McConico, Andrea L.

AbstractAirway smooth muscle (ASM) cells modulate the local airway milieu via production of inflammatory mediators and growth factors including classical neurotrophins, such as brain‐derived neurotrophic factor (BDNF). The glial cell‐derived neurotrophic factor (GDNF) family of ligands (GFLs) are nonclassical neurotrophins and their role in the airway is barely understood. The major GFLs, GDNF and Neurturin (NRTN) bind to GDNF family receptor (GFR) α1 and α2 respectively that pair with Ret receptor to accomplish signaling. In this study, we found GDNF is expressed in human lung and increased in adult asthma, while human ASM expresses GDNF and its receptors. Accordingly, we used human ASM cells to test the hypothesis that ASM expression and autocrine signaling by GFLs regulate [Ca2+]i. Serum‐deprived ASM cells from non‐asthmatics were exposed to 10 ng/ml GDNF or NRTN for 15 min (acute) or 24 h (chronic). In fura‐2 loaded cells, acute GDNF or NRTN alone induced [Ca2+]i responses, and further enhanced responses to 1 µM ACh or 10 µM histamine. Ret inhibitor (SPP86; 10 µM) or specific GDNF chelator GFRα1‐Fc (1 µg/ml) showed roles of these receptors in GDNF effects. In contrast, NRTN did not enhance [Ca2+]i response to histamine. Furthermore, conditioned media of nonasthmatic and asthmatic ASM cells showed GDNF secretion. SPP86, Ret inhibitor and GFRα1‐Fc chelator markedly decreased [Ca2+]i response compared with vehicle, highlighting autocrine effects of secreted GDNF. Chronic GDNF treatment increased histamine‐induced myosin light chain phosphorylation. These novel data demonstrate GFLs particularly GDNF/GFRα1 influence ASM [Ca2+]i and raise the possibility that GFLs are potential targets of airway hyperresponsiveness.

Cervical vagal nerve stimulation (VNS) is an FDA approved treatment for epilepsy and depression and is currently being evaluated for treatment of numerous other disorders including pain, anxiety, and inflammation. VNS also received European market approval for heart failure, bronchoconstriction, and diabetes; however, approval for these indications was based on studies in animals, such as dogs and rats, whose vagal nerve size may not have been representative of humans. When subjected to double‐blinded, randomized, sham controlled studies for subsequent U.S. approval, many of these CE‐marked VNS indications failed to meet their primary efficacy endpoints. More than 100,000 patients have undergone VNS, but despite the efficacy of vagal nerve stimulation in a subset of patients, many fail to reach stimulation levels that result in alleviation of symptoms without triggering unwanted side effects. Post hoc studies of the patient population suggested that VNS may not engage the intended fiber pathways in humans, despite engagement in preclinical studies in mouse, rat, or dog. Stimulation parameters were limited in these studies as higher parameters caused side effects such as cough, voice alteration, and dyspnea. Discrepancies between stimulation amplitude needed for intended effect versus unwanted side effect could be directly related to the relative size of the vagus nerve across animal models, variations in fascicle organization, or variations in branching patterns. To address translating improvements in VNS from the lab to the clinic, we evaluated the swine as a potential model for approximating the clinical environment. The size and fascicular organization of the swine vagus nerve is more representative of the human nerve than previously used models. We used a combination of histology, microdissection, and ultrasound to establish a distinct bimodal organization of groups of fascicles stemming from one large fascicle at the inferior (nodose) ganglion, an organization we have termed ‘vagotopy’. We demonstrated that with the use of ultrasound we can visualize the fascicular organization in vivo , an approach that could be used clinically to target stimulation of either sensory fibers or parasympathetic efferents, depending on therapeutic intent. These advances in a clinically relevant model not only inform electrode placement for target engagement, but provide an approach for reduction of side effects. Support or Funding Information Acknowledgements Funded by TL1TR002380 andThe Defense Advanced Research Projects Agency (DARPA) Biological Technologies Office (BTO) Targeted Neuroplasticity Training Program under the auspices of Doug Weber and Tristan McClure‐Begley through the Space and Naval Warfare Systems Command (SPAWAR) Systems Center with (SSC) Pacific grants no. N66001‐17‐2‐4010, and the NIH SPARC Program Award OT2 OD025340.

Abstract Implanted neural stimulation and recording devices hold vast potential to treat a variety of neurological conditions, but the invasiveness, complexity, and cost of the implantation procedure greatly reduce access to an otherwise promising therapeutic approach. To address this need, a novel electrode that begins as an uncured, flowable prepolymer that can be injected around a neuroanatomical target to minimize surgical manipulation is developed. Referred to as the Injectrode, the electrode conforms to target structures forming an electrically conductive interface which is orders of magnitude less stiff than conventional neuromodulation electrodes. To validate the Injectrode, detailed electrochemical and microscopy characterization of its material properties is performed and the feasibility of using it to stimulate the nervous system electrically in rats and swine is validated. The silicone‐metal‐particle composite performs very similarly to pure wire of the same metal (silver) in all measures, including exhibiting a favorable cathodic charge storage capacity (CSC C ) and charge injection limits compared to the clinical LivaNova stimulation electrode and silver wire electrodes. By virtue of its simplicity, the Injectrode has the potential to be less invasive, more robust, and more cost‐effective than traditional electrode designs, which could increase the adoption of neuromodulation therapies for existing and new indications.

The sequence of events that lead to inflammation and fibrosing nonalcoholic steatohepatitis (NASH) is incompletely understood. Hence, we investigated the chronology of whole body, tissue, and cellular events that occur during the evolution of diet-induced NASH. Male C57Bl/6 mice were assigned to a fast-food (FF; high calorie, high cholesterol, high fructose) or standard-chow (SC) diet over a period of 36 wk. Liver histology, body composition, mitochondrial respiration, metabolic rate, gene expression, and hepatic lipid content were analyzed. Insulin resistance [homeostasis model assessment-insulin resistance (HOMA-IR)] increased 10-fold after 4 wk. Fibrosing NASH was fully established by 16 wk. Total hepatic lipids increased by 4 wk and remained two- to threefold increased throughout. Hepatic triglycerides declined from sixfold increase at 8 wk to threefold increase by 36 wk. In contrast, hepatic cholesterol levels steadily increased from baseline at 8 wk to twofold by 36 wk. The hepatic immune cell population altered over time with macrophages persisting beyond 16 wk. Mitochondrial oxygen flux rates of FF mice diet were uniformly lower with all the tested substrates (13–276 pmol·s−1·ml−1 per unit citrate synthase) than SC mice (17–394 pmol·s−1·ml−1 per unit citrate synthase) and was accompanied by decreased mitochondrial:nuclear gene copy number ratios after 4 wk. Metabolic rate was lower in FF mice. Mitochondrial glutathione was significantly decreased at 24 wk in FF mice. Expression of dismutases and catalase was also decreased in FF mice. The evolution of NASH in the FF diet-induced model is multiphasic, particularly in terms of hepatic lipid composition. Insulin resistance precedes hepatic inflammation and fibrosis. Mitochondrial dysfunction and depletion occur after the histological features of NASH are apparent. Collectively, these observations provide a unique overview of the sequence of changes that coevolve with the histological evolution of NASH. NEW & NOTEWORTHY This study demonstrates in a first of kind longitudinal analysis, the evolution of nonalcoholic steatohepatitis (NASH) on a fast-food diet-induced model. Key findings include 1) hepatic lipid composition changes in a multiphasic fashion as NASH evolves; 2) insulin resistance precedes hepatic inflammation and fibrosis, answering a longstanding chicken-and-egg question regarding the relationship of insulin resistance to liver histology in NASH; and 3) mitochondrial dysfunction and depletion occur after the histological features of NASH are apparent.

Free vascular endothelial growth factor (VEGF) is undetectable in plasma during human pregnancy. However, studies examining pregnant rats have reported both low (8–29 pg/ml) and high (527–1,030 pg/ml) free VEGF. These discrepancies cast uncertainty over the use of rat models to study angiogenic factors in pregnancy and preeclampsia. This study investigates methodological factors that may explain these discrepancies. Plasma VEGF in nonpregnant, day 7 pregnant, and day 19 pregnant rats was measured using rat and mouse ELISAs (R&D Systems). The rat ELISA detected VEGF in plasma from nonpregnant rats but not in plasma from day 19 pregnant rats. The mouse ELISA detected higher VEGF concentrations than the rat ELISA in every sample tested. This discrepancy was greater in day 19 pregnant rats (median: 2,273 vs. 0 pg/ml) than in nonpregnant (97 vs. 20 pg/ml) and day 7 pregnant (66 vs. 2 pg/ml) rats. Recovery of recombinant rat VEGF (rrVEGF) spiked into plasma from nonpregnant and day 7 pregnant rats was high for the rat ELISA (82–105%) but low for the mouse ELISA (17–22%). The rat ELISA did not recover rrVEGF in plasma from day 19 pregnant rats, suggesting that this ELISA measures free VEGF. The use of the rat versus mouse ELISA likely explains the differences in reported VEGF concentrations in pregnant rats. While the rat ELISA appears to measure free VEGF, plasma concentrations in nonpregnant and pregnant rats are below the assay sensitivity limit. As most previous studies of pregnant rats used the mouse VEGF ELISA, these data should be interpreted cautiously.

Although there are small animal platforms that recapitulate some of the histological features of nonalcoholic fatty liver disease, there are no small animal models of nonalcoholic steatohepatitis (NASH) with consistent hepatocellular ballooning and progressive fibrosis that also exhibit fidelity to the human condition physiologically. We examined the metabolic and histological effects of a diet on the basis of the composition of “fast food” (high saturated fats, cholesterol, and fructose). Mice ( n = 8 in each group) were assigned to diets as follows: 1) standard chow (SC), i.e., 13% energy as fat [1% saturated fatty acids (SFA)], 2) high fat (HF), i.e., 60% energy as fat (1% SFA), and 3) fast food (FF), i.e., 40% energy as fat (12% SFA, 2% cholesterol). All three diets were supplemented with high fructose. All diets produced obesity. The HF and FF diets produced insulin resistance. Liver histology was normal in animals fed the SC diet. Steatohepatitis with pronounced ballooning and progressive fibrosis (stage 2) was observed in mice fed the FF diet. Although the HF diet produced obesity, insulin resistance, and some steatosis; inflammation was minimal, and there was no increase in fibrosis. The FF diet produced a gene expression signature of increased fibrosis, inflammation, and endoplasmic reticulum stress and lipoapoptosis. A diet based on high cholesterol, high saturated fat, and high fructose recapitulates features of the metabolic syndrome and NASH with progressive fibrosis. This represents a novel small animal model of fibrosing NASH with high fidelity to the human condition. These results highlight the contribution of dietary composition to the development of nonalcoholic fatty liver disease and NASH.

Trafficking of cells between mother and fetus during the course of normal pregnancy is well documented. Similarly, cells are known to travel between twins that share either a placenta (i.e. monozygotic) or associated chorion (i.e. monochorionic). Transferred cells are thought to be channelled via the vessels of the placenta or vascular connections established via the chorion and the long-term presence of these cells (i.e. microchimerism) can have important consequences for immune system function and reparative capacity of the host. Whether cells can be transferred between twins with separate placentas and separate chorions (i.e. no vascular connections between placentas) has not been investigated nor have the biological consequences of such a transfer. In the present study, we tested the possibility of this type of cell transfer by injecting human cord blood-derived cells into a portion of the littermates of swine and probing for human cells in the blood and tissues of unmanipulated littermates. Human cells were detected in the blood of 78% of unmanipulated littermates. Human cells were also detected in various tissues of the unmanipulated littermates, including kidney (56%), spleen (33%), thymus (11%) and heart (22%). Human cells were maintained in the blood until the piglets were sacrificed (8 months after birth), suggesting the establishment of long-term microchimerism. Our findings show that the transfer of cells between fetuses with separate placentas and separate chorions is significant and thus such twins may be subject to the same consequences of microchimerism as monozygotic or monochorionic counterparts.