Cholinergic Stimulation Improves Hemostasis in a Hemophilia Mouse Model
Authors: Shukla Shilpa , Fritz Jason R. , Kyaw Crystal A. , Valentino Sara P. , Imossi Catherine W. , Sarangi Susmita N. , Tracey Kevin J. , Czura Christopher J. , Huston Jared M. 
Published: October 2019

Abstract
Background: Management of bleeding in hemophilia patients that develop inhibitors remains a challenging clinical problem. Current bypassing agents are expensive, not universally effective and have pro-thrombotic potential. New approaches, including bispecific antibodies and others have shown potential but still require additional testing in clinical trials. As such, there is a clear need for alternative therapeutic options.
The inflammatory reflex is an endogenous neuro-immune pathway that monitors and regulates systemic inflammation via the vagus nerve. The afferent vagus nerve can inform the brain of increased peripheral inflammation. The brain then down-regulates systemic inflammation via increased efferent vagus nerve signaling to the spleen, a mechanism referred to as the cholinergic anti-inflammatory pathway (KJ Tracey, Nat Rev Imm 9(6), 2009). Our previous work has demonstrated that activation of the cholinergic anti-inflammatory pathway via direct electrical vagus nerve stimulation (VNS) or by systemic administration of a pharmacological cholinergic agonist inhibits proinflammatory cytokine production and protects against lethal systemic inflammation (JR Fritz, Bioelectron Med 1(1), 2014).
While studying the cholinergic anti-inflammatory pathway in mice, we observed that cholinergic stimulation reduces traumatic hemorrhage. To explore this observation further, we developed a porcine model of soft tissue trauma and hemorrhage. Electrical stimulation of the cervical vagus nerve significantly reduces total blood loss and bleeding time in pigs. Electrical vagus nerve stimulation also significantly increases local thrombin generation at the site of injury, whereas systemic thrombin concentrations remain unchanged (CJ Czura, Shock 33(6), 2010).
Aim: To evaluate the beneficial hemostatic effects of cholinergic stimulation on tail hemorrhage in a mouse model of hemophilia A.
Methods: Male 8-12-week-old, factor VIII knockout mice (B6;129S-F8<tm1Kaz>/J, Jackson Labs) receive either electrical cervical vagus nerve stimulation (1 V, 30 Hz, 2 ms pulse for 5 minutes) or sham stimulation under anesthesia (ketamine/xylazine 140/16 mg/kg, ip) five minutes before 2 mm distal tail transection. In separate experiments, nicotine (2 mg/kg, ip) or saline is administered 17 minutes before injury. Mouse tails are placed into a 37¡ÆC normal saline bath for five minutes prior to injury. After injury, tails are placed into 50 mL conical tubes filled with 37¡ÆC normal saline and allowed to bleed freely for a total of ten minutes. Total shed blood volume and active bleeding time are recorded, and systemic and local thrombin generation (thrombin-antithrombin complex) is measured via ELISA. Complete blood counts are measured via standard clinical assay.
Results: Vagus nerve stimulation significantly reduces total blood loss and bleeding time by 75% and 18%, respectively. Administration of nicotine significantly reduces total blood loss and bleeding time by 79% and 41%, respectively (Fig. 1). Nicotine significantly increases local thrombin generation compared with vehicle controls, whereas systemic thrombin generation is unchanged. There are no differences in circulating platelet counts or hematocrit levels.
Conclusions: Cholinergic stimulation results in rapid and specific improvements in hemostasis during traumatic hemorrhage in a mouse model of hemophilia A. The molecular mechanisms of localized thrombin generation remain an active area of research. Future clinical trials are necessary to determine if cholinergic stimulation is an efficacious, safe and cost-effective therapy for hemophilia A patients.
Figure 1. Vagus nerve stimulation (A) and nicotine administration (B) significantly reduce blood loss following traumatic hemorrhage in hemophilia A mice. Figure 1. Vagus nerve stimulation (A) and nicotine administration (B) significantly reduce blood loss following traumatic hemorrhage in hemophilia A mice. Figure 2. Nicotine administation significantly increases thrombin generation specifically at the site of injury and not systemically. Figure 2. Nicotine administation significantly increases thrombin generation specifically at the site of injury and not systemically.

Disclosures
No relevant conflicts of interest to declare.