Unlike accentuating cardiac parasympathetic evocation during CRD-induced autonomic dysreflexia, the β₁-adrenergic receptor agonist, dobutamine, is an inotrope that simulates cardiac sympathetic activation to increase the magnitude and rate of myocardial contractility, mimicking exercise-related myocardial stress (Ghio et al., 2018; Kieu et al., 2018). As a standardized procedure, infusion of dobutamine is used to examine cardiac load and identify various forms of heart diseases during echocardiography (Krahwinkel et al., 1997; Kieu et al., 2018), which has been well established in multiple rat models of cardiac disease (Plante et al., 2005; Hazari et al., 2012). However, the detection of arrhythmias using this test during ECG has rarely been tried in SCI rats. Delivery of dobutamine significantly elevated HR in T2/3 injured rats but not in other two groups, suggesting increased sympathetic sensitivity in the heart after high-level SCI. Nevertheless, all groups exhibited a reduction in MAP. Initially we were surprised of this unexpected data. However, many previous studies reported a similar phenomenon of hypotension during dobutamine administration in able-bodied subjects or those with cardiac disease (Lieberman et al., 1995; Rallidis et al., 1998; Enrico et al., 2012). Although the exact mechanism remains unclear, it has been postulated that the excessive stimulation of cardiac vagal afferents innervating mechanoreceptors in the left ventricle, such as forceful systolic contraction, mainly contributes to the hypotension. This may occur during dobutamine infusion to squeeze and activate these receptors, which could trigger sympathoinhibition and increased parasympathetic discharge, resulting in decrease in systemic vascular resistance or/and bradycardia (Mazeika et al., 1992). It was noted that hypotension often occurred in the setting of good left ventricular function (Lieberman et al., 1995). Because SCI rats in the current study were in the subacute stage, their left ventricle might not progressprogress to dysfunctional state yet, thereby leading to hypotension via this mechanism. During dobutamine infusion, various cardiac arrhythmias occurred, such as AV block, ventricular ectopic, junctional complex, and ventricular bigeminies and trigeminies. The incidence of these arrhythmias was higher in T2/3 injured group compared to T9 injured or naïve rats. Following SCI, the changes in sensitivity and dysregulation of cycling of β₁-adrenergic receptors, due to reduced sympathetic control of the heart, are one of the possible underlying mechanisms of the increased arrhythmic events in T2/3 SCI rats. Although this hypothesis has not been tested in the heart, α-adrenergic receptors present on the arteries exhibit supersensitivity towards norepinephrine and other α-adrenergic mimetics after SCI (Mathias et al., 1976; Yeoh et al., 2004). Additionally, previous studies have shown that, in naïve rats, dobutamine increases end diastolic volume, ejection fraction, stroke volume, and overall cardiac output (DeVeau et al., 2018). In rats with T2 contusion, stroke volume and the overall cardiac output are further augmented following dobutamine infusion. This suggests that SCI causes the heart to work at maximum capacity, which could promote cardiac arrhythmogenesis (Pierard et al., 1989). In the present study, various arrhythmias were triggered in either high cardiac vagal activity during autonomic dysreflexia or β₁-adrenergic receptor stimulation with dobutamine. This suggests arrhythmogenesis is not attributable to dysregulated sympathetic outflow alone but autonomic imbalance between these two components.

There are certain limitations in the methodologies employed to study SCI-induced arrhythmias. First, the large variation in different types of arrhythmias between individual rats makes it difficult for quantitative analysis. Though increasing the size of groups may overcome this difficulty, a study for spontaneous arrhythmias that used approximately 55 healthy, freely moving rats still had results with high variability (Pereira et al., 2019). Considering the number of sophisticated surgeries, recordings, and analytical procedures, it is impractical in this study to utilizeutilize such large sample sizes for the research on SCI rats. Second, respiratory rhythm might affect HR and certainly then HRV spectral analysis, but our current techniques cannot link them together. Furthermore, it is uncertain if there is plasticity of sympathetic terminals in the heart after SCI at the T2/3 level which may be related to conduction problems. Despite these caveats, this study provides important neuronal machinery and methods to investigate the complicated plethora of cardiac electrical dysfunction.

5. Conclusions

Following high-level thoracic spinal cord crush in rats, total HRV was significantly reduced in which time and frequency domain analyses illustrated reduced sympathetic and increased parasympathetic input to the heart. During CRD-induced episodes of autonomic dysreflexia, activation of cardiac parasympathetic activity triggered various arrhythmia events. Similarly, stimulation of cardiac sympathetic branch with the β1-adrenergic receptor agonist dobutamine caused numerous incidences of arrhythmias. This indicates that cardiac electrical disorders result from the imbalance of autonomic function rather than a single component dysfunction after SCI. Ultimately, the susceptibility to cardiac arrhythmias is prevalent in rats with T2/3 spinal cord crush injury due to compromised autonomic homeostasis.

Data availability

Data will be made available on request.