Cutaneous collateral axonal sprouting re-innervates the skin component and restores sensation of denervated Swine osteomyocutaneous alloflaps.

PMC3799840Reconstructive transplantation such as extremity and face transplantation is a viable treatment option for select patients with devastating tissue loss. Sensorimotor recovery is a critical determinant of overall success of such transplants. Although motor function recovery has been extensively studied, mechanisms of sensory re-innervation are not well established. Recent clinical reports of face transplants confirm progressive sensory improvement even in cases where optimal repair of sensory nerves was not achieved. Two forms of sensory nerve regeneration are known. In regenerative sprouting, axonal outgrowth occurs from the transected nerve stump while in collateral sprouting, reinnervation of denervated tissue occurs through growth of uninjured axons into the denervated tissue. The latter mechanism may be more important in settings where transected sensory nerves cannot be re-apposed. In this study, denervated osteomyocutaneous alloflaps (hind- limb transplants) from Major Histocompatibility Complex (MHC)-defined MGH miniature swine were performed to specifically evaluate collateral axonal sprouting for cutaneous sensory re-innervation. The skin component of the flap was externalized and serial skin sections extending from native skin to the grafted flap were biopsied. In order to visualize regenerating axonal structures in the dermis and epidermis, 50 um frozen sections were immunostained against axonal and Schwann cell markers. In all alloflaps, collateral axonal sprouts from adjacent recipient skin extended into the denervated skin component along the dermal-epidermal junction from the periphery towards the center. On day 100 post-transplant, regenerating sprouts reached 0.5 cm into the flap centripetally. Eight months following transplant, epidermal fibers were visualized 1.5 cm from the margin (rate of regeneration 0.06 mm per day). All animals had pinprick sensation in the periphery of the transplanted skin within 3 months post-transplant. Restoration of sensory input through collateral axonal sprouting can revive interaction with the environment; restore defense mechanisms and aid in cortical re-integration of vascularized composite allografts.JH Libraries Open Access Fun

Injectable bioadhesive hydrogels with innate antibacterial properties.

Surgical site infections cause significant postoperative morbidity and increased healthcare costs. Bioadhesives used to fill surgical voids and support wound healing are typically devoid of antibacterial activity. Here we report novel syringe-injectable bioadhesive hydrogels with inherent antibacterial properties prepared from mixing polydextran aldehyde and branched polyethylenimine. These adhesives kill both Gram-negative and Gram-positive bacteria, while sparing human erythrocytes. An optimal composition of 2.5 wt% oxidized dextran and 6.9 wt% polyethylenimine sets within seconds forming a mechanically rigid (~1,700 Pa) gel offering a maximum adhesive stress of ~2.8 kPa. A murine infection model showed that the adhesive is capable of killing Streptococcus pyogenes introduced subcutaneously at the bioadhesive\u27s surface, with minimal inflammatory response. The adhesive was also effective in a cecal ligation and puncture model, preventing sepsis and significantly improving survival. These bioadhesives represent novel, inherently antibacterial materials for wound-filling applications

Monocytes loaded with indocyanine green as active homing contrast agents permit optical differentiation of infectious and non-infectious inflammation.

Distinguishing cutaneous infection from sterile inflammation is a diagnostic challenge and currently relies upon subjective interpretation of clinical parameters, microbiological data, and nonspecific imaging. Assessing characteristic variations in leukocytic infiltration may provide more specific information. In this study, we demonstrate that homing of systemically administered monocytes tagged using indocyanine green (ICG), an FDA-approved near infrared dye, may be assessed non-invasively using clinically-applicable laser angiography systems to investigate cutaneous inflammatory processes. RAW 264.7 mouse monocytes co-incubated with ICG fluoresce brightly in the near infrared range. In vitro, the loaded cells retained the ability to chemotax toward monocyte chemotactic protein-1. Following intravascular injection of loaded cells into BALB/c mice with induced sterile inflammation (Complete Freund's Adjuvant inoculation) or infection (Group A Streptococcus inoculation) of the hind limb, non-invasive whole animal imaging revealed local fluorescence at the inoculation site. There was significantly higher fluorescence of the inoculation site in the infection model than in the inflammation model as early as 2 hours after injection (p<0.05). Microscopic examination of bacterial inoculation site tissue revealed points of near infrared fluorescence, suggesting the presence of ICG-loaded cells. Development of a non-invasive technique to rapidly image inflammatory states without radiation may lead to new tools to distinguish infectious conditions from sterile inflammatory conditions at the bedside

Near-infrared lymphography as a minimally invasive modality for imaging lymphatic reconstitution in a rat orthotopic hind limb transplantation model.

Wider application of vascularized composite allotransplantation (VCA) is limited by the need for chronic immunosuppression. Recent data suggest that the lymphatic system plays an important role in mediating rejection. This study used near-infrared (NIR) lymphography to describe lymphatic reconstitution in a rat VCA model. Syngeneic (Lewis-Lewis) and allogeneic (Brown Norway-Lewis) rat orthotopic hind limb transplants were performed without immunosuppression. Animals were imaged pre- and postoperatively using indocyanine green (ICG) lymphography. Images were collected using an NIR imaging system. Co-localization was achieved through use of an acrylic paint/hydrogen peroxide mixture. In all transplants, ICG first crossed graft suture lines on postoperative day (POD) 5. Clinical signs of rejection also appeared on POD 5 in allogeneic transplants, with most exhibiting Grade 3 rejection by POD 6. Injection of an acrylic paint/hydrogen peroxide mixture on POD 5 confirmed the existence of continuous lymphatic vessels crossing the suture line and draining into the inguinal lymph node. NIR lymphography is a minimally invasive imaging modality that can be used to study lymphatic vessels in a rat VCA model. In allogeneic transplants, lymphatic reconstitution correlated with clinical rejection. Lymphatic reconstitution may represent an early target for immunomodulation

De novo variants in SUPT16H cause neurodevelopmental disorders associated with corpus callosum abnormalities

IntroductionWhole-exome sequencing (WES) has identified de novo variants in chromatin remodelling genes in patients with neurodevelopmental disorders (NDD). We report on a novel genetic discovery in chromatin remodelling in patients with NDD who also have corpus callosum (CC) anomalies.ObjectiveTo discover novel genes linked to both CC anomalies and NDD.MethodsClinical WES was performed for evaluation of NDD, identifying five patients with de novo variants in SUPT16H, a subunit of the FACT (facilitates chromatin transcription) complex. The clinical phenotypes, genetic results and brain MRIs were obtained and systematically reviewed. In silico protein function predictions were assessed and allele frequencies in control populations were compared.ResultsWe identified four patients with de novo missense variants in SUPT16H and one patient with a de novo deletion including SUPT16H. These variants were not reported in the updated Genome Aggregation Database. When assayable, all protein products were predicted to be damaging. Symptoms included intellectual disability, autistic features, minor dysmorphic features and seizures. Anomalies of the CC were seen in all three patients with available brain imaging.ConclusionOur findings implicate the gene SUPT16H in a novel disorder characterised by neurodevelopmental deficits and CC anomalies

Mesenchymal Stem Cells Enhance Nerve Regeneration in a Rat Sciatic Nerve Repair and Hindlimb Transplant Model.

This study investigates the efficacy of local and intravenous mesenchymal stem cell (MSC) administration to augment neuroregeneration in both a sciatic nerve cut-and-repair and rat hindlimb transplant model. Bone marrow-derived MSCs were harvested and purified from Brown-Norway (BN) rats. Sciatic nerve transections and repairs were performed in three groups of Lewis (LEW) rats: negative controls (n = 4), local MSCs (epineural) injection (n = 4), and systemic MSCs (intravenous) injection (n = 4). Syngeneic (LEW-LEW) (n = 4) and allogeneic (BN-LEW) (n = 4) hindlimb transplants were performed and assessed for neuroregeneration after local or systemic MSC treatment. Rats undergoing sciatic nerve cut-and-repair and treated with either local or systemic injection of MSCs had significant improvement in the speed of recovery of compound muscle action potential amplitudes and axon counts when compared with negative controls. Similarly, rats undergoing allogeneic hindlimb transplants treated with local injection of MSCs exhibited significantly increased axon counts. Similarly, systemic MSC treatment resulted in improved nerve regeneration following allogeneic hindlimb transplants. Systemic administration had a more pronounced effect on electromotor recovery while local injection was more effective at increasing fiber counts, suggesting different targets of action. Local and systemic MSC injections significantly improve the pace and degree of nerve regeneration after nerve injury and hindlimb transplantation

In vitro studies of ICG loaded monocytes.

<p>(<i>A</i>, <i>B</i>) Representative images, RAW 264.7 mouse monocytes incubated in media alone and stained with DAPI. (<i>A</i>) Brightfield image. (<i>B</i>) DAPI blue nuclear staining and no NIR fluorescence (pseudocolor green). (<i>C</i>, <i>D</i>) Representative images, Monocytes after incubation in media with ICG solution and stained with DAPI. (<i>C</i>) Brightfield image. (<i>D</i>) DAPI fluorescence (blue) and NIR ICG fluorescence (pseudocolor green). (<i>E</i>) <i>Ex </i><i>vivo</i> cellular fluorescence over time. Monocytes incubated in media with ICG display an average NIR fluorescence of 5.22±0.34 arbitrary units (a.u.) above background (control non-ICG loaded cells) following the loading procedure, decreasing toward background over the following 12 hours (N=10). (<i>F</i>, <i>G</i>) Yield and viability of monocytes after ICG loading procedure. (<i>F</i>) Cell yield, after maintaining the cells <i>ex </i><i>vivo</i> in culture media and washing and centrifuging at each time point, decreases over the first 24 hours after loading with ICG. (<i>G</i>) Viability of cells remains greater than 80% for more than 12 hours (N=10). (<i>H</i>) ICG loaded monocyte chemotactic capacity. Chemotactic index is the number of cells migrating through the chemotaxis filter toward media with the chemoattractant MCP-1 relative to the number of cells migrating through the chemotaxis filter toward media alone. The average number of migrated ICG-loaded monocytes per five high powered fields was 86.0±27.8 with MCP-1 in the bottom well, and 48.9±16.5 with chemotaxis media alone in the bottom well (N=3). All error bars represent SEM. *<i>P</i><0.05.</p