Microchannel Electrode Stimulation of Deep Peroneal Nerve Fascicles Induced Mean Arterial Depressor Response in Hypertensive Rats
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Hypertension (HTN) affects over 1 billion people in the world, and while most are treated effectively with pharmacological regimens, 10–30% of them do not show a beneficial response. Electrical stimulation of the renal sympathetic, vagus and carotid sinus nerves has shown depressor effects and has been proposed as an alternative treatment for resistant hypertension (R-HTN). However, these nerves are heterogeneous in afferent/efferent composition, and their stimulation often results in unwanted side effects. We evaluated the possibility of eliciting a depressor response from stimulation of single fascicle of the somatic deep peroneal nerve (fDPN). A microchannel electrode array (µCEA) was used to stimulate the fDPN at low frequency, which induced a significant (p ≤ 0.03) transient reduction in mean arterial pressure (MAP) with no significant effects on heart rate. The depressor response was prolonged for several hours by extending the fDPN stimulation to 5 min, which induced significant reduction (17–25%) in MAP for up to 4 h. Immunofluorescence evaluation of the axonal marker, myelin, and active macrophages in the fDPN revealed no indication of nerve damage or overt inflammation in response to the procedure. This study provides evidence supporting the use of µCEA interfacing of small somatic nerve fascicles associated with cardiovascular relevant acupoints to induce significant reductions in MAP and opens the possibility of neuromodulation of small fascicles as an alternative strategy to treat R-HTN with minimal side effects. Further, the µCEA multielectrode array offers an effective tool for neuromodulation of small nerve fascicles, enabling a number of possible future medical bioelectronic applications.
Hypertension (HTN) affects over 1 billion people around the world (1). The current management of hypertension relies on thiazide-type diuretics, calcium channel blockers, angiotensin-converting enzyme inhibitors and/or angiotensin receptor blockers to maintain normal blood pressure and prevent myocardial infarction, stroke, renal failure and death (2). However, appropriate dosing is challenging because of the high variability in the patient response, narrow therapeutically acceptable range, adverse reactions and toxicity (3). Moreover, approximately 10–30% of the affected population is resistant to pharmacological treatments, despite the use of combinatorial drug treatments and maximum doses (4,5). Individuals suffering from resistant hypertension (R-HTN) have high rates of cardiovascular complications and few treatment options. Unfortunately, some promising research candidates for the treatment of R-HTN such as renal artery denervation have failed to show efficacy (6,7). Therefore, there is much need for developing alternative methods for R-HTN regulation.
Vagal nerve stimulation (VNS) was first proposed as a possible method for the treatment of HTN in the late 1990s, but reports of bradycardia and apnea side effects limited its application (8). Recent studies have shown that selective VNS using tripolar cuff electrode stimulation reduces blood pressure from 100 to 80 mmHg in normal rats with minimal side effects (that is, 5–10% in apnea) using 40 Hz, 1 mA and 0.3-ms pulse stimulation for 4 ms (135 pulses) (9). However, apnea increased to 15% when stimulation was applied at 50 Hz. Furthermore, whether this approach can achieve the same therapeutic effect in hypertensive animals was not investigated. In addition, vagal nerve is highly heterogeneous in axonal composition and side effects including cough, hoarseness and voice alteration, and paresthesias have been reported in humans (9,10). Thus, alternative peripheral nerves that can achieve regulation of HTN with significantly less side effects might offer a more appealing treatment for R-HTN.
A recent randomized blind clinical trial demonstrated that active traditional Chinese acupuncture significantly lowers blood pressure in HTN patients compared with sham acupuncture (11), and some report the effect of acupuncture and pharmacological treatment (12). While the working mechanism of this therapeutic effect is unknown, increasing evidence suggests that the acupuncture therapy benefit is based on neuromodulation of the underlying nervous system. Indeed, depressor responses of ~20 mmHg have been reported in hypertensive rats after stimulation of the sciatic nerve (13). Recently, it was shown that electropuncture on a fascicle of the sciatic nerve (the common peroneal nerve, which overlays the acupoint Zuzanli [ST36] and Shangjuxu [ST37]) significantly reduces blood pressure 10–18 mmHg without affecting heart rate (HR) (14).
We hypothesized that electrical stimulation of a fascicle of the somatic deep peroneal nerve (fDPN) provides an effective substrate to induce a significant depressor response in HTN with minimal side effects. A microchannel electrode array (µCEA) specifically designed and fabricated for interfacing with small nerves (that is, 50–300 µm in diameter; autonomic nerve or nerve fascicles) was used to stimulate the fDPN (~150 µm in diameter) (15). We demonstrate that a significant reduction in mean arterial pressure (MAP) in hypertensive rats can be achieved through microelectrical stimulation of a somatic nerve fascicle for up to 4 h without significantly altering HR.
Materials and Methods
Microchannel Electrode Array (µCEA)
Acute µCEA Implantation onto the Ventral Fascicle of the DPN
Six normotensive Wistar Kyoto (WKY, n = 6) rats and nine spontaneously hypertensive rats (SHR, Harlan, n = 9) were used in this study. Because hypertensive SHR and normotensive WKY rats were both established from the same parental line, WKY rats were used as control rats in studies of SHR (16). The animals were anesthetized with inhaled isoflurane (1–3% in 100% oxygen) from a vaporizer with a scavenger system. An appropriate level of anesthesia was confirmed by the lack of response to noxious stimuli. As previously described (17), intubation via tracheotomy and cannulation of the left carotid artery and right jugular vein was performed. The arterial cannula was interfaced with a flow pressure sensor (Model DTX plus-DT, NN12, Ohmeda) for continuous MAP and HR monitoring (Figure 1B). To stabilize fluid balance and maintain baseline MAP, a continuous infusion of physiological saline (2 mL, 1 mol/L NaHCO3; 10 mL 5% dextrose) was administered into the jugular vein (3–5 mL · h−1 · kg−1).
The DPN was exposed in the right hind limb by lateral spreading of the gastrocnemius and peroneus muscles, and a 150–200 µm diameter ventral fascicle (fDPN) was gently separated with a glass rod (Figures 1C, D). The fascicle was then easily inserted into the groove of the µCEA and secured by using a medical grade silicone elastomer (Kwik-Sil, World Precision Instruments).
Animals were then transferred to urethane-based anesthesia and stabilized for 1 h before stimulation, and arterial blood gases and body temperature were maintained at normal values throughout the experiment. The UTSW Institutional Animal Care and Use Committee (IACUC) approved all procedures, and the studies were conducted in accordance with the Guide for the Care and Use of Laboratory Animals, used by the U.S. Department of Health and Human Services National Institutes of Health.
Short- and Long-Duration fDPN Electrical Stimulation
We first evaluated the response of the entire DPN to electrical stimulation for the MAP depressor response in WKY and SHR animals by using custom-made hooks with multistranded stainless steel wires (Bioflex wire AS633, Cooner Wire, CA), to determine the optimal range of current intensities (0.1, 0.3 and 0.5 mA), number of pulses (1 and 10 pulses) and stimulation frequency (1 and 100 Hz).
Subsequently, a set of identified conditions (0.1 mA, 10 pulses, 1–2 Hz) were used to stimulate the fDPN by using the µCEA either for 10 s (short duration, n = 6 SHR and WKY) or 5 min (long duration, n = 3 SHR). The biphasic cathodic leading current stimulation pulses were given by using a Plexon Stimulator module (Plexon), and the resultant effects were monitored continuously for either 40 s (short duration) or up to 4 h after stimulation. For consecutive trials, a minimum of 15–20 min of recovery time was maintained to (a) avoid cross-contamination between trials and (b) stabilize the MAP. Continuous MAP and HR data acquisition and analysis were done by using data acquisition software (LabChart, AD Instruments) with the Powerlab analog-to-digital convertor at a 1-kHz sampling rate. Basal measurements were obtained by taking the mean value over 30 s of baseline data immediately before nerve stimulation.
Immunohistochemical Analysis for fDPN upon Repeated Electrical Stimulation
At the end of the recording sessions, the animals were euthanized with an overdose of urethane. The fDPN tissue was collected, post-fixed, cryopreserved in 30% sucrose in 1× phosphate-buffered saline and cryosectioned at 20-µm sections for immunohistochemical analysis. Contralateral fDPN tissue was collected and used as a control (that is, no electrical stimulation). Potential stimulation-related acute axonal damage was evaluated by triple labeling immunofluorescence studies by using antibodies against the axonal marker β-III tubulin (1:500, mouse IgG; Sigma), myelin (P0) and activated macrophages (ED-1). The tissue sections were blocked with 5% normal goat serum incubated overnight with the primary antibodies and reacted with Cy2 donkey anti-rabbit (1:250) and Cy3 donkey anti-mouse (1:500); Cy5 donkey anti-goat-stained (1:400) (Jackson labs) tissue sections were imaged by using an inverted Nikon Ti Eclipse confocal microscope with a 20× objective.
MAP and HR from all the trials among different animals were expressed as mean ± standard error of the mean. The data were analyzed by parametric two-tailed Student t test followed by Mann-Whitney post hoc evaluation using the Prism 4 software (GraphPad Software). Values with p ≤ 0.05 were considered to be statistically significant.
Cardiovascular Depressor Response by DPN Stimulation
Significant Depressor Response by µCEA Stimulation of the fDPN without Bradycardia
Prolonged Depressor Response by Extended µCEA Stimulation of the fDPN
Repeated Electrical Stimulation Does Not Induce Axonal Damage
Refractory HTN is characterized by blood pressure that remains above 140/90 mmHg, despite treatments with at least two antihypertensive drugs. Among the surgical therapeutic strategies proposed are renal sympathetic denervation (18), carotid sinus baroreceptor stimulation (19,20) and deep brain stimulation (21). All have shown some promise, but require complicated surgical interventions. Stimulation of autonomic nerves such as the vagal nerve or the carotid sinus nerve is particularly effective. However, these nerves are heterogeneous in afferent/efferent composition, and their stimulation often results in unwanted side effects such as bradycardia and bradypnea (9,22).
In 1982, Yao et al. reported that electrical stimulation of somatic nerves might explain and reproduce the depressor effects of acupuncture in SHR rats. Indeed, acupuncture-like stimulation of the sciatic nerve in SHR animals was reported to induce an initial increase in HR and MAP during constant stimulation at 0.1 mA, 3 Hz for 30 min and a subsequent decrease in MAP from 160 to 135 mmHg, which lasted several hours after stimulation (23). However, this stimulation paradigm also elicited leg twitches, and more specific stimulation strategies were needed. The ST36 acupoint receives sensory innervation from saphenous, superficial peroneal, and lateral sural cutaneous nerves and motor innervation from the deep peroneal and anterior tibialis nerves (24). Stimulation of the DPN for 8 wks has been shown to reduce MAP by 10–18 mmHg without affecting HR (14). In humans, the peroneal nerve has approximately 25–38 fascicles (25) and whether stimulation of a single fascicle can significantly reduce MAP has not been evaluated. Here, we demonstrate that stimulating a single fascicle of the DPN (that is, fDPN) can be used to induce a depressor response in hypertensive SHR without significantly affecting HR. We showed that low-frequency stimulation (1 Hz) of 10 pulses at 0.1 mA induces an immediate MAP drop in a similar range to that observed after sciatic nerve stimulation or ST36-37 electroacupuncture, despite only interfacing a relatively small axonal population. The duration of the depressor response was significantly prolonged by extending the stimulation paradigm for 5 min. This brief stimulation of the fDPN was found to be effective in reducing the MAP in SRH animals for up to 4 h.
The mechanism of action of the observed depressor response in this study remains to be elucidated. However, the acupuncture stimulation of the ST36 acupoint has been shown to stimulate the DPN and to evoke neural afferent activity from the skin, muscles and joints. These axons terminate in the dorsal horn of the lumbar spinal cord, where they activate GABA-ergic interneurons, which in turn mediate synaptic inhibition of second-order neurons projecting to the brain stem periaqueductal gray, hypothalamic nuclei and rostral ventrolateral medulla (26) regions. Thus, a complex heterosynaptic pathway is likely involved in eliciting the hypotensive effect induced by stimulation of this nerve. This result has been confirmed by recent studies that have demonstrated that damage to the DPN abolishes the depressor response (27). Others have reported that DPN stimulation at 12 Hz and 0.15–0.3 mA excites only Aα, Aβ and Aδ fibers, whereas 0.5–0.8 mA evokes activity in C-fibers (28). Thus, it is likely that the effect that others and we have observed relates to the autonomic regulation of vasodilation in the hind limb.
One of the limitations of the present study is that all experiments were done in anesthetized animals, and thus further studies are warranted to determine whether the fDPN stimulation can be achieved in conscious animals. Such experiments will require not only the use of microelectrode arrays such as the µCEA, but also the development of fully implantable systems. We are currently working on the development of wireless stimulating microelectrodes, which can be used in the future to evaluate the effect of fDPN stimulation in fully awake animals.
In summary, this study provides evidence supporting the use of µCEA interfacing of small somatic nerve fascicles associated with cardiovascular relevant acupoints to induce significant reductions in MAP. The findings open the possibility of clinically relevant neuromodulation of small fascicles, which can be used as a strategy to achieve selective R-HTN therapy with minimal side effects.
The authors declare that they have no competing interests as defined by Bioelectronic Medicine, or other interests that might be perceived to influence the results and discussion reported in this paper.
This study was supported by the Texas Medical Research Collaborative Fund (TxMRC; MI Romero-Ortega) and the University of Texas at Arlington Research Institute (UTARI) at UT Arlington.
- 15.Kim Y, et al. (c2014) A novel microchannel electrode array: towards bioelectronic medical interfacing of small peripheral nerves. In: Engineering in Medicine and Biology Society (EMBC), 2014 36th Annual International Conference of the IEEE; 2014 Aug 24–30; Chicago (IL). p. 1981–4. Available from: https://doi.org/ieeexplore.ieee.org/xpl/freeabs_all.jsp?arnumber=6944002&tag=1&abstractAccess=no&userType=inst
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