An implant for long-term cervical vagus nerve stimulation in mice
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (eLife)
- Bioengineering (eLife)
Abstract
Vagus nerve stimulation (VNS) is a neuromodulation therapy with the potential to treat a wide range of chronic conditions in which inflammation is implicated, including type 2 diabetes, obesity, atherosclerosis and heart failure. Many of these diseases have well-established mouse models but due to the significant surgical and engineering challenges that accompany a reliable interface for long-term VNS in mice, the therapeutic implications of this bioelectronic approach remain unexplored. Here, we describe a long-term VNS implant in mice, developed at 3 research laboratories and validated for between-lab reproducibility. Implant functionality was evaluated over 3-8 weeks in 81 anesthetized or conscious mice by determining the stimulus intensity required to elicit a change in heart rate (heart rate threshold, HRT). HRT was also used as a method to standardize stimulation dosing across animals. Overall, 60-90% of implants produced stimulus-evoked physiological responses for at least 4 weeks, with HRT values stabilizing after the second week of implantation. Furthermore, stimulation delivered through 6-week-old implants decreased TNF levels in a subset of mice with acute inflammation caused by endotoxemia. Histological examination of 4- to 6-week-old implants revealed fibrotic encapsulation and no gross fiber loss. This implantation and dosing approach provide a tool to systematically investigate the therapeutic potential of long-term VNS in chronic diseases modeled in the mouse, the most widely used vertebrate species in biomedical research.
Article activity feed
-
This manuscript is in revision at eLife
The decision letter after peer review, sent to the authors on August 20 2020, follows.
Summary:
In this manuscript, Mughrabi et al reported a technical advance of long term vagus nerve stimulation (VNS) in mice. VNS has been used in clinics for treating certain patients with epilepsy and depression and pioneered in clinical trials for a number of disorders including inflammation. Yet, VNS has not been widely used in mice for mechanistic studies largely due to technical challenges dealing with the small size. Here, the authors developed a method for chronic implantation of VNS stimulator in mice, and tested the effectiveness of the method using measurements of heart rate changes and effects on inflammation. This method is potentially useful to investigate the therapeutic potential of long-term VNS …
This manuscript is in revision at eLife
The decision letter after peer review, sent to the authors on August 20 2020, follows.
Summary:
In this manuscript, Mughrabi et al reported a technical advance of long term vagus nerve stimulation (VNS) in mice. VNS has been used in clinics for treating certain patients with epilepsy and depression and pioneered in clinical trials for a number of disorders including inflammation. Yet, VNS has not been widely used in mice for mechanistic studies largely due to technical challenges dealing with the small size. Here, the authors developed a method for chronic implantation of VNS stimulator in mice, and tested the effectiveness of the method using measurements of heart rate changes and effects on inflammation. This method is potentially useful to investigate the therapeutic potential of long-term VNS in chronic disease models in mice. While reviewers were positive about the work performed in this study including that it was carried out by multiple labs, there are major concerns about certain points and additional essential experiments are needed. These include the need for robust data related to the LPS inflammation studies and histological analysis. There were also missing details of methodologies that decrease the enthusiasm for this study.
Essential Revisions:
At least two papers (PMID: 28628030, 32521521) have reported implants usable for the same application (long-term VNS in mice) although more extensive validation and characterization were performed in this manuscript. A comparison between those implants and the one in this manuscript needs to be discussed. As the authors stated, one technical challenge is that the vague nerve in mice is very small and fragile. However, it is unclear how the approach presented here is different from previous designs, and in particular, how mechanical damage is reduced using the reported apparatus.
If the paper is going to be a resource, the authors should provide detailed descriptions of the materials and construction of the electrode. Currently the details are sparse and the photos of poor resolution. It is unclear how the custom cuff was built (no details provided in the method section), what materials were used, and whether these materials are bio-compatible. Also, it is not clear whether and how the cuff electrode is appropriately insulated to prevent stimulation of surrounding muscles/nerves. In addition, the touching point between the nerve and the cuff is very easy to be damaged. With the description of the implantation procedure, it should also be made clearer as to when the cuff electrode is place on the nerve. A clear description could prevent torsion or other injury to the nerve.
LPS experiments: All reviewers thought the LPS experiment needed improvement. This study is under-powered and lacks a control group (saline + Sham stim). The LPS study is inconclusive due to a small number of animals. Increasing N to get conclusive data is important because this implant will be very useful to investigate the anti-inflammatory effect of long-term VNS in chronic disease models in mice. Related to this point, out of the 4 animals with bradycardia, 2 animals did not show a decrease in serum TNF. This raises a concern that using heart rate threshold may not be appropriate to deliver a consistent stimulation dose within/across animals if the goal is to get a consistent anti-inflammatory effect. It is likely that vagus efferent fibers responsible for HR decrease (innervating the sinoatrial and atrioventricular nodes) and those responsible for an anti-inflammatory effect are different populations. Those two populations might be differently affected by the implantation surgery and repetitive stimulation. In addition, performing VNS in awake animals is closer to the human situation.
Please confirm that 0.1mg/kg is the correct dose, this seems low to induce this amount of TNFa.
The histology of the vagus nerve raised questions and needs to be addressed. Here were relevant comments by reviewers.
In fig 4b, the vagus nerve in the cuff is quite clear, as is the carotid artery. But there are other nerve fragments and/or auto-fluorescent tissue immediately adjacent. What are these? Leads one to wonder if they only stimulated the vagus? The cervical sympathetic travels with the cervical vagus and care is needed to separate them from the carotid sheath. On the right side of fig 4b, the "control" side, they highlight a nerve nowhere near the carotid artery. This is intact tissue, so the vagus has to be next to the carotid artery. There is a big nerve next to the right carotid that I would bet is the vagus. I think they've got it wrong. It is not clear at what level these photos are taken, is it the cervical vagus? The authors should indicate the left and right carotid in these figures.
Figure 4. I do not see how fibrosis is determined. Is this actually collagen? Can the sections in B be stained with mason's trichrome. In "B" I am not sure that I see that the indicated regions are in fact the vagus nerve. It is hard to tell what other nerves would be present as there are few indications of the anatomical area these sections are from other than neck. Thus it Is hard to discern if this really is the vagus or not. I would have thought that the carotid artery should be visible in close proximity to the nerve bundle, this seems not to be the case and leads to uncertainty that this is the correct nerve.
Was there any difference in histology between mice with functioning and non-functioning cuffs? As stated in Discussion, left VN without surgery in different animals would be a better control than right VN in the same animals.
In the data presented in fig 2 or any of the studies where the kent scientific pulse/ox was used, Did O2 saturation decrease with the change in breathing?
Why didn't animals receiving awake VNS show visible changes in BR, which is in contrast to remarkable changes in BR in anesthetized animals?
In video 1, it is unclear when the stimulation starts or stops. As a result, it is uncertain if the mouse scratching is due to stimulation. Is this a pain/nociceptive response?
Fig 3 is presented in a confusing manner. In "A", I'm not sure why two mice are presented for different days post implantation and what this is showing. There is a clear effect of VNS on the heart rate and breathing (rate, and air flow), is this the minimum current for each day that was found to induce the heart rate threshold change. While I appreciate that the longer pulse widths are less susceptible to the effect of bio-encapsulation of the electrode over time, I'm not sure how one compares 100 uA at 100 us to 400 uA at 600 us. In B how is the HRT achieved without damaging the electrode as the ICIC is exceeded, or are we not understanding this graph correctly? In C there are days that seem to be missing given the legend. The supplementary figure also appears to have data points missing or obscured?
Success rate tops out at 75% with a skilled surgeon, and ranges between 40-60% for your average player. I'd say this is not too good.
It would be nice to show that the implant does not cause chronic inflammation as this would impact its usefulness as a method. The authors should measure tnfa 14 days Post implanted in cuff implanted and sham implanted mice.
What behavioral experiments were done, and what were the results? These are mentioned in several places (line 172, line 279 etc) but not reported.
The vagus nerve is critically involved in many essential body functions. Chronic implantation of the VNS stimulator may cause severe inflammation, nerve damage, and neuronal dysfunction. Therefore, it is critical to demonstrate that the chronic implantation does not alter nerve function. The chronic effect of the VNS stimulator implantation needs to be carefully monitored. For example, whether there is any change in body weight, food intake, as well as the sensitivity of diverse physiological reflexes such as the baroreflex, the Hering-Breuer reflex, and the stomach accommodation reflex.
-
-