Opposing Effects of Neuropilin-1 and -2 on Sensory Nerve Regeneration in Wounded Corneas: Role of Sema3C in Ameliorating Diabetic Neurotrophic Keratopathy.
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ABSTRACT:
SUBMITTER: Lee PS
PROVIDER: S-EPMC6425876 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The flat bones of the skull are densely innervated during development, but little is known regarding their role during repair. We describe a neurotrophic mechanism that directs sensory nerve transit in the mouse calvaria. Patent cranial suture mesenchyme represents an NGF (nerve growth factor)-rich domain, in which sensory nerves transit. Experimental calvarial injury upregulates Ngf in an IL-1β/TNF-α-rich defect niche, with consequent axonal ingrowth. In calvarial osteoblasts, IL-1β and TNF-α stimulate Ngf and downstream NF-κB signaling. Locoregional deletion of Ngf delays defect site re-innervation and blunted repair. Genetic disruption of Ngf among LysM-expressing macrophages phenocopies these observations, whereas conditional knockout of Ngf among Pdgfra-expressing cells does not. Finally, inhibition of TrkA catalytic activity similarly delays re-innervation and repair. These results demonstrate an essential role of NGF-TrkA signaling in bone healing and implicate macrophage-derived NGF-induced ingrowth of skeletal sensory nerves as an important mediator of this repair.
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Project description:BACKGROUND:Neurotrophic keratopathy/keratitis (NK) is a rare disease of the cornea that can lead to anatomical loss of the eye. Little is known about the NK experience from the patients' perspective. The objectives of this study were to examine the symptomatic experience and impacts of NK on patients and assess the overall comprehension, relevance, and content validity of a new questionnaire. METHODS:This was a cross-sectional, qualitative study conducted with NK patients with varying levels of disease severity, recruited from one clinical site. One-on-one interviews using concept elicitation and cognitive interviewing techniques were conducted. RESULTS:Fourteen NK patients participated; 64.3% were female (n?=?9), mean age was 65.7?±?13.3, and 14.3% (n?=?2), 21.4% (n?=?3), and 64.3% (n?=?9) were classified as Mackie stage I, stage II, or stage III, respectively. Participants reported 24 concepts, including: redness (n?=?12, 86%), sensitivity to light (n?=?11, 79%), general discomfort (n?=?9, 64%), dry eye (n?=?9, 64%), reduced visual acuity (n?=?9, 64%), blurred vision (n?=?8, 57%), and eye fatigue (n?=?8, 57%). No new concepts were reported after the 13th interview. The most frequently reported impacts included frustration (n?=?10, 71%), driving impairment (n?=?8, 57%), reading impairment (n?=?7, 50%), difficulty watching television (n?=?7, 50%), and concern with potentially losing their eyesight due to NK (n?=?6, 43%). Participants provided positive feedback on the draft NK Questionnaire (NKQ) and felt that it was comprehensive and relevant to their experience with NK. Additionally, the recall period, instructions, item concepts, and response options were well-understood by participants. Minor revisions were made to the tool for consistency (i.e., the timeframe "in the past 7 days" was added to items 12-14); item 14 was modified to include "how often"; examples were added to item 9. CONCLUSIONS:The results of the concept elicitation portion of the qualitative study support the content validity of the draft NKQ. The clinically significant concepts identified in the literature and raised during concept elicitation are included as items in the questionnaire. Further assessment of the psychometric properties should be conducted in support of this new tool to measure the effect of new treatments on symptoms and impacts associated with NK.
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Project description:Neurotrophic Keratopathy (NK), classified as an orphan disease (ORPHA137596), is a rare degenerative corneal disease characterized by epithelial instability and decreased corneal sensitivity caused by the damage to the corneal nerves. The administration of human recombinant nerve growth factor (rhNGF) eye drops, as a licensed-in-Europe specific medication for treatment of moderate and severe NK, has added promising perspectives to the management of this disorder by providing a valid alternative to the neurotization surgery. However, few studies have been conducted to the molecular mechanism underlying the response to the treatment. Here, we carried out tears proteomics to highlight the protein expression during pharmacological treatment of NK (Data are available via ProteomeXchange with identifier PXD025408).Our data emphasized a proteome modulation during rhNGF treatment related to an increase in DNA synthesis, an activation of both BDNF signal and IL6 receptor. Furthermore, the amount of neuronal Extracellular Vesicles EVs (CD171+) correlated with the EVs carrying IL6R (CD126+) together associated to the inflammatory EVs (CD45+) in tears. Such scenario determined drug response, confirmed by an in vivo confocal microscopy analysis, showing an increase in length, density and number of nerve fiber branches during treatment. In summary, rhNGF treatment seems to determine an inflammatory micro-environment, mediated by functionalized EVs, defining the drug response by stimulating protein synthesis and fiber regeneration.
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Project description:Neurotrophic Keratopathy (NK),classified as an orphan disease (ORPHA137596), is a rare degenerative corneal disease characterized by epithelial instability and decreased corneal sensitivity caused by the damage to the corneal nerves. The administration of human recombinant nerve growth factor (rhNGF) eye drops, as a licensed-in-Europe specific medication for treatment of moderate and severe NK, has added promising perspectives to the management of this disorder by providing a valid alternative to the neurotization surgery. However, few studies have been conducted to the molecular mechanism underlying the response to the treatment. Here, we carried out tears proteomics to highlight the protein expression during pharmacological treatment of NK. Our data emphasized a proteome modulation duringrhNGF treatment related to an increase in DNA synthesis, an activation ofboth BDNF signaland IL6 receptor.Furthermore, the amount of neuronal Extracellular Vesicles EVs (CD171+)correlated with the EVs carrying IL6R (CD126+) togetherassociated to the inflammatory EVs (CD45+) in tears. Such scenario determined drug response, confirmed by an in vivo confocal microscopy analysis, showing an increase in length, density and number of nerve fiber branches during treatment. In summary, rhNGF treatment seems to determine an inflammatory micro-environment, mediated by functionalized EVs, defining the drug response by stimulating protein synthesis and fiber regeneration.
2022-02-14 | PXD025408 | Pride
Project description:ObjectiveTo investigate the entire human corneal nerve architecture of donors with different durations of insulin-dependent diabetes mellitus (IDDM).DesignExperimental study.Participants and controlsSixteen fresh human eyes from 8 diabetic donors (aged 43-66 years, with IDDM for 2-17 years) and 12 eyes from 6 normal donors (aged from 44-67 years) were obtained from the National Disease Research Interchange (NDRI).MethodsAfter fixation, corneas were stained with mouse monoclonal anti-?-Tubulin III antibody, and images were acquired to build a whole view of the corneal nerve architecture. The same corneas were used for both whole-mount and cross-section examination.Main outcome measuresCorneal epithelial nerve density was calculated on the basis of the whole-mount view of the central area. The number of stromal nerves was calculated by counting the nerve trunks at the corneoscleral limbus of the entire cornea. Differences between diabetic and normal corneas in epithelial nerve densities and main stromal nerve numbers were compared by paired-samples t test.ResultsThe diabetic eyes presented numerous neuropathies in areas where the epithelial nerve bundles emerged. A striking pathologic change was the presence of abundant nerve fiber loops in the stroma. These loops seemed to form by resistance presented by the basement membrane, which may prevent penetration of stromal nerve branches into epithelia. There was no difference in the numbers of main stromal nerve trunks between corneas from diabetic and normal donors, but there was a significant decrease in central epithelial nerve density in the diabetic corneas. We did not find an age effect on this decrease. Instead, it was significantly affected by 5 or more years of IDDM.ConclusionsThis is the first study to show an entire view of the nerve architecture in human diabetic corneas. The decreased epithelial nerve density may result from the abnormalities of stromal nerve architecture and is affected by 5 or more years of IDDM. Although compensation for some nerve regeneration takes place, the alterations in the stromal nerves can explain the poor healing and persistent epithelial defects seen in diabetic patients.
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Project description:We sought to determine if connective tissue growth factor (CTGF) is necessary for the formation of corneal haze after corneal injury. Mice with post-natal, tamoxifen-induced, knockout of CTGF were subjected to excimer laser phototherapeutic keratectomy (PTK) and the corneas were allowed to heal. The extent of scaring was observed in non-induced mice, heterozygotes, and full homozygous knockout mice and quantified by macrophotography. The eyes from these mice were collected after euthanization for re-genotyping to control for possible Cre-mosaicism. Primary corneal fibroblasts from CTGF knockout corneas were established in a gel plug assay. The plug was removed, simulating an injury, and the rate of hole closure and the capacity for these cells to form light reflecting cells in response to CTGF and platelet-derived growth factor B (PDGF-B) were tested and compared to wild-type cells. We found that independent of genotype, each group of mice was still capable of forming light reflecting haze in the cornea after laser ablation (p = 0.40). Results from the gel plug closure rate in primary cell cultures of knockout cells were not statistically different from serum starved wild-type cells, independent of treatment. Compared to the serum starved wild-type cells, stimulation with PDGF-BB significantly increased the KO cell culture's light reflection (p = 0.03). Most interestingly, both reflective cultures were positive for ?-SMA, but the cellular morphology and levels of ?-SMA were distinct and not in proportion to the light reflection seen. This new work demonstrates that corneas without CTGF can still form sub-epithelial haze, and that the light reflecting phenotype can be reproduced in culture. These data support the possibilities of growth factor redundancy and that multiple pro-haze pathways exist.
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Project description:Glial cell line-derived neurotrophic factor (GDNF) has potent trophic effects on adult sensory neurons after nerve injury and is one of a family of proteins that includes neurturin, persephin, and artemin. Sensitivity to these factors is conferred by a receptor complex consisting of a ligand binding domain (GFRalpha1-GFRalpha4) and a signal transducing domain RET. We have investigated the normal expression of GDNF family receptor components within sensory neurons and the response to nerve injury. In normal rats, RET and GFRalpha1 were expressed in a subpopulation of both small- and large-diameter afferents projecting through the sciatic nerve [60 and 40% of FluoroGold (FG)-labeled cells, respectively]. GFRalpha2 and GFRalpha3 were both expressed principally within small-diameter DRG cells (30 and 40% of FG-labeled cells, respectively). Two weeks after sciatic axotomy, the expression of GFRalpha2 was markedly reduced (to 12% of sciatic afferents). In contrast, the proportion of sciatic afferents that expressed GFRalpha1 increased (to 66% of sciatic afferents) so that virtually all large-diameter afferents expressed this receptor component, and the expression of GFRalpha3 also increased (to 66% of sciatic afferents) so that almost all of the small-diameter afferents expressed this receptor component after axotomy. There was little change in RET expression. The changes in the proportions of DRG cells expressing different receptor components were mirrored by alterations in the total RNA levels within the DRG. The changes in GFRalpha1 and GFRalpha2 expression after axotomy could be largely reversed by treatment with GDNF.
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Project description:The cornea has unique features that make it a useful model for regenerative medicine studies. It is an avascular, transparent, densely innervated tissue and any pathological changes can be easily detected by slit lamp examination. Corneal sensitivity is provided by the ophthalmic branch of the trigeminal nerve that elicits protective reflexes such as blinking and tearing and exerts trophic support by releasing neuromediators and growth factors. Corneal nerves are easily evaluated for both function and morphology using standard instruments such as corneal esthesiometer and in vivo confocal microscope. All local and systemic conditions that are associated with damage of the trigeminal nerve cause the development of neurotrophic keratitis, a rare degenerative disease. Neurotrophic keratitis is characterized by impairment of corneal sensitivity associated with development of persistent epithelial defects that may progress to corneal ulcer, melting and perforation. Current neurotrophic keratitis treatments aim at supporting corneal healing and preventing progression of corneal damage. Novel compounds able to stimulate corneal nerve recovery are in advanced development stage. Among them, nerve growth factor eye drops showed to be safe and effective in stimulating corneal healing and improving corneal sensitivity in patients with neurotrophic keratitis. Neurotrophic keratitis represents an useful model to evaluate in clinical practice novel neuro-regenerative drugs.
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Project description:Peripheral nerve repair and functional recovery depend on the rate of nerve regeneration and the quality of target reinnervation. It is important to fully understand the cellular and molecular basis underlying the specificity of peripheral nerve regeneration, which means the achieving of respective correct pathfinding and accurate target reinnervation for regrowing motor and sensory axons. In this study, a quantitative proteomic technique, based on isobaric tags for relative and absolute quantitation (iTRAQ) was used to profile the protein expression pattern between single motor and sensory nerves at 14 days after peripheral nerve transection. Among a total of 1259 proteins identified, 176 proteins showed the differential expressions between injured motor and sensory nerves. Quantitative real-time RT-PCR and Western blot analysis were applied to validate the proteomic data on representative differentially expressed proteins. Functional categorization indicated that differentially expressed proteins were linked to a diverse array of molecular functions, including axonogenesis, response to axon injury, tissue remodeling, axon ensheathment, cell proliferation and adhesion, vesicle-mediated transport, response to oxidative stress, internal signal cascade, and macromolecular complex assembly, which might play an essential role in peripheral motor and sensory nerve regeneration. Overall, we hope that the proteomic database obtained in this study could serve as a solid foundation for the comprehensive investigation of differentially expressed proteins between injured motor and sensory nerves and for the mechanism elucidation of the specificity of peripheral nerve regeneration.
2020-12-10 | PXD022097 | Pride