A critical role for erythropoietin on vagus nerve Schwann cells in intestinal motility.

BACKGROUND: Dysmotility and postoperative ileus (POI) are frequent major clinical problems post-abdominal surgery. Erythropoietin (EPO) is a multifunctional tissue-protective cytokine that promotes recovery of the intestine in various injury models. While EPO receptors (EPOR) are present in vagal Schwann cells, the role of EPOR in POI recovery is unknown because of the lack of EPOR antagonists or Schwann-cell specific EPOR knockout animals. This study was designed to explore the effect of EPO via EPOR in vagal nerve Schwann cells in a mouse model of POI. RESULTS: The structural features of EPOR and its activation by EPO-mediated dimerization were understood using structural analysis. Later, using the Cre-loxP system, we developed a myelin protein zero (Mpz) promoter-driven knockout mouse model of Schwann cell EPOR (MpzCre-EPORflox/flox / Mpz-EPOR-KO) confirmed using PCR and qRT-PCR techniques. We then measured the intestinal transit time (ITT) at baseline and after induction of POI with and without EPO treatment. Although we have previously shown that EPO accelerates functional recovery in POI in wild type mice, EPO treatment did not improve functional recovery of ITT in POI of Mpz-EPOR-KO mice. CONCLUSIONS: To the best of our knowledge, this is the first pre-clinical study to demonstrate a novel mouse model of EPOR specific knock out on Schwan cells with an effect in the gut. We also showed novel beneficial effects of EPO through vagus nerve Schwann cell-EPOR in intestinal dysmotility. Our findings suggest that EPO-EPOR signaling in the vagus nerve after POI is important for the functional recovery of ITT.

Functional recovery and muscle atrophy in pre-clinical models of peripheral nerve transection and gap-grafting in mice: effects of 4-aminopyridine.

We recently demonstrated a repurposing beneficial effect of 4-aminopyridine (4-AP), a potassium channel blocker, on functional recovery and muscle atrophy after sciatic nerve crush injury in rodents. However, this effect of 4-AP is unknown in nerve transection, gap, and grafting models. To evaluate and compare the functional recovery, nerve morphology, and muscle atrophy, we used a novel stepwise nerve transection with gluing (STG), as well as 7-mm irreparable nerve gap (G-7/0) and 7-mm isografting in 5-mm gap (G-5/7) models in the absence and presence of 4-AP treatment. Following surgery, sciatic functional index was determined weekly to evaluate the direct in vivo global motor functional recovery. After 12 weeks, nerves were processed for whole-mount immunofluorescence imaging, and tibialis anterior muscles were harvested for wet weight and quantitative histomorphological analyses for muscle fiber cross-sectional area and minimal Feret's diameter. Average post-injury sciatic functional index values in STG and G-5/7 models were significantly greater than those in the G-7/0 model. 4-AP did not affect the sciatic functional index recovery in any model. Compared to STG, nerve imaging revealed more misdirected axons and distorted nerve architecture with isografting. While muscle weight, cross-sectional area, and minimal Feret's diameter were significantly smaller in G-7/0 model compared with STG and G-5/7, 4-AP treatment significantly increased right TA muscle mass, cross-sectional area, and minimal Feret's diameter in G-7/0 model. These findings demonstrate that functional recovery and muscle atrophy after peripheral nerve injury are directly related to the intervening nerve gap, and 4-AP exerts differential effects on functional recovery and muscle atrophy.

Effects of 4-Aminopyridine on Combined Nerve and Muscle Injury and Bone Loss.

PURPOSE: Musculoskeletal injuries are common, and peripheral nerve injury (PNI) causes significant muscle and bone loss within weeks. After PNI, 4-aminopyridine (4-AP) improves functional recovery and muscle atrophy. However, it is unknown whether 4-AP has any effect on isolated traumatic muscle injury and PNI-induced bone loss. METHODS: A standardized crush injury was performed on the sciatic nerve and muscles in mice, and the mice were assigned to receive normal saline or 4-AP treatment daily for 21 days. The postinjury motor and sensory function recovery was assessed, injured muscles were processed for histomorphometry, and the tibial bone was scanned for bone density. RESULTS: 4-Aminopyridine significantly accelerated the postinjury motor and sensory function recovery, improved muscle histomorphometry, increased muscle satellite cell numbers, and shifted muscle fiber types after combined nerve and muscle injury. Importantly, the 4-AP treatment significantly reduced PNI-induced bone loss. In contrast, in the case of isolated muscle injury, 4-AP had no effect on functional recovery and bone density, but it improved muscle-specific histomorphometry to a limited extent. CONCLUSIONS: These findings demonstrate the potential beneficial effects of 4-AP on the recovery of muscle morphology and bone density after combined muscle and nerve injury. CLINICAL RELEVANCE: Nerve injuries frequently involve muscle and result in rapid muscle and bone atrophy. In this scenario, 4-AP, in addition to accelerating nerve functional recovery, might work as an adjunctive agent to improve the recovery of injured muscle and attenuate PNI-induced bone loss.

Erythropoietin promotes M2 macrophage phagocytosis of Schwann cells in peripheral nerve injury.

Following acute sciatic nerve crush injury (SNCI), inflammation and the improper phagocytic clearance of dying Schwann cells (SCs) has effects on remodeling that lead to morbidity and incomplete functional recovery. Therapeutic strategies like the use of erythropoietin (EPO) for peripheral nerve trauma may serve to bring immune cell phagocytotic clearance under control to support debris clearance. We evaluated EPO's effect on SNCI and found EPO treatment increased myelination and sciatic functional index (SFI) and bolstered anti-apoptosis and phagocytosis of myelin debris via CD206+ macrophages when compared to saline treatment. EPO enhanced M2 phenotype activity, both in bone marrow-derived macrophages (BMMØs) and peritoneal-derived macrophages (PMØs) in vitro, as well as in PMØs in vivo. EPO increased efferocytosis of apoptotic sciatic nerve derived Schwann cells (SNSCs) in both settings as demonstrated using immunofluorescence (IF) and flow cytometry. EPO treatment significantly attenuated pro-inflammatory genes (IL1ß, iNOS, and CD68) and augmented anti-inflammatory genes (IL10 and CD163) and the cell-surface marker CD206. EPO also increased anti-apoptotic (Annexin V/7AAD) effects after lipopolysaccharide (LPS) induction in macrophages. Our data demonstrate EPO promotes the M2 phenotype macrophages to ameliorate apoptosis and efferocytosis of dying SCs and myelin debris and improves SN functional recovery following SNCI.

(4-Aminopyridine)-PLGA-PEG as a Novel Thermosensitive and Locally Injectable Treatment for Acute Peripheral Nerve Injury.

Traumatic peripheral nerve injury (TPNI) represents a major medical problem that results in loss of motor and sensory function, and in severe cases, limb paralysis and amputation. To date, there are no effective treatments beyond surgery in selective cases. In repurposing studies, we found that daily systemic administration of the FDA-approved drug 4-aminopyridine (4-AP) enhanced functional recovery after acute peripheral nerve injury. This study was aimed at constructing a novel local delivery system of 4-AP using thermogelling polymers. We optimized a thermosensitive (4-AP)-poly(lactide-co-glycolide)-b-poly(ethylene glycol)-b-poly(lactide-co-glycolide) (PLGA-PEG-PLGA) block copolymer formulation. (4-AP)-PLGA-PEG exhibited controlled release of 4-AP both in vitro and in vivo for approximately 3 weeks, with clinically relevant safe serum levels in animals. Rheological investigation showed that (4-AP)-PLGA-PEG underwent a solution to gel transition at 32 °C, a physiologically relevant temperature, allowing us to administer it to an injured limb while subsequently forming an in situ gel. A single local administration of (4-AP)-PLGA-PEG remarkably enhanced motor and sensory functional recovery on post-sciatic nerve crush injury days 1, 3, 7, 14, and 21. Moreover, immunohistochemical studies of injured nerves treated with (4-AP)-PLGA-PEG demonstrated an increased expression of neurofilament heavy chain (NF-H) and myelin protein zero (MPZ) proteins, two major markers of nerve regeneration. These findings demonstrate that (4-AP)-PLGA-PEG may be a promising long-acting local therapeutic agent in TPNI, for which no pharmacologic treatment exists.

An effective erythropoietin dose regimen protects against severe nerve injury-induced pathophysiological changes with improved neural gene expression and enhances functional recovery.

The functional recovery following non-severing peripheral nerve injury (PNI) is often incomplete. Erythropoietin (EPO) is a pleiotropic hormone and it has been shown to protect peripheral nerves following mild and even moderate severity injuries. However, the effectiveness of EPO in severe PNI is largely unknown. In this study, we sought to investigate the neuroprotective effect of a new dose regimen of EPO in severe sciatic nerve crush injury (SSCI). Adult male mice (8 animals/group) were randomly assigned to sham (normal saline, 0.1 ml/mouse), SSCI (normal saline, 0.1 ml/mouse) and SSCI with EPO (5000 IU/kg) groups. SSCI was performed using calibrated forceps for 30 sec. EPO or normal saline was administered intraperitoneally immediately after the SSCI and at post-injury day1 and 2. The functional recovery after injury was assessed by sciatic function index (SFI), von Frey Test (VFT), and grip strength test. Mice were euthanized on day 7 and 21 and nerves at injury/peri-injury site were processed for gene (quantitative real-time PCR) and protein (immunohistochemistry) expression analysis. EPO significantly improved SFI, VFT, and hind limb paw grip strength from post-injury day 7. EPO demonstrated significant regulatory effects on mRNA expression of inflammatory (IL-1ß and TNF-α), anti-inflammatory (IL-10), angiogenesis (VEGF and eNOS), and myelination (MBP) genes. The protein expression of IL-1ß, F4/80, CD31, NF-κB p65, NF-H, MPZ, and DHE (redox-sensitive probe) was also significantly modulated by EPO treatment. In conclusion, the new dose regimen of EPO augments sciatic nerve functional recovery by mitigating inflammatory, anti-inflammatory, oxidative stress, angiogenesis, and myelination components of SSCI.

Anticancer efficacy of 6-thioguanine loaded chitosan nanoparticles with or without curcumin.

6-Thioguanine encapsulated chitosan nanoparticles (6-TG-CNPs) has formulated by the ionic-gelation method. Morphologically, the 6-TG-CNPs were spherical and showed mean size, PDI, zeta potential, and entrapment efficiency of 261.63 ± 6.01 nm, 0.34 ± 0.10, +15.97 ± 0.46 mV and 44.27%, respectively. The IR spectra confirmed the 6-TG complex with chitosan. The in vitro drug release profile of 6-TG-CNPs revealed an increase in sustained-release (91.40 ± 1.08% at 48 h) at pH 4.8 compared to less sustained-release (73.96 ± 1.12% at 48 h) at pH 7.4. The MTT assay was conducted on MCF-7 and PA-1 cell lines at 48 h incubation to determine % cell viability. The IC50 values of 6-TG, 6-TG-CNPs, and curcumin for MCF-7 were 23.09, 17.82, and 15.73 µM, respectively. Likewise, IC50 values of 6-TG, 6-TG-CNPs, and curcumin for PA-1 were 5.81, 3.92, and 12.89 µM, respectively. A combination of 6-TG-CNPs (IC25) with curcumin (IC25) on PA-1 and MCF-7 showed % cell viability of 43.67 ± 0.02 and 49.77 ± 0.05, respectively. The in vitro cytotoxicity potential in terms of % cell viability, early apoptosis, G2/M phase arrest, and DNA demethylating activity of 6-TG-CNPs alone and combination with curcumin proved to be more effective than that of 6-TG on PA-1 cells.

Toxicity evaluation of 6-mercaptopurine-Chitosan nanoparticles in rats.

BACKGROUND: The 6-mercaptopurine (6-MP) is an effective immunosuppressant and anti-cancer drug. However, the usage of 6-MP is limited due to its well-known side effects, such as myelotoxicity and hepato-renal toxicity. To curtail the potential toxic effects, we have used chitosan as a natural biodegradable and biocompatible polysaccharide to synthesize 6-Mercaptopurine-Chitosan Nanoparticles (6-MP-CNPs). METHODS: The 6-MP-CNPssize, morphology, physicochemical interactions, and thermal stability were characterized using Dynamic Light Scattering (DLS), Scanning Electron Microscopy (SEM), Fourier Transform Infrared Spectroscopy (FTIR), and Differential Scanning Calorimetry (DSC), respectively. The loading efficiency of the 6-MP in CNPs was estimated using LCMS/MS. Then, the 6-MP-CNPs were subjected to in vivo acute and sub-acute oral toxicity evaluations. RESULTS: The DLS and SEM analysis respectively indicated size (70.0 nm to 400.0 nm), polydispersity index (0.462), and zeta potential (54.9 mV) with improved morphology of 6-MP-CNPs. The FTIR and DSC results showed the efficient interactive and stable nature of the 6-MP-CNPs, which sustained the drug-delivery process. The loading efficiency of 6-MP-CNPs was found to be 25.23%. The chitosan improved the lethal dose (LD50 cut off) of 6-MP-CNPs (1000 mg/kg b.w) against 6-MP (500 mg/kg b.w) and also significantly (p ≤ 0.05) reduces the toxic adverse effect (28-day repeated oral dose) on hemato-biochemical and hepato-renal histological profiles. CONCLUSION: The findings suggest that chitosan, as a prime drug-delivery carrier, significantly alleviates the acute and sub-acute toxic effects of 6-MP.

Targeting exosome-associated human antigen R attenuates fibrosis and inflammation in diabetic heart.

RNA-binding proteins like human antigen R (HuR) are key regulators in post-transcriptional control of gene expression in several pathophysiological conditions. Diabetes adversely affects monocyte/macrophage biology and function. It is not known whether diabetic milieu affects cellular/exosome-HuR and its implications on cardiac inflammation and fibrosis. Here, we evaluate in vitro and in vivo effects of diabetic milieu on macrophage cellular/exosome-HuR, alterations in intercellular cross talk with fibroblasts, and its impact on cardiac remodeling. Human failing hearts show higher HuR levels. Diabetic milieu activates HuR expression in cardiac- and cultured bone marrow-derived macrophages (BMMØ) and stimulates HuR nuclear-to-cytoplasmic translocation and exosome transfer. Exosomes from macrophages exposed to diabetic milieu (high glucose or db/db mice) significantly increase inflammatory and profibrogenic responses in fibroblast (in vitro) and cardiac fibrosis in mice. Intriguingly, Exo-HuR deficiency (HuR knockdown in macrophage) abrogates the above effects. In diabetic mice, macrophage depletion followed by reconstitution with BMMØ-derived HuR-deficient exosomes inhibits angiotensin II-induced cardiac fibrosis response and preserves left ventricle function as compared to control-exosome administration. To the best of our knowledge, this is the first study to demonstrate that diabetes activates BMMØ HuR expression and its transfer into exosome. The data suggest that HuR might be targeted to alleviate macrophage dysfunction and pathological fibrosis in diabetes.

Effect of Withania somnifera on gentamicin induced renal lesions in rats

ABSTRACT Gentamicin induced renal complications are well known in humans and animals. Medicinal properties of Withania somnifera (L.) Dunal, Solanaceae, are recognized to improve renal functions. However, the pharmacological function of W. somnifera is not completely understood. We sought to unravel medicinal therapeutic function of W. somnifera on gentamicin-induced nephrotoxicity in wistar rats. Twenty-four adult male wistar rats evenly divided into four groups to evaluate in vivo nephroprotective and nephrocurative function of W. somnifera in gentamicin induced nephrotoxic rats. Experimental design as follows: Group I, saline control for 21 days; Group II, gentamicin nephrotoxic control for eight days; Group III, alcoholic extract of W. somnifera for 13 days + simultaneous administration of gentamicin and W. somnifera, from day 14 to 21 (nephroprotective) and Group IV, gentamicin for 8 days + alcoholic extract of W. somnifera from day 9 to 21 (nephrocurative). End of experiment, respective serum and kidney tissue samples used to analyze renal function. Withania somnifera as a nephroprotective and nephrocurative molecule significantly restore the renal function on gentamicin-induced nephrotoxicity. This phenomenon is accompanied with significantly reduced blood urea nitrogen, creatine, alkaline phosphatase, gamma-glutamyl transferase, albumin, total protein, calcium, potassium and kidney malondialdehyde concentrations. Additionally, W. somnifera significantly increased antioxidant activities of glutathione and superoxide dismutase to protect renal tissue damage from gentamicin in wistar rats. Over all, W. somnifera treated nephroprotective animal shows improved recovery compared to nephrocuartive. The nephroprotective or nephrocurative effect of W. somnifera could be due to inherent antioxidant and free-radical-scavenging principle(s). In the near future, biologically active compounds of W. somnifera (withanolides) could appear as a novel therapeutic molecule for renal disorders.