MAB1435 Sigma-AldrichAnti-Macrophages/Monocytes Antibody, clone ED-1

Neuregulin-1 (NRG-1) has been shown to act as a neuroprotectant in animal models of nerve agent intoxication and other acute brain injuries. We recently demonstrated that NRG-1 blocked delayed neuronal death in rats intoxicated with the organophosphate (OP) neurotoxin diisopropylflurophosphate (DFP). It has been proposed that inflammatory mediators are involved in the pathogenesis of OP neurotoxin-mediated brain damage.We examined the influence of NRG-1 on inflammatory responses in the rat brain following DFP intoxication. Microglial activation was determined by immunohistchemistry using anti-CD11b and anti-ED1 antibodies. Gene expression profiling was performed with brain tissues using Affymetrix gene arrays and analyzed using the Ingenuity Pathway Analysis software. Cytokine mRNA levels following DFP and NRG-1 treatment was validated by real-time reverse transcription polymerase chain reaction (RT-PCR).DFP administration resulted in microglial activation in multiple brain regions, and this response was suppressed by treatment with NRG-1. Using microarray gene expression profiling, we observed that DFP increased mRNA levels of approximately 1,300 genes in the hippocampus 24 h after administration. NRG-1 treatment suppressed by 50% or more a small fraction of DFP-induced genes, which were primarily associated with inflammatory responses. Real-time RT-PCR confirmed that the mRNAs for pro-inflammatory cytokines interleukin-1β (IL-1β) and interleukin-6 (IL-6) were significantly increased following DFP exposure and that NRG-1 significantly attenuated this transcriptional response. In contrast, tumor necrosis factor α (TNFα) transcript levels were unchanged in both DFP and DFP + NRG-1 treated brains relative to controls.Neuroprotection by NRG-1 against OP neurotoxicity is associated with the suppression of pro-inflammatory responses in brain microglia. These findings provide new insight regarding the molecular mechanisms involved in the neuroprotective role of NRG-1 in acute brain injuries.

Deep brain stimulation (DBS) of the superolateral branch of the medial forebrain bundle (MFB) has provided rapid and dramatic reduction of depressive symptoms in a clinical trial. Early intracranial self-stimulation experiments of the MFB suggested detrimental side effects on the animals' health; therefore, the current study looked at the viability of chronic and continuous MFB-DBS in rodents, with particular attention given to welfare issues and identification of stimulated pathways.Sprague-Dawley female rats were submitted to stereotactic microelectrode implantation into the MFB. Chronic continuous DBS was applied for 3-6 weeks. Welfare monitoring and behavior changes were assessed. Postmortem histological analysis of c-fos protein expression was carried out.MFB-DBS resulted in mild and temporary weight loss in the animals, which was regained even with continuing stimulation. MFB-DBS led to increased and long-lasting c-fos expression in target regions of the mesolimbic/mesocortical system.Bilateral continuous chronic MFB-DBS is feasible, safe, and without impact on the rodent's health. MFB-DBS results in temporary increase in exploration, which could explain the initial weight loss, and does not produce any apparent behavioral abnormalities. This platform represents a powerful tool for further preclinical investigation of the MFB stimulation in the treatment of depression.

Pt/Ir electrodes have been extensively used in neurophysiology research in recent years as they provide a more inert recording surface as compared to tungsten or stainless steel. While floating microelectrode arrays (FMA) consisting of Pt/Ir electrodes are an option for neuroprosthetic applications, long-term in vivo functional performance characterization of these FMAs is lacking. In this study, we have performed comprehensive abiotic-biotic characterization of Pt/Ir arrays in 12 rats with implant periods ranging from 1 week up to 6 months. Each of the FMAs consisted of 16-channel, 1.5 mm long, and 75 μm diameter microwires with tapered tips that were implanted into the somatosensory cortex. Abiotic characterization included (1) pre-implant and post-explant scanning electron microscopy (SEM) to study recording site changes, insulation delamination and cracking, and (2) chronic in vivo electrode impedance spectroscopy. Biotic characterization included study of microglial responses using a panel of antibodies, such as Iba1, ED1, and anti-ferritin, the latter being indicative of blood-brain barrier (BBB) disruption. Significant structural variation was observed pre-implantation among the arrays in the form of irregular insulation, cracks in insulation/recording surface, and insulation delamination. We observed delamination and cracking of insulation in almost all electrodes post-implantation. These changes altered the electrochemical surface area of the electrodes and resulted in declining impedance over the long-term due to formation of electrical leakage pathways. In general, the decline in impedance corresponded with poor electrode functional performance, which was quantified via electrode yield. Our abiotic results suggest that manufacturing variability and insulation material as an important factor contributing to electrode failure. Biotic results show that electrode performance was not correlated with microglial activation (neuroinflammation) as we were able to observe poor performance in the absence of neuroinflammation, as well as good performance in the presence of neuroinflammation. One biotic change that correlated well with poor electrode performance was intraparenchymal bleeding, which was evident macroscopically in some rats and presented microscopically by intense ferritin immunoreactivity in microglia/macrophages. Thus, we currently consider intraparenchymal bleeding, suboptimal electrode fabrication, and insulation delamination as the major factors contributing toward electrode failure.

Numerous studies have reported a beneficial impact of neural progenitor cell transplantation on functional outcome after traumatic brain injury (TBI) during short and medium follow-up periods. However, our knowledge regarding long-term functional effects is fragmentary while a direct comparison between local and systemic transplantation is missing so far.This study investigated the long-term (12 week) impact of human fetal neuronal progenitor cell (hNPC) transplantation 24 h after severe TBI in rats.Cells were either transplanted stereotactically (1 × 10(5)) into the putamen or systemically (5 × 10(5)) via the tail vein. Control animals received intravenous transplantation of vehicle solution.An overall functional benefit was observed after systemic, but not local hNPC transplantation by area under the curve analysis (p less than 0.01). Surprisingly, this effect vanished during later stages after TBI with all groups exhibiting comparable functional outcomes 84 days after TBI. Investigation of cell-mediated inflammatory processes revealed increasing microglial activation and macrophage presence during these stages, which was statistically significant after systemic cell administration (p less than 0.05). Intracerebral hNPC transplantation slightly diminished astrogliosis in perilesional areas (p less than 0.01), but did not translate into a permanent functional benefit. No significant effects on angiogenesis were observed among the groups.Our results suggest the careful long-term assessment of cell therapies for TBI, as well as to identify potential long-term detrimental effects of such therapies before moving on to clinical trials. Moreover, immunosuppressive protocols, though widely used, should be rigorously assessed for their applicability in the respective setup.

Patients with osteoporosis who receive tooth extractions are typically on either oral bisphosphonate or parathyroid hormone (PTH) therapy. Currently, the consequence of these therapies on hard- and soft-tissue healing in the oral cavity is not clearly defined. The aim of this study is to determine the differences in the therapeutic effect on tooth-extraction wound healing between bisphosphonate and PTH therapies.Maxillary second molars were extracted in Sprague Dawley rats (n = 30), and either bisphosphonate (zoledronate [Zol]), PTH, or saline (vehicle control [VC]) was administered for 10 days (n = 10 per group). Hard-tissue healing was evaluated by microcomputed tomography and histomorphometric analyses. Collagen, blood vessels, inflammatory cell infiltration, and cathepsin K expression were assessed in soft tissue using immunohistochemistry, quantitative polymerase chain reaction, and immunoblotting.Both therapies significantly increased bone fill and suppressed vertical bone loss. However, considerably more devital bone was observed in the sockets of rats on Zol versus VC. Although Zol increased the numbers of blood vessels, the total blood vessel area in soft tissue was significantly smaller than in VC. PTH therapy increased osteoblastic bone formation and suppressed osteoclasts. PTH therapy promoted soft-tissue maturation by suppressing inflammation and stimulating collagen deposition.Zoledronate therapy deters whereas PTH therapy promotes hard- and soft-tissue healing in the oral cavity, and both therapies prevent vertical bone loss.

To investigate whether the P2X₇ receptor is involved in retinal ganglion cell (RGC) death after the intraocular pressure (IOP) is elevated in rats.After the IOP was elevated to 90 mmHg for 1 h, the rats were subsequently administered oxidized adenosine triphosphate (OxATP) and brilliant blue G (BBG) as P2X₇ antagonists. The rats were euthanized 7 days after IOP elevation for histologic evaluation and at 1, 3, and 7 days after IOP elevation to immunostain for the P2X₇ receptor and neuron-specific class III β-tubulin in the retina. Changes in P2X₇ receptor expression were measured in total retina extracts using western blot analysis. Quantitative real-time PCR was also performed using the entire retina to determine whether the P2X₇ receptor is involved in upregulating tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 at 1, 2, and 3 days after the IOP was elevated.RGC density and the inner plexiform layer thickness significantly decreased 7 days after IOP elevation, but were dose-dependently preserved when treated with OxATP or BBG. P2X₇ immunoreactivity in the RGCs increased after IOP elevation, with the peak occurring from day 1 through day 3. Protein levels of P2X₇ receptor were significantly increased 1, 2, and 3 days after IOP elevation. The messenger ribonucleic acid expression of the P2X₇ receptor, TNF-α, IL-1β, and IL-6 was significantly upregulated in the retina after IOP elevation, and was suppressed by treatment with OxATP.These results suggest the expression of the P2X₇ receptor is upregulated in the retina after IOP elevation, leading to RGC death. Upregulation of TNF-α, IL-1β, and IL-6 might be involved in this mechanism of RGC death. Furthermore, P2X₇ antagonists may prevent RGC death after IOP elevation.

Bladder sensation is mediated by lumbosacral dorsal root ganglion neurons and is essential for normal voiding and nociception. Numerous electrophysiological, structural, and molecular changes occur in these neurons following inflammation. Defining which neurons undergo these changes is critical for understanding the mechanism underlying bladder pain and dysfunction. Our first aim was to define the chemical classes of bladder sensory neurons that express receptors for the endogenous modulators of nociceptor sensitivity, glial cell line-derived neurotrophic factor (GDNF), the related neurotrophic factor, artemin, and estrogens. Bladder sensory neurons of adult female Sprague-Dawley rats were identified with retrograde tracer. Diverse groups of neurons express these receptors, and some neurons express receptors for both neurotrophic factors and estrogens. Lumbar and sacral sensory neurons showed some distinct differences in their expression profile. We also distinguished the chemical profile of myelinated and unmyelinated bladder sensory neurons. Our second aim was to identify bladder sensory neurons likely to be undergoing structural remodeling during inflammation. Following systemic administration of cyclophosphamide (CYP), its renal metabolite acrolein causes transient urothelial loss, exposing local afferent terminals to a toxic environment. CYP induced expression of the injury-related immediate-early gene product, activating transcription factor-3 (ATF-3), in a small population of sacral nitrergic bladder sensory neurons. In conclusion, we have defined the bladder sensory neurons that express receptors for GDNF, artemin and estrogens. Our study has also identified a sub-population of sacral sensory neurons that are likely to be undergoing structural remodeling during acute inflammation of the bladder. Together these results contribute to increased understanding of the neurons that are known to be involved in pain modulation and hyperreflexia during inflammation.

We examined the relationship of musculoskeletal risk factors underlying force and repetition on tissue responses in an operant rat model of repetitive reaching and pulling, and if force x repetition interactions were present, indicative of a fatigue failure process. We examined exposure-dependent changes in biochemical, morphological and sensorimotor responses occurring with repeated performance of a handle-pulling task for 12 weeks at one of four repetition and force levels: 1) low repetition with low force, 2) high repetition with low force, 3) low repetition with high force, and 4) high repetition with high force (HRHF).Rats underwent initial training for 4-6 weeks, and then performed one of the tasks for 12 weeks, 2 hours/day, 3 days/week. Reflexive grip strength and sensitivity to touch were assayed as functional outcomes. Flexor digitorum muscles and tendons, forelimb bones, and serum were assayed using ELISA for indicators of inflammation, tissue stress and repair, and bone turnover. Histomorphometry was used to assay macrophage infiltration of tissues, spinal cord substance P changes, and tissue adaptative or degradative changes. MicroCT was used to assay bones for changes in bone quality.Several force x repetition interactions were observed for: muscle IL-1alpha and bone IL-1beta; serum TNFalpha, IL-1alpha, and IL-1beta; muscle HSP72, a tissue stress and repair protein; histomorphological evidence of tendon and cartilage degradation; serum biomarkers of bone degradation (CTXI) and bone formation (osteocalcin); and morphological evidence of bone adaptation versus resorption. In most cases, performance of the HRHF task induced the greatest tissue degenerative changes, while performance of moderate level tasks induced bone adaptation and a suggestion of muscle adaptation. Both high force tasks induced median nerve macrophage infiltration, spinal cord sensitization (increased substance P), grip strength declines and forepaw mechanical allodynia by task week 12.Although not consistent in all tissues, we found several significant interactions between the critical musculoskeletal risk factors of force and repetition, consistent with a fatigue failure process in musculoskeletal tissues. Prolonged performance of HRHF tasks exhibited significantly increased risk for musculoskeletal disorders, while performance of moderate level tasks exhibited adaptation to task demands.

There is a major need for scaffold-based tissue engineered vascular grafts and heart valves with long-term patency and durability to be used in diabetic cardiovascular patients. We hypothesized that diabetes, by virtue of glycoxidation reactions, can directly crosslink implanted scaffolds, drastically altering their properties. In order to investigate the fate of tissue engineered scaffolds in diabetic conditions, we prepared valvular collagen scaffolds and arterial elastin scaffolds by decellularization and implanted them subdermally in diabetic rats. Both types of scaffolds exhibited significant levels of advanced glycation end products (AGEs), chemical crosslinking and stiffening -alterations which are not favorable for cardiovascular tissue engineering. Pre-implantation treatment of collagen and elastin scaffolds with penta-galloyl glucose (PGG), an antioxidant and matrix-binding polyphenol, chemically stabilized the scaffolds, reduced their enzymatic degradation, and protected them from diabetes-related complications by reduction of scaffold-bound AGE levels. PGG-treated scaffolds resisted diabetes-induced crosslinking and stiffening, were protected from calcification, and exhibited controlled remodeling in vivo, thereby supporting future use of diabetes-resistant scaffolds for cardiovascular tissue engineering in patients with diabetes.

We used morphological, immunohistochemical and functional assessments to determine the impact of genetically-modified peripheral nerve (PN) grafts on axonal regeneration after injury. Grafts were assembled from acellular nerve sheaths repopulated ex vivo with Schwann cells (SCs) modified to express brain-derived neurotrophic factor (BDNF), a secretable form of ciliary neurotrophic factor (CNTF), or neurotrophin-3 (NT3). Grafts were used to repair unilateral 1 cm defects in rat peroneal nerves and 10 weeks later outcomes were compared to normal nerves and various controls: autografts, acellular grafts and grafts with unmodified SCs. The number of regenerated βIII-Tubulin positive axons was similar in all grafts with the exception of CNTF, which contained the fewest immunostained axons. There were significantly lower fiber counts in acellular, untransduced SC and NT3 groups using a PanNF antibody, suggesting a paucity of large caliber axons. In addition, NT3 grafts contained the greatest number of sensory fibres, identified with either IB4 or CGRP markers. Examination of semi- and ultra-thin sections revealed heterogeneous graft morphologies, particularly in BDNF and NT3 grafts in which the fascicular organization was pronounced. Unmyelinated axons were loosely organized in numerous Remak bundles in NT3 grafts, while the BDNF graft group displayed the lowest ratio of umyelinated to myelinated axons. Gait analysis revealed that stance width was increased in rats with CNTF and NT3 grafts, and step length involving the injured left hindlimb was significantly greater in NT3 grafted rats, suggesting enhanced sensory sensitivity in these animals. In summary, the selective expression of BDNF, CNTF or NT3 by genetically modified SCs had differential effects on PN graft morphology, the number and type of regenerating axons, myelination, and locomotor function.