Preparation and Activity of Hemostatic and Antibacterial Dressings with Greige Cotton/Zeolite Formularies Having Silver and Ascorbic Acid Finishes.

The need for prehospital hemostatic dressings that exert an antibacterial effect is of interest for prolonged field care. Here, we consider a series of antibacterial and zeolite formulary treatment approaches applied to a cotton-based dressing. The design of the fabric formulations was based on the hemostatic dressing TACGauze with zeolite Y incorporated as a procoagulant with calcium and pectin to facilitate fiber adherence utilizing silver nanoparticles, and cellulose-crosslinked ascorbic acid to confer antibacterial activity. Infra-red spectra were employed to characterize the chemical modifications on the dressings. Contact angle measurements were employed to document the surface hydrophobicity of the cotton fabric which plays a role in the contact activation of the coagulation cascade. Ammonium Y zeolite-treated dressings initiated fibrin equal to the accepted standard hemorrhage control dressing and showed similar improvement with antibacterial finishes. The antibacterial activity of cotton-based technology utilizing both citrate-linked ascorbate-cellulose conjugate analogs and silver nanoparticle-embedded cotton fibers was observed against Staphylococcus aureus and Klebsiella pneumoniae at a level of 99.99 percent in the AATCC 100 assay. The hydrogen peroxide levels of the ascorbic acid-based fabrics, measured over a time period from zero up to forty-eight hours, were in line with the antibacterial activities.

Selective inhibition of soluble TNF using XPro1595 relieves pain and attenuates cerulein-induced pathology in mice.

BACKGROUND: Symptoms associated with acute pancreatitis can be debilitating, and treatment remains a challenge. This study aimed to investigate the efficacy of selectively inhibiting the soluble form of TNF (solTNF) using the biologic XPro1595 in a mouse model of acute pancreatitis. METHODS: Acute pancreatitis was induced in adult male C57Bl/6J mice by administering cerulein (8 injections of 50 µg/kg I.P., spaced an hour apart), with XPro1595 (10 mg/kg, S.C.) or vehicle being administered approximately 18 h after the last injection. Serum was collected 6 or 18 h after the last cerulein injection, pancreatic tissue was collected 2 and 7 days post-induction, and brain hippocampal tissue was collected at 7 days post-induction. The animal's pain level was assessed 3, 5 and 7 days post-induction. RESULTS: The induction of acute pancreatitis promoted a strong increase in serum amylase levels, which had receded back to baseline levels by the next morning. XPro1595 treatment began after amylase levels had subsided at 18 h, and prevented pancreatic immune cell infiltration, that subsequently prevented tissue disruption and acinar cell death. These improvements in pathology were associated with a significant reduction in mechanical hypersensitivity (neuropathic pain). XPro1595 treatment also prevented an increase in hippocampal astrocyte reactivity, that may be associated with the prevention of neuropathic pain in this mouse model. CONCLUSION: Overall, we observed that selectively inhibiting solTNF using XPro1595 improved the pathophysiological and neurological sequelae of cerulein-induced pancreatitis in mice, which provides support of its use in patients with pancreatitis.

The Rho kinase pathway regulates mouse adult neural precursor cell migration.

Adult neural precursor cells (NPCs) in the subventricular zone (SVZ) normally migrate via the rostral migratory stream (RMS) to the olfactory bulb (OB). Following neural injury, they also migrate to the site of damage. This study investigated the role of Rho-dependent kinase (ROCK) on the migration of NPCs in vitro and in vivo. In vitro, using neurospheres or SVZ explants, inhibition of ROCK using Y27632 promoted cell body elongation, process protrusion, and migration, while inhibiting NPC chain formation. It had no effect on proliferation, apoptosis, or differentiation. Both isoforms of ROCK were involved. Using siRNA, knockdown of both ROCK1 and ROCK2 was required to promote NPC migration and morphological changes; knockdown of ROCK2 alone was partially effective, with little/no effect of knockdown of ROCK1 alone. In vivo, infusion of Y27632 plus Bromodeoxyuridine (BrdU) into the lateral ventricle for 1 week reduced the number of BrdU-labeled NPCs in the OB compared with BrdU infusion alone. However, ROCK inhibition did not affect the tangential-to-radial switch of NPC migration, as labeled cells were present in all OB layers. The decrease in NPC number at the OB was not attributed to a decrease in NPCs at the SVZ. However, ROCK inhibition decreased the density of BrdU-labeled cells in the RMS and increased the distribution of these cells to ectopic brain regions, such as the accessory olfactory nucleus, where the majority differentiated into neurons. These findings suggest that ROCK signaling regulates NPC migration via regulation of cell-cell contact and chain migration.

Species differences in reactivity of mouse and rat astrocytes in vitro.

Reactive astrogliosis constitutes a major obstacle to neuronal regeneration and is characterized by rearrangement and upregulation of expression of cytoskeletal proteins, increased proliferation and hypertrophy. Many approaches have been attempted to mimic astrogliosis by inducing reactive astrocytes in vitro. Such research is usually performed using astrocytes derived from Mus musculus or Rattus norvegicus, and results compared between species on the assumption that these cells behave equivalently. Therefore, we compared reactivity between mouse and rat astrocytes in scratch wound assays to gain further insight into how comparable these cell culture models are. Proliferation and migration, as well as expression of the cytoskeletal proteins glial fibrillary acidic protein (GFAP) and vimentin, were compared by immunocytochemistry and immunoblot. Further, we investigated migration of proliferating cells by 5-ethynyl-2'-deoxyuridine staining. Substantial differences in GFAP expression and proliferation between astrocytes of the two species were found: rat astrocytes showed different cytoskeletal morphology, expressed significantly more GFAP and vimentin of different molecular size and were more proliferative than comparable mouse astrocytes. Our results suggest that rat and mouse astrocytes may respond differently to various reactivity-triggering stimuli, which needs to be considered when general conclusions are drawn regarding effects of factors regulating astrocyte reactivity.

Pathophysiology of Traumatic Brain Injury.

Traumatic brain injury (TBI) has become the signature injury of the military conflict in Iraq and Afghanistan and also has a high rate of occurrence in civilian populations in the United States. Although the effects of a moderate to severe brain injury have been investigated for decades, the chronic effects of single and repetitive mild TBI are just beginning to be investigated. Data suggest that the different types and severities of TBI have unique long-term outcomes and thus may represent different types of diseases. Therefore, this review outlines the causes, incidence, symptoms, and pathophysiology of mild, moderate, and severe TBI.

Explant Methodology for Analyzing Neuroblast Migration.

The subventricular zone (SVZ) in the mammalian forebrain contains stem/progenitor cells that migrate through the rostral migratory stream (RMS) to the olfactory bulb throughout adulthood. SVZ-derived explant cultures provide a convenient method to assess factors regulating the intermediary stage of neural stem/progenitor cell migration. Here, we describe the isolation of SVZ-derived RMS explants from the neonatal mouse brain, and the conditions required to culture and evaluate their migration.

EphrinB3 restricts endogenous neural stem cell migration after traumatic brain injury.

Traumatic brain injury (TBI) leads to a series of pathological events that can have profound influences on motor, sensory and cognitive functions. Conversely, TBI can also stimulate neural stem/progenitor cell proliferation leading to increased numbers of neuroblasts migrating outside their restrictive neurogenic zone to areas of damage in support of tissue integrity. Unfortunately, the factors that regulate migration are poorly understood. Here, we examine whether ephrinB3 functions to restrict neuroblasts from migrating outside the subventricular zone (SVZ) and rostral migratory stream (RMS). We have previously shown that ephrinB3 is expressed in tissues surrounding these regions, including the overlying corpus callosum (CC), and is reduced after controlled cortical impact (CCI) injury. Our current study takes advantage of ephrinB3 knockout mice to examine the influences of ephrinB3 on neuroblast migration into CC and cortex tissues after CCI injury. Both injury and/or ephrinB3 deficiency led to increased neuroblast numbers and enhanced migration outside the SVZ/RMS zones. Application of soluble ephrinB3-Fc molecules reduced neuroblast migration into the CC after injury and limited neuroblast chain migration in cultured SVZ explants. Our findings suggest that ephrinB3 expression in tissues surrounding neurogenic regions functions to restrict neuroblast migration outside the RMS by limiting chain migration.

Endogenous neural stem/progenitor cells stabilize the cortical microenvironment after traumatic brain injury.

Although a myriad of pathological responses contribute to traumatic brain injury (TBI), cerebral dysfunction has been closely linked to cell death mechanisms. A number of therapeutic strategies have been studied in an attempt to minimize or ameliorate tissue damage; however, few studies have evaluated the inherent protective capacity of the brain. Endogenous neural stem/progenitor cells (NSPCs) reside in distinct brain regions and have been shown to respond to tissue damage by migrating to regions of injury. Until now, it remained unknown whether these cells have the capacity to promote endogenous repair. We ablated NSPCs in the subventricular zone to examine their contribution to the injury microenvironment after controlled cortical impact (CCI) injury. Studies were performed in transgenic mice expressing the herpes simplex virus thymidine kinase gene under the control of the nestin(δ) promoter exposed to CCI injury. Two weeks after CCI injury, mice deficient in NSPCs had reduced neuronal survival in the perilesional cortex and fewer Iba-1-positive and glial fibrillary acidic protein-positive glial cells but increased glial hypertrophy at the injury site. These findings suggest that the presence of NSPCs play a supportive role in the cortex to promote neuronal survival and glial cell expansion after TBI injury, which corresponds with improvements in motor function. We conclude that enhancing this endogenous response may have acute protective roles after TBI.

EphA4 receptor tyrosine kinase is a modulator of onset and disease severity of experimental autoimmune encephalomyelitis (EAE).

The EphA4 receptor tyrosine kinase is a major regulator of axonal growth and astrocyte reactivity and is a possible inflammatory mediator. Given that multiple sclerosis (MS) is primarily an inflammatory demyelinating disease and in mouse models of MS, such as experimental autoimmune encephalomyelitis (EAE), axonal degeneration and reactive gliosis are prominent clinical features, we hypothesised that endogenous EphA4 could play a role in modulating EAE. EAE was induced in EphA4 knockout and wildtype mice using MOG peptide immunisation and clinical severity and histological features of the disease were then compared in lumbar spinal cord sections. EphA4 knockout mice exhibited a markedly less severe clinical course than wildtype mice, with a lower maximum disease grade and a slightly later onset of clinical symptoms. Numbers of infiltrating T cells and macrophages, the number and size of the lesions, and the extent of astrocytic gliosis were similar in both genotypes; however, EphA4 knockout mice appeared to have decreased axonal pathology. Blocking of EphA4 in wildtype mice by administration of soluble EphA4 (EphA4-Fc) as a decoy receptor following induction of EAE produced a delay in onset of clinical symptoms; however, most mice had clinical symptoms of similar severity by 22 days, indicating that EphA4 blocking treatment slowed early EAE disease evolution. Again there were no apparent differences in histopathology. To determine whether the role of EphA4 in modulating EAE was CNS mediated or due to an altered immune response, MOG primed T cells from wildtype and EphA4 knockout mice were passively transferred into naive recipient mice and both were shown to induce disease of equivalent severity. These results are consistent with a non-inflammatory, CNS specific, deleterious effect of EphA4 during neuroinflammation that results in axonal pathology.

Partial change in EphA4 knockout mouse phenotype: loss of diminished GFAP upregulation following spinal cord injury.

In a previous study we found that the EphA4 receptor inhibits regeneration following spinal cord injury by blocking regrowth of axons and regulation of astrocyte reactivity. In our original studies using EphA4 null mice [Goldshmit et al., J. Neurosci., 2004] we found attenuated astrocyte reactivity following spinal cord injury. Several other studies have now supported the role of EphA4 in regulating neural regeneration but a recent study [Herrmann et al., Exp. Neurol., 2010] did not find an effect of EphA4 on astrocyte reactivity. Re-examination of astrocytic gliosis following injury in our current cohort of EphA4 null mice revealed that they no longer showed attenuation of astrocyte reactivity, however other EphA4 null mouse phenotypes, such as decreased size of the dorsal funiculus were unaltered. We hypothesised that long-term breeding on the C57Bl/6 background may influence the EphA4-mediated astrocyte phenotype and compared astrocytic gliosis at 4 days following spinal cord injury in wildtype and EphA4 null mice on the C57Bl/6 background and backcrossed C57Bl/6×129Sv(F2) mice, as well as wildtype 129Sv mice. 129Sv mice had increased GFAP expression and increased numbers of reactive GFAP astrocytes compared to C57Bl/6 mice. There was no significant effect of EphA4 deletion on GFAP expression in C57Bl/6 mice or the F2 crosses other than a moderately decreased number of EphA4 null astrocytes in C57Bl/6 mice using one of two antibodies. Therefore, there has been an apparent change in EphA4-mediated astroglial phenotype associated with long term breeding of the EphA4 colony but it does not appear to be influenced by background mouse strain.

Differential expression of TrkB isoforms switches climbing fiber-Purkinje cell synaptogenesis to selective synapse elimination.

Correct neural function depends on precisely organized connectivity, which is refined from broader projections through synaptic/collateral elimination. In the rat, olivocerebellar topography is refined by regression of multiple climbing fiber (CF) innervation of Purkinje cells (PC) during the first two postnatal weeks. The molecules that initiate this regression are not fully understood. We assessed the role of cerebellar neurotrophins by examining tropomycin receptor kinase (Trk) receptor expression in the inferior olive and cerebellum between postnatal days (P)3-7, when CF-PC innervation changes from synapse formation to selective synapse elimination, and in a denervation-reinnervation model when synaptogenesis is delayed. Trks A, B, and C are expressed in olivary neurons; although TrkA was not transported to the cerebellum and TrkC was unchanged during innervation and reinnervation, suggesting that neither receptor is involved in CF-PC synaptogenesis. In contrast, both total and truncated TrkB (TrkB.T) increased in the olive and cerebellum from P4, whereas full-length and activated phosphorylated TrkB (phospho-TrkB) decreased from P4-5. This reveals less TrkB signaling at the onset of CF regression. This expression pattern was reproduced during CF-PC reinnervation: in the denervated hemicerebellum phospho-TrkB decreased as CF terminals degenerated, then increased in parallel with the delayed neosynaptogenesis as new CFs reinnervated the denervated hemicerebellum. In the absence of this signaling, CF reinnervation did not develop. Our data reveal that olivocerebellar TrkB activity parallels CF-PC synaptic formation and stabilization and is required for neosynaptogenesis. Furthermore, TrkB.T expression rises to reduce TrkB signaling and permit synapse elimination.

Brain-derived neurotrophic factor induces post-lesion transcommissural growth of olivary axons that develop normal climbing fibers on mature Purkinje cells.

In the adult mammalian central nervous system, reinnervation and recovery from trauma is limited. During development, however, post-lesion plasticity may generate alternate paths providing models to investigate factors that promote reinnervation to appropriate targets. Following unilateral transection of the neonatal rat olivocerebellar pathway, axons from the remaining inferior olive reinnervate the denervated hemicerebellum and develop climbing fiber arbors on Purkinje cells. However, the capacity to recreate this accurate target reinnervation in a mature system remains unknown. In rats lesioned on day 15 (P15) or 30 and treated with intracerebellar injection of brain-derived neurotrophic factor (BDNF) or vehicle 24 h later, the morphology and organisation of transcommissural olivocerebellar reinnervation was examined using neuronal tracing and immunohistochemistry. In all animals BDNF, but not vehicle, induced transcommissural olivocerebellar axonal growth into the denervated hemicerebellum. The distribution of reinnervating climbing fibers was not confined to the injection sites but extended throughout the denervated hemivermis and, less densely, up to 3.5 mm into the hemisphere. Transcommissural olivocerebellar axons were organised into parasagittal microzones that were almost symmetrical to those in the right hemicerebellum. Reinnervating climbing fiber arbors were predominantly normal, but in the P30-lesioned group 10% were either branched within the molecular layer forming a smaller secondary arbor or were less branched, and in the P15 lesion group the reinnervating arbors extended their terminals almost to the pial surface and were larger than control arbors (P < 0.02). These results show that BDNF can induce transcommissural olivocerebellar reinnervation, which resembles developmental neuroplasticity to promote appropriate target reinnervation in a mature environment.

Post-lesion transcommissural olivocerebellar reinnervation improves motor function following unilateral pedunculotomy in the neonatal rat.

In the adult mammalian central nervous system, reinnervation and recovery from trauma are limited. During development, however, post-lesion plasticity may generate alternate paths providing models to investigate reinnervation and repair. Sometimes, these paths are maladaptive, although the relationship between dysfunction and anatomical abnormality remains unknown. After unilateral transection of the neonatal rat olivocerebellar path (pedunculotomy), axons from the remaining inferior olive reinnervate Purkinje cells in the denervated hemicerebellum with appropriate topography and synaptic function. However, whether this new pathway confers beneficial behavioural effects remains unknown. We studied the behavioural sequelae in rats with and without transcommissural reinnervation using righting and vestibular-drop reflexes, simple locomotion (bridge), complex locomotion (wire) and motor coordination (rotarod) tests. In animals pedunculotomised on day 3 (Px3), which develop olivocerebellar reinnervation, dynamic postural adjustments and complex motor skills develop normally, whereas simple gait is broad-based and slightly delayed. In contrast, Px11 animals, which do not develop reinnervation, have delayed maturation of postural reflexes, gait and complex locomotor skills. In addition, when compared to control animals, their performance in locomotory tasks was slower and the complex task impaired. On the rotarod, control and Px3 animals learned to coordinate their gait and walked for longer at 10 and 20 rpm than Px11 animals. These results show that transcommissural olivocerebellar reinnervation is associated with almost normal motor development and the ability to synchronise gait at slow and moderate speeds, i.e. this reinnervation confers significant behavioural function and is therefore truly compensatory.