Positive Fibers Research Articles

Electroacupuncture Zusanli (ST36) Relieves Somatic Pain in Colitis Rats by Inhibiting Dorsal Root Ganglion Sympathetic-Sensory Coupling and Neurogenic Inflammation.

Referred somatic pain triggered by hyperalgesia is common in patients with inflammatory bowel disease (IBD). It was reported that sprouting of sympathetic nerve fibers into the dorsal root ganglion (DGR) and neurogenic inflammation were related to neuropathic pain, the excitability of neurons, and afferents. The purpose of the study was to explore the potential and mechanism of electroacupuncture (EA) at Zusanli (ST36) for the intervention of colon inflammation and hyperalgesia. Sprague-Dawley (SD) was randomly divided into four groups, including control, model, EA, and sham-EA. Our results showed EA treatment significantly attenuated dextran sulfate sodium- (DSS-) induced colorectal lesions and inflammatory cytokine secretion, such as TNF-α, IL-1β, PGE2, and IL-6. EA also inhibited mechanical and thermal pain hypersensitivities of colitis rats. Importantly, EA effectively abrogated the promotion effect of DSS on ipsilateral lumbar 6 (L6) DRG sympathetic-sensory coupling, manifested as the sprouting of tyrosine hydroxylase- (TH-) positive sympathetic fibers into sensory neurons and colocalization of and calcitonin gene-related peptide (CGRP). Furthermore, EA at Zusanli (ST36) activated neurogenic inflammation, characterized by decreased expression of substance P (SP), hyaluronic acid (HA), bradykinin (BK), and prostacyclin (PGI2) in colitis rat skin tissues corresponding to the L6 DRG. Mechanically, EA treatment reduced the activation of the TRPV1/CGRP, ERK, and TLR4 signaling pathways in L6 DRG of colitis rats. Taken together, we presumed that EA treatment improved colon inflammation and hyperalgesia, potentially by suppressing the sprouting of sympathetic nerve fibers into the L6 DGR and neurogenic inflammation via deactivating the TRPV1/CGRP, ERK, and TLR4 signaling pathways.

Rodent behavior following a dural inflammation model with anti-CGRP migraine medication treatment.

Migraine is a widespread and prevalent disease with a complex pathophysiology, of which neuroinflammation and increased pain sensitivity have been suggested to be involved. Various studies have investigated the presence of different inflammatory markers in migraineurs and investigated the role of inflammation in inflammatory models with complete Freund's adjuvant (CFA) or inflammatory soup added to the dura mater. The aim of the current study was to examine whether application of CFA to the dura mater would cause behavioral alterations that are migraine relevant. In addition, we investigated the potential mitigating effects of fremanezumab, a CGRP (calcitonin gene-related peptide) specific antibody, following CFA application. Male Sprague-Dawley rats were randomly divided into six groups: fresh (n = 7), fresh + carprofen (n = 6), fresh + anti-CGRP (n = 6), sham (n = 7), CFA (n = 16), CFA + anti-CGRP (n = 8). CFA was applied for 15 min on a 3 × 3 mm clearing of the skull exposing the dura mater of male Sprague-Dawley rats. We applied the Light/Dark box and Open Field test, combined with the electronic von Frey test to evaluate outcomes. Finally, we observed CGRP immunoreactivity in the trigeminal ganglion. No differences were observed in the Light/Dark box test. The Open Field test detected behavior differences, notably that sham rats spend less time in the central zone, reared less and groomed more than fresh + carprofen rats. The other groups were not significantly different compared to sham rats, indicating that activation of the TGVS is present in sham surgery and cannot be exacerbated by CFA. However, for the allodynia, we observed specific periorbital sensitization, not observed in the sham animals. This could not be mitigated by fremanezumab, although it clearly reduced the amount of CGRP positive fibers. CFA surgically administered to the dura causes periorbital allodynia and increases CGRP positive fibers in the trigeminal ganglion. Fremanezumab does not reduce periorbital allodynia even though it reduces CGRP positive fibers in the TG. Further work is needed to investigate whether CFA administered to the dura could be used as a non-CGRP inflammatory migraine model.

Bioactive self-healing hydrogel based on tannic acid modified gold nano-crosslinker as an injectable brain implant for treating Parkinson’s disease

BackgroundParkinson’s disease (PD) is one of the most common long-term neurodegenerative diseases. Current treatments for PD are mostly based on surgery and medication because of the limitation and challenges in selecting proper biomaterials. In this study, an injectable bioactive hydrogel based on novel tannic acid crosslinker was developed to treat PD.MethodsThe oxidized tannic acid modified gold nano-crosslinker was synthesized and used to effectively crosslink chitosan for preparation of the bioactive self-healing hydrogel. The crosslinking density, conductivity, self-healing ability, and injectability of the hydrogel were characterized. Abilities of the hydrogel to promote the proliferation and differentiation of neural stem cells (NSCs) were assessed in vitro. Anti-inflammatory property was analyzed on J774A.1 macrophages. The hydrogel was injected in the PD rat model for evaluation of the motor function recovery, electrophysiological performance improvement, and histological repair.ResultsThe hydrogel exhibited self-healing property and 34G (~ 80 μm) needle injectability. NSCs grown in the hydrogel displayed long-term proliferation and differentiation toward neurons in vitro. Besides, the hydrogel owned strong anti-inflammatory and antioxidative capabilities to rescue inflamed NSCs (~ 90%). Brain injection of the bioactive hydrogel recovered the motor function of PD rats. Electrophysiological measurements showed evident alleviation of irregular discharge of nerve cells in the subthalamic nucleus of PD rats administered with the hydrogel. Histological examination confirmed that the hydrogel alone significantly increased the density of tyrosine hydroxylase positive neurons and fibers as well as reduced inflammation, with a high efficacy similar to drug-loaded hydrogel.ConclusionThe new bioactive hydrogel serves as an effective brain injectable implant to treat PD and a promising biomaterial for developing novel strategies to treat brain diseases.Graphical

Structure and Function Adaption of Lung in Phrynocephalus vlangalii by Multiple Mechanisms

Respiration and water-liquid transportation are controlled by many factors in the lung. The aim of this study was to explore the structure and proteins expression in lungs of Phrynocephalus vlangalii by means of gross anatomy, light microscope observation, scanning electron microscope and immunohistochemistry. Results show that there were many alveoli in the lung and the walls of alveoli and capillaries were very thin. The inner surface of the lung was divided into many cystic chambers by reticular diaphragm, and the network of pulmonary capillaries was dense. Immunohistochemistry showed that AQP1 was mainly expressed in the epithelium of interstitial bronchi, parabronchiole endothelium, capillary endothelium and alveolar epithelial cells. VIP positive nerve fibers are mainly distributed in trachea, bronchial smooth muscle layer, the walls of pulmonary vessels and bronchial vessels and around submucosal glands. CECR2 is distributed in peripheral capillaries and small. Investigations of structure and proteins biology could be relevant with the adaptive strategy to drought and hypoxia environment in Phrynocephalus vlangalii.

Histology of the optic nerve head with special reference to the layer-specific distribution of composite fibers at and near the lamina cribrosa: An immunohistochemical study using specimens from elderly donated cadavers

BackgroundThis study aimed to demonstrate the composite fibers of the lamina cribrosa (LC) and their layer-specific distributions. The elastic fiber-rich septa, showing a cribriform arrangement in the optic nerve, may continue into the LC. MethodsOrbital content, including the long course of the optic nerve, was obtained from 25 elderly cadavers. Sagittal and cross-sections were prepared from each specimen. In addition to elastica Masson staining, immunohistochemistry was performed for elastin, glial fibrillary acidic protein (GFAP), S100 protein (S100), and CD68 in microglia. ResultsThe LC beam usually had fewer elastic fibers than the septa, but an elastic fiber-rich zone was observed along the scleral flange. GFAP-positive fibers were rich in the prelaminar area, whereas S100-positive fibers were rich in all layers of the LC. Double-positive (GFAP+/S100+) fibers were present in the prelaminar area. In contrast, S100-single positive fibers were evident in the LC and retrolaminar areas and were likely to insert into a sclera-choroid border area. The density of macrophages and microglia was not different between the septa and LC. Individual variations were observed in the distribution and density of the nerve-associated fibrous tissues. ConclusionThe LC beam was quite different from the septa in the composite fibers and architecture. Transverse fibers, dominant in the LC beam, corresponded to fibrous processes of astrocytes and other nerve-associated fibrous tissues. Many of these nerve elements suggest low mechanical properties of the LC.

TRPV4-induced Neurofilament Injury Contributes to Memory Impairment after High Intensity and Low Frequency Noise Exposures

ObjectiveExposure to high intensity, low frequency noise (HI-LFN) causes vibroacoustic disease (VAD), with memory deficit as a primary non-auditory symptomatic effect of VAD. However, the underlying mechanism of the memory deficit is unknown. This study aimed to characterize potential mechanisms involving morphological changes of neurons and nerve fibers in the hippocampus, after exposure to HI-LFN. MethodsAdult wild-type and transient receptor potential vanilloid subtype 4 knockout (TRPV4-/-) mice were used for construction of the HI-LFN injury model. The new object recognition task and the Morris water maze test were used to measure the memory of these animals. Hemoxylin and eosin and immunofluorescence staining were used to examine morphological changes of the hippocampus after exposure to HI-LFN. ResultsThe expression of TRPV4 was significantly upregulated in the hippocampus after HI-LFN exposure. Furthermore, memory deficits correlated with lower densities of neurons and neurofilament-positive nerve fibers in the cornu ammonis 1 (CA1) and dentate gyrus (DG) hippocampal areas in wild-type mice. However, TRPV4-/- mice showed better performance in memory tests and more integrated neurofilament-positive nerve fibers in the CA1 and DG areas after HI-LFN exposure. ConclusionTRPV4 up-regulation induced neurofilament positive nerve fiber injury in the hippocampus, which was a possible mechanism for memory impairment and cognitive decline resulting from HI-LFN exposure. Together, these results identified a promising therapeutic target for treating cognitive dysfunction in VAD patients.

Anatomic and histomorphometric study of the nerve to the vastus lateralis in cadaver for its clinical application in facial reanimation.

The innervated vastus lateralis flap (IVLF) is a barely used possibility for facial palsy reconstruction because of its thickness compared to the gracilis, latissimus dorsi, and pectoralis minor flaps. The aim of this study is to perform a precise description of the intramuscular distribution of the nerve motor branches and its relationship with the vascular pedicle in order to harvest a segmental muscle flap with the best contractile strength to restore facial reanimation. The study was performed on 16 adult cadaver thighs identifying the vastus lateralis muscle and the distribution and relationships of its neurovascular pedicle and branches. We evaluated where the nerve pierced the muscle and the course of the nerve within it. Transverse segments of the nerve were obtained from the proximal and distal ends of the nerve and stained using anti-ChAT (Choline acetyltransferase) antibodies which are specific of motor neurons. A nerve for the vastus lateralis from the posterior division of the femoral nerve divided into 2 branches in 56% of cases; the principal branch coursed along the vascular pedicle and pierced the muscle more proximally than the respective vessels, and a minor branch that pierced the muscle 25-60 mm proximally. There were 3 main intramuscular branches. The nerve length (mean 132.65 ± 22.89 mm) allowed to reach the contralateral side of the face in almost all cases (95%). The mean ChAT positive fibers was 351.0± 92.4/mm2 at the proximal end, and 270.3± 87.9/mm2 at the distal end (p= 0.49). The number of ChAT negative fibers was higher than ChAT positive in both proximal and distal ends of the nerve. We propose the IVLF as a one-step surgical flap for facial paralysis reanimation due to the constant neurovascular pattern and lengthy pedicle. The amount of motor fibers in several segments of the nerve is appropriate to produce a powerful contraction for dynamic reconstruction.

Reduced density of cholinergic fibers in App knock‐in mouse models of Aβ amyloidosis

AbstractBackgroundCholinergic system is critical for learning, memory, attention, and other cognitive functions. Nucleus basalis of Meynert (NBM) is a nucleus containing the cell bodies of cholinergic neurons in the brain and is severely affected by Alzheimer’s disease (AD). Significant loss of forebrain cholinergic projections is evident at prodromal stage of AD, and, along with locus coeruleus (LC), NBM neurons are among the first to display neurofibrillary tangle, suggesting that abnormality in cholinergic system is involved in the early stage of AD pathogenesis. Acetylcholinesterase inhibitors have been used for symptomatic treatment to improve cognitive function, however, mechanisms underlying neurodegeneration in cholinergic system remain elusive. We have previously reported that AppNL‐G‐F mice, which harbor three familial AD mutations (Swedish, Beyreuther/Iberian, and Arctic) exhibited cognitive deficits, severe neuroinflammation and significant loss of LC noradrenergic fibers accompanied by massive amyloid‐β (Aβ) pathology. In this study, we asked whether and how cholinergic system is altered in AppNL‐G‐F mice.MethodTo ask whether AppNL‐G‐F mice exhibit cholinergic degeneration, we compared the density of vesicular acetylcholine transporter (VAChT)‐positive fibers in the cortex and hippocampus as well as the number of choline acetyltransferase (ChAT)‐positive neurons in the NBM between AppNL‐G‐F and wild‐type (WT) C57BL/6J mice at 24 months of age. We also examined whether tau pathology occurs in the cholinergic system in these mice.ResultHistochemical analyses revealed that 24‐month‐old AppNL‐G‐F mice exhibited significant decreases in the density of VAChT‐positive fibers in the cortex and the hippocampal CA1, the regions associated with learning and memory, compared to WT mice.ConclusionThis study demonstrates that Aβ pathology is sufficient to reduce the density of cholinergic fibers. Quantitative analyses of cell loss and tau accumulation in ChAT‐positive neurons in the NBM are currently underway, and we will present these results at the meeting.

The Distribution of Innervation and Immune Cell Infiltration Is Different in Genital and Extragenital Variants of Lichen Sclerosus.

Lichen sclerosus (LS) is a progressive skin disease that is characterized by chronic inflammation of either genital or extragenital skin, and it disproportionately affects women. We analyzed the distribution of nerve fibers, vanilloid receptors, cell proliferation, mast cells and macrophages in genital and extragenital LS samples, as well as in healthy skin, by using immunohistochemistry. The total amount of intraepidermal nerve fibers was lower in LS samples compared to healthy controls, while the total amount of subepidermal nerve fibers and calcitonin gene-related peptide (CGRP) positive fibers was higher in genital LS samples compared to both extragenital LS and healthy controls. Cell proliferation, macrophage and mast cell density were increased in LS samples compared to healthy controls. Genital LS had a higher macrophage density compared to the extragenital variant. Mast cell distribution significantly differed between genital and extragenital LS samples, even though their total mast cell densities were similar. These findings could explain the differences between pruritic symptoms of genital and extragenital LS and provide targets for the research of novel therapeutic strategies for LS management.

IL-10 is not anti-fibrotic but pro-fibrotic in endometriosis: IL-10 treatment of endometriotic stromal cells in vitro promotes myofibroblast proliferation and collagen type I protein expression.

Is interleukin-10 (IL-10) anti-fibrotic in endometriosis? IL-10 is not anti-fibrotic but pro-fibrotic in endometriosis, because IL-10 treatment of endometriotic stromal cells in vitro promotes myofibroblast proliferation and collagen type I protein expression. We previously showed that persistent activation of signal transducer and activator of transcription 3 (STAT3) via IL-6 trans-signaling promotes fibrosis of endometriosis. Studies showed marked anti-fibrotic effects of IL-10 via the STAT3 signaling pathway, which is generally considered to be anti-inflammatory, in various organs. Endometrial and/or endometriotic samples of 54 patients who had histological evidence of deep endometriosis, and endometrial samples from 30 healthy fertile women were analyzed. The effects of IL-10/STAT3 signaling as well as inhibition of STAT3 activation by knockdown of STAT3 gene on the pro-fibrotic phenotype in endometrial and endometriotic stromal cells in vitro were investigated. Then, the effects of various time points of IL-10 treatment in combination with transforming growth factor (TGF)-β1 and/or IL-6/soluble IL-6 receptor (sIL-6R) on the profibrotic phenotype of endometrial and endometriotic stromal cells were investigated. IL-10 induced pro-fibrotic phenotype (cell proliferation, collagen type I synthesis, α-smooth muscle actin positive stress fibers and collagen gel contraction) of endometriotic stromal cells. Knockdown of STAT3 gene decreased the IL-10 induced pro-fibrotic phenotype of endometriotic stromal cells. In contrast, IL-10 had no significant effects on pro-fibrotic phenotype of endometrial stromal cells of healthy women. Sequential IL-10 treatment with or without TGF-β1 and/or IL-6/sIL-6R induced persistent activation of STAT3 and significantly increased proliferation of myofibroblasts (cells with α-smooth muscle actin positive stress fibers) and protein expression of collagen type I in endometriotic stromal cells. TGF-β1 and/or IL-6/sIL6RIL-6/sIL6R treatment significantly increased tissue inhibitor of metalloproteinase 1 (TIMP1) protein expression, whereas IL-10 had no significant effects. Knockdown of STAT3 gene significantly decreased the TGF-β1 and/or IL-6/sIL6R induced TIMP1 protein expression. In contrast, pre-treatment with IL-10 before TGF-β1 and/or IL-6/sIL-6R treatment and sequential IL-10 treatment with or without TGF-β1 and/or IL-6/sIL-6R significantly decreased proliferation of fibroblasts (cells without α-smooth muscle actin positive stress fibers) and collagen type I protein expression in endometrial stromal cells of healthy women. N/A. Given the large number of complex interactions and signaling pathways of pro- and anti-inflammatory mediators that are involved in the pathophysiology of endometriosis, the present study investigated only a very small portion of the whole. Further in vivo studies are required to validate the present findings. Inflammatory mediators in the pathophysiology of endometriosis have been extensively investigated as potential therapeutic targets. However, the present study showed that anti-inflammatory signals of IL-10 and IL-6 through persistent STAT3 activation may promote endometriosis fibrosis. Therapeutic strategies, such as suppression of 'inflammation', might dysregulate the cross-regulation of 'pro- and anti-inflammatory mediators', leading to detrimental effects in patients with endometriosis, such as fibrosis. To develop new, but not deleterious, therapeutic strategies, studies are required to investigate whether, how and what 'anti-inflammatory mediators' along with pro-inflammatory mediators are involved in individual patients with endometriosis. This study was supported in part by KARL STORZ SE & Co. KG (Tuttlingen, Germany). The authors have no conflict of interest to disclose.

413 Role of calcitonin gene-related peptide in atopic dermatitis

Atopic dermatitis (AD) is an often severely pruritic chronic inflammatory disease often worsened by psychological stress. Calcitonin gene-related peptide (CGRP) is a sensory neuropeptide, which may cause pruritus. Earlier studies indicate a special pathogenetic role for CGRP in AD. However, somewhat contradictory results regarding CGRP positive nerve fibers in AD skin, have been reported. We have studied skin biopsies from 26 patients with AD. The biopsies were taken from lesional and non-lesional skin, in addition, normal skin biopsies from 10 healthy individuals were investigated for expression of CGRP, using a biotinylated–streptavidine technique and fluorescence microscopy.

Cutaneous expression of growth-associated protein 43 is not a compelling marker for human nerve regeneration in carpal tunnel syndrome.

Growth-associated protein 43 (GAP-43) has long been used as a marker for nerve regeneration following nerve injury, with numerous in vitro and animal studies showing its upregulation in regenerating neurons. In humans, expression of GAP-43 has predominantly been examined in skin biopsies from patients with peripheral neuropathies; with several studies showing a reduction in GAP-43 immunoreactive cutaneous nerve fibres. However, it remains elusive whether cutaneous GAP-43 is a valid marker for human nerve regeneration. Here, we present a cohort of 22 patients with electrodiagnostically confirmed carpal tunnel syndrome (CTS), used as a model system for focal nerve injury and neural regeneration after decompression surgery. We evaluate GAP-43 immunoreactivity and RNA expression levels in finger skin biopsies taken before and 6 months after surgery, relative to healthy controls. We further classify patients as 'regenerators' or 'non-regenerators' based on post-surgical epidermal re-innervation. We demonstrate that patients with CTS have lower GAP-43 positive intra-epidermal nerve fibre density (IENFD) before surgery than healthy controls. However, this difference disappears when normalising for total IENFD. Of note, we found surgery did not change GAP-43 expression in IENF, with no differences both in patients who were classified as regenerators and non-regenerators. We also did not identify pre-post surgical differences in cutaneous GAP-43 gene expression or associations with regeneration. These findings suggest cutaneous GAP-43 may not be a compelling marker for nerve regeneration in humans.

Neonatal ketamine exposure impairs infrapyramidal bundle pruning and causes lasting increase in excitatory synaptic transmission in hippocampal CA3 neurons

Preclinical models demonstrate that nearly all anesthetics cause widespread neuroapoptosis in the developing brains of infant rodents and non-human primates. Anesthesia-induced developmental apoptosis is succeeded by prolonged neuropathology in the surviving neurons and lasting cognitive impairments, suggesting that anesthetics interfere with the normal developmental trajectory of the brain. However, little is known about effects of anesthetics on stereotyped axonal pruning, an important developmental algorithm that sculpts neural circuits for proper function. Here, we proposed that neonatal ketamine exposure may interfere with stereotyped axonal pruning of the infrapyramidal bundle (IPB) of the hippocampal mossy fiber system and that impaired pruning may be associated with alterations in the synaptic transmission of CA3 neurons. To test this hypothesis, we injected postnatal day 7 (PND7) mouse pups with ketamine or vehicle over 6 h and then studied them at different developmental stages corresponding to IPB pruning (PND20–40). Immunohistochemistry with synaptoporin (a marker of mossy fibers) revealed that in juvenile mice treated with ketamine at PND7, but not in vehicle-treated controls, positive IPB fibers extended farther into the stratum pyramidale of CA3 region. Furthermore, immunofluorescent double labeling for synaptoporin and PSD-95 strongly suggested that the unpruned IPB caused by neonatal ketamine exposure makes functional synapses. Importantly, patch-clamp electrophysiology for miniature excitatory postsynaptic currents (mEPSCs) in acute brain slices ex vivo revealed increased frequency and amplitudes of mEPSCs in hippocampal CA3 neurons in ketamine-treated groups when compared to vehicle controls. We conclude that neonatal ketamine exposure interferes with normal neural circuit development and that this interference leads to lasting increase in excitatory synaptic transmission in hippocampus.

Connections between postparotid terminal branches of the facial nerve: An immunohistochemistry study.

It has been assumed that connections between the postparotid terminal branches of the facial nerve are purely motor. However, the nature of their fibers remains unexplored. The aim of this study is to determine whether these connections comprise motor fibers exclusively. In total 17 connections between terminal facial nerve branches were obtained from 13 different facial nerves. Choline acetyltransferase antibody (ChAT) was used to stain the fibers in the connections and determine whether or not all of them were motor. All connections contained ChAT positive and negative fibers. The average number of fibers overall was 287 (84-587) and the average proportion of positive fibers was 63% (37.7%-91.5%). In 29% of the nerves, >75% of the fibers were ChAT+ (strongly positive); in 52.94%, 50%-75% were ChAT+ (intermediately positive); and in 17.65%, <50% were ChAT+ (weakly positive). Fibers traveling inside the postparotid terminal cranial nerve VII branch connections are not exclusively motor.

Abstract 10134: Role of Cardiac Sympathetic Nerves in Mediating the Central Nervous System Cardioprotective Effects of Leptin After Ischemia/Reperfusion Injury

We previously demonstrated that central nervous system (CNS) administration of leptin improves left ventricular (LV) function after myocardial infarction through mechanisms that are poorly understood. Increased cardiac sympathetic activity is an important compensatory mechanism to maintain cardiac output after ischemic injury, and previous studies showed that centrally-administered leptin increases sympathetic outflow to various tissues. In the present study, we tested the hypothesis that leptin’s CNS-mediated cardioprotective effects are mediated via cardiac sympathetic nerves. Male and female Wistar rats (~8 weeks of age) were submitted to cervical ganglia denervation (CGx) and instrumented with an intracerebroventricular (ICV) cannula in the brain lateral ventricle. After recovery and baseline assessment of cardiac function by echocardiography (ECHO) and an exercise performance test (Vmax), myocardial ischemia/reperfusion (I/R) was induced by temporary (60 min) ligation of the left anterior descending coronary artery. Vehicle (saline, 0.5 μL/hr) or leptin (0.62 μg/hr) was infused chronically for 28 days starting 20 min after reperfusion using osmotic pumps implanted subcutaneously in the scapular region and connected to the ICV cannula. Cardiac function was assessed weekly by ECHO. At the end of week 4, a final Vmax was performed and +dP/dt max was accessed by LV catheterization using a Millar catheter. Despite ~70% reduction in cardiac tyrosine hydroxylase positive fibers compared to sham, CGx did not prevent leptin’s CNS-mediated cardioprotective effects as indicated by improved ejection fraction (55±1 vs. 29±3 %), global longitudinal strain (-19±2 vs. -3±2 %), +dP/dt max (10214±614 vs. 7174±716 mmHg/s) and exercise performance (1.3±14 vs. -46.3±14 Δ% compared to baseline before I/R) when compared with CGx vehicle-treated animals. CGx also did not prevent the effect of ICV leptin to reduce septal collagen deposition and heart weight/tibia length post-I/R. These results suggests that leptin, via its CNS actions, improves cardiac function and remodeling after I/R injury independent of cardiac sympathetic innervation.

Studies on the interactions of retinal dopamine with choroidal thickness in the chicken

PurposeRecently, an increasing number of studies relied on the assumption that visually induced changes in choroidal thickness can serve as a proxy to predict future axial eye growth. The retinal signals controlling choroidal thickness are, however, not well defined. We have studied the potential roles of dopamine, released from the retina, in the choroidal response in the chicken.MethodsChanges in retinal dopamine release and choroidal thickness changes were induced by intravitreal injections of either atropine (250 µg or 360 nMol), atropine combined with a dopamine antagonist, spiperone (500 µMol), or spiperone alone and were tracked by optical coherence tomography (OCT). To visually stimulate dopamine release, other chicks were exposed to flicker light of 1, 10, or 400 Hz (duty cycle 0.2) and choroidal thickness was tracked. In all experiments, dopamine and 3,4-Dihydroxyphenylacetic acid (DOPAC) were measured in vitreous, retina, and choroid by high-performance liquid chromatography with electrochemical detection (HLPC-ED). The distribution of the rate-limiting enzyme of dopamine synthesis, tyrosine hydroxylase (TH), neuronal nitric oxide synthase (nNOS), vascular endothelial growth factor (VEGF), and alpha2A adrenoreceptors (alpha2A-ADR) was studied in the choroid by immunofluorescence.ResultsThe choroid thickened strongly in atropine-injected eyes, less so in atropine + spiperone–injected eyes and became thinner over the day in spiperone alone-, vehicle-, or non-injected eyes. Flickering light at 20 lx, both 1 and 10 Hz, prevented diurnal choroidal thinning, compared to 400 Hz, and stimulated retinal dopamine release. Correlation analysis showed that the higher retinal dopamine levels or release, the thicker became the choroid. TH-, nNOS-, VEGF-, and alpha2A adrenoreceptor–positive nerve fibers were localized in the choroid around lacunae and in the walls of blood vessels with colocalization of TH and nNOS, and TH and VEGF.ConclusionsRetinal DOPAC and dopamine levels were positively correlated with choroidal thickness. TH-positive nerve fibers in the choroid were closely associated with peptides known to play a role in myopia development. Findings are in line with the hypothesis that dopamine is related to retinal signals controlling choroidal thickness.

FP.14 Dystrophin and satellite cell quantification in Duchenne and Becker muscular dystrophies

The dystrophinopathies are a group of muscular dystrophies stemming from mutations in the <i>DMD</i> gene. We have developed a series of sensitive and reproducible dystrophin and satellite cell quantification methods in order to better define associations between dystrophin expression and disease severity, as well as the molecular mechanisms of mutations in the N-terminal region of dystrophin. In a pilot study of 18 dystrophinopathy patient muscle biopsies, we employed western blot, capillary western immunoassay, and automated quantitative immunofluorescence analysis to quantify dystrophin expression and Pax7-positive satellite cells. Among 9 patients with a Duchenne muscular dystrophy phenotype, the mean dystrophin protein level by capillary western was 1.8% of normal. These biopsies displayed a mean of 6.8% immunofluorescence staining intensity of individual fibers and 26% dystrophin positive fibers (PDPF). This high PDPF reflects the use of a sensitive cutoff to improve the discriminating power of the assay at low expression levels. By contrast, patients with intermediate or Becker muscular dystrophy had 11% dystrophin expression by capillary western and 70% PDPF. Several truncating mutations in the first 4 exons of DMD were associated with milder phenotypes and significantly higher dystrophin expression (16% by capillary western and 79% PDPF), in keeping with activation of an internal ribosome entry site in exon 5. There was poor overall correlation between Pax7-positive satellite cell count and dystrophin expression. Analysis of individual muscle fibers however demonstrated that fibers with one or more adjacent Pax7-positive satellite cells had a lower mean dystrophin expression, indicating concentration of satellite cells around dystrophin-deficient fibers. This study lays the groundwork for a comprehensive characterization of genotype, phenotype, and molecular features in a broader cohort of dystrophinopathy patients.

O.03 Nemaline myopathy type 6: From pathology to therapeutics

Patients harboring variants in <i>KBTBD13</i> (NEM6) display impaired muscle relaxation, which compromises normal muscle function and daily-life activities. Histopathologically, NEM6 is characterized by the presence of myofibers containing nemaline bodies and a predominance of type I fibers. The majority of NEM6 patients harbor the Dutch founder mutation (c.1222C>T, p.Arg408Cys). Recently, we revealed that the R408C variantin <i>KBTBD13</i> slows muscle relaxation through an actin-based mechanism. To date, no therapy is available to treat NEM6. To fill this void, we developed a Kbtbd13^R408C knock-in mouse model. To identify a therapeutic window, we first performed a natural history study to characterize time of disease onset and progression in homozygous Kbtbd13^R408C knock-in at 1, 3, 9 and 18 months of age. Morphological and functional assays of soleus muscle showed that Kbtbd13^R408C knock-in mice closely recapitulate the human NEM6 phenotype: slow relaxation kinetics and muscle weakness was observed, in addition to the presence of nemaline bodies, type I fiber predominance, and hypertrophy of type I and IIa myofibers. This phenotype was absent at 1 month, developed between 1-3 months, and showed little progression after 9 months of age. Our treatment of choice was the knock-down of mutant <i>Kbtbd13</i> transcript. This choice was based on experiments on homozygous Kbtbd13-KO mice, which showed no overt phenotype at 3 and 9 months of age. Thus, Kbtbd13-deficiency is well tolerated. We treated soleus with a single intramuscular injection with an AAV9 containing a short-hairpin RNA for Kbtbd13 (AAV9-shKbtbd13). We hypothesized that a reduction in mutant transcript level would prevent or reverse disease development. Our data demonstrate that a single injection knocked down mutant <i>Kbtbd13</i> transcript levels by >90% in 3 (injected at 1-month, prior to disease development) and 9 month (injected at 7-months, at which an overt disease phenotype was present) old mice. Importantly, in treated mice, the relaxation kinetics were normalized to those of wild-type mice. Additionally, in treated mice, we observed a >70% reduction in the number of nemaline rod positive fibers. In conclusion, our results show the natural history of NEM6 in a novel mouse model and the promise of knocking down mutant transcript to prevent and reverse the NEM6 phenotype.

VP.82 PK/PD modelling to inform clinical development of an adeno-associated virus gene transfer therapy for Duchenne muscular dystrophy

Delandistrogene moxeparvovec (SRP-9001) is an investigational gene transfer therapy for Duchenne muscular dystrophy (DMD) developed for targeted skeletal and cardiac muscle expression of micro-dystrophin—a shortened, functional dystrophin protein. We evaluated vector biodistribution, expression, and clinical dose selection of delandistrogene moxeparvovec using a novel application of a pharmacokinetic (PK) and pharmacodynamic (PD) modelling approach applied to data collected from an animal model of DMD (DMD^MDX mice). We analysed PK/PD relationships between dose, tissue vector genome exposure, micro-dystrophin protein expression (percentage dystrophin positive fibres [PDPF] and western blot), and functional improvement (relative specific force from tibialis anterior and diaphragm). Linear kinetics with a dose-proportional increase in tissue drug exposure was demonstrated across the nearly 10-fold dose range (4.43x10¹³–4.01x10¹⁴ vg/kg), and in all tissues. The relationship between tissue vector exposure and PD endpoints (PDPF, motor function outcome) showed a saturable response across a wide range of vector exposures. The vector exposure at 1.33x10¹⁴ vg/kg (the clinical dose) approached the maximum treatment response. Relative specific force and PDPF were significantly correlated (P=4.43x10^-6). However, the relationship appeared to be nonlinear, with increased PDPF expression approaching the maximal functional improvement. Relative specific force and western blot were not significantly correlated. For the first time, biodistribution, biomarker, and functional efficacy data were used to quantify and demonstrate PK/PD relationships for an adeno-associated virus (AAV)-based gene transfer therapy in a DMD animal model. The results continue to support the expected therapeutic benefit and clinical dose of delandistrogene moxeparvovec, an AAV-based gene transfer therapy.

Study on Dynamic Splitting Properties of S-PP Hybrid Fiber Concrete after High Temperatures

To study the dynamic tensile mechanical properties of steel polypropylene hybrid fiber reinforced concrete (SP-HFRC) after high temperature, split Hopkinson pressure bar (SHPB) dynamic splitting tests were carried out, and the optimal fiber content combination was obtained. With the plain concrete (PC) as the control, the effects of fiber addition on energy dissipation and failure forms of concrete specimens after high temperatures were analyzed. LS-DYNA software was used to simulate the dynamic splitting test. The results show that the splitting strength of specimens increases first and then deteriorates with the increase of temperature. After high temperatures, HFRC has a positive and negative fiber hybrid effect. Among the studied fiber mixture combinations, S1PP0.2 (1 vol% steel fiber + 0.2 vol% polypropylene fiber) concrete has the best splitting resistance. Compared with PC, the splitting strength increases by 106.8% at 25 °C and 128.2% at 800 °C. From the perspective of energy, we can conclude that adding hybrid fiber can significantly improve the dynamic splitting and tensile toughness of concrete after high temperatures, and defining damage variables can better characterize the damage degree of concrete. PC cracks seriously after high temperatures, while S1PP0.2 concrete cracks but does not disperse at 800 °C, showing ductile failure characteristics. By modifying some parameters of the HJC model, the state of high-temperature concrete mechanical properties can be better characterized after deterioration. The simulated failure process shows an excellent agreement with the experimental results.