Neuromodulation of the immune system has been proposed as a novel therapeutic strategy for the treatment of inflammatory conditions. We recently demonstrated that stimulation of near-organ autonomic nerves to the spleen can be harnessed to modulate the inflammatory response in an anesthetized pig model. The development of neuromodulation therapy for the clinic requires chronic efficacy and safety testing in a large animal model. This manuscript describes the effects of longitudinal conscious splenic nerve neuromodulation in chronically-implanted pigs. Firstly, clinically-relevant stimulation parameters were refined to efficiently activate the splenic nerve while reducing changes in cardiovascular parameters. Subsequently, pigs were implanted with a circumferential cuff electrode around the splenic neurovascular bundle connected to an implantable pulse generator, using a minimally-invasive laparoscopic procedure. Tolerability of stimulation was demonstrated in freely-behaving pigs using the refined stimulation parameters. Longitudinal stimulation significantly reduced circulating tumor necrosis factor alpha levels induced by systemic endotoxemia. This effect was accompanied by reduced peripheral monocytopenia as well as a lower systemic accumulation of CD16+CD14high pro-inflammatory monocytes. Further, lipid mediator profiling analysis demonstrated an increased concentration of specialized pro-resolving mediators in peripheral plasma of stimulated animals, with a concomitant reduction of pro-inflammatory eicosanoids including prostaglandins. Terminal electrophysiological and physiological measurements and histopathological assessment demonstrated integrity of the splenic nerves up to 70 days post implantation. These chronic translational experiments demonstrate that daily splenic nerve neuromodulation, via implanted electronics and clinically-relevant stimulation parameters, is well tolerated and is able to prime the immune system toward a less inflammatory, pro-resolving phenotype.

Neuromodulation of immune function by stimulating the autonomic connections to the spleen has been demonstrated in rodent models. Consequently, neuroimmune modulation has been proposed as a new therapeutic strategy for the treatment of inflammatory conditions. However, demonstration of the translation of these immunomodulatory mechanisms in anatomically and physiologically relevant models is still lacking. Additionally, translational models are required to identify stimulation parameters that can be transferred to clinical applications of bioelectronic medicines. Here, we performed neuroanatomical and functional comparison of the mouse, rat, pig, and human splenic nerve using in vivo and ex vivo preparations. The pig was identified as a more suitable model of the human splenic innervation. Using functional electrophysiology, we developed a clinically relevant marker of splenic nerve engagement through stimulation-dependent reversible reduction in local blood flow. Translation of immunomodulatory mechanisms were then assessed using pig splenocytes and two models of acute inflammation in anesthetized pigs. The pig splenic nerve was shown to locally release noradrenaline upon stimulation, which was able to modulate cytokine production by pig splenocytes. Splenic nerve stimulation was found to promote cardiovascular protection as well as cytokine modulation in a high- and a low-dose lipopolysaccharide model, respectively. Importantly, splenic nerve–induced cytokine modulation was reproduced by stimulating the efferent trunk of the cervical vagus nerve. This work demonstrates that immune responses can be modulated by stimulation of spleen-targeted autonomic nerves in translational species and identifies splenic nerve stimulation parameters and biomarkers that are directly applicable to humans due to anatomical and electrophysiological similarities.

A nine-year-old female alpaca with a history of a recurrent tooth root abscess presented for further investigation of a swelling of the left mandible and possible tooth extraction. During the manipulation of the mandible in surgery, the mandibular body fractured, and due to active infection it was left to heal by secondary intention. After surgery, the alpaca became dull and inappetent. Analgesic drugs included buprenorphine and meloxicam, which were ineffective according to the alpaca’s clinical appearance. An inferior alveolar nerve block was achieved by repeated administration of local anaesthetic (ropivacaine 0.75 per cent) every six hours via an epidural catheter placed in the mandibular foramen under CT guidance. Despite the adequate level of comfort achieved, discharge from surgical site resumed and the owner elected euthanasia. In conclusion, the placement of an epidural catheter in the mandibular canal, under CT guidance, was proven to successfully provide analgesia to an alpaca suffering from mandibular fractures.

Four captive reindeer underwent anaesthesia to allow dehorning or drainage of lymph nodes abscessation. Premedication was based on xylazine (dose range: 0.075- 0.5 mg/kg, IM or IV), with or without ketamine (dose range: 1-2 mg/kg, IM or IV), all of which failed to produce effective sedation without side effects. During anaesthesia, 2 reindeer experienced severe hypoxaemia and hypoventilation. Recovery was smooth in 3 out 4 animals, but delayed in one reindeer sedated with 0.5 mg/kg of xylazine IV; this patient required repeated atipamezole administrations (0.01 mg/kg IM given 3 times) to regain normal locomotion. Anaesthesia of reindeer is challenging and useful dose ranges for safe and effective anaesthesia are mostly unknown.