Browsing by Author "Gordon, Tessa"
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Item 72. The Role of TrkA And P75NTR NGF Receptors in Corneal Wound Healing(Wolters Kluwer, 2022) Tajdaran, Kiana; Feinberg, Konstantin; Mirmoeini, Seyed K.; Zhang, Jennifer; Gordon, Tessa; Ali, Asim; Borschel, Gregory; Surgery, School of MedicinePurpose: The cornea is the window through which we see the world and is one of the most densely innervated structures in the body. Besides providing protective sensory input, corneal nerves may also stimulate limbal stem cells (LSCs), governing corneal epithelial maintenance and recovery. Loss of corneal innervation, through injury, diabetes, tumors, infections, and even improper contact lens use, leads to neurotrophic keratopathy (NK), a degenerative corneal disease that is characterized by corneal epithelial breakdown, scarring, and permanent vision loss1. The only non-invasive treatment option for NK is human recombinant nerve growth factor (rhNGF), but the short half-life of exogenous neurotrophins-based therapies limits their effecacy2. Development of small molecule ligands for neurotrophin receptors that have more favorable pharmacokinetics and plasma stability showed promising results in the treatment of several neurodegenerative conditions in recent years3. In this study, we investigated the molecular mechanism of NK and the role of the NGF receptors, TrkA and p75NTR, in corneal healing. We hypothesized that TrkA inhibition would delay corneal wound healing and p75NTR inhibition would accelerate corneal healing. Establishing the roles of these receptors may enable novel topical therapeutics for NK. Methods: We used commercially available Ntrk1 mutant mice, whose TrkA receptors are inhibited by a mammalian kinase inhibitor (1-NM-PP1)4. Ntrk1 mice (n=20) were divided into three groups, which received saline injection as a control. In one experimental group animals received TrkA inhibitor and the other group received both TrkA and p75 inhibitor for 5 days. On day six we removed the corneal epithelium with a 0.5 mm rotating brush. To measure epithelial healing, we performed digital imaging of fluorescein staining daily for four days after injury. We then harvested the corneas for immunofluorescent and biochemical analyses. Results: We observed a significant delay in corneal epithelial healing following TrkA inhibition. Further, we observed that topical p75NTR inhibition accelerated corneal wound healing. Conclusion: A selective TrkA agonist or p75NTR inhibition could represent new topical therapeutics for NK.Item A functional tacrolimus-releasing nerve wrap for enhancing nerve regeneration following surgical nerve repair(Wolters Kluwer, 2024) Daeschler, Simeon C.; So, Katelyn J. W.; Feinberg, Konstantin; Manoraj, Marina; Cheung, Jenny; Zhang, Jennifer; Mirmoeini, Kaveh; Santerre, J. Paul; Gordon, Tessa; Borschel, Gregory H.; Surgery, School of MedicineAxonal regeneration following surgical nerve repair is slow and often incomplete, resulting in poor functional recovery which sometimes contributes to lifelong disability. Currently, there are no FDA-approved therapies available to promote nerve regeneration. Tacrolimus accelerates axonal regeneration, but systemic side effects presently outweigh its potential benefits for peripheral nerve surgery. The authors describe herein a biodegradable polyurethane-based drug delivery system for the sustained local release of tacrolimus at the nerve repair site, with suitable properties for scalable production and clinical application, aiming to promote nerve regeneration and functional recovery with minimal systemic drug exposure. Tacrolimus is encapsulated into co-axially electrospun polycarbonate-urethane nanofibers to generate an implantable nerve wrap that releases therapeutic doses of bioactive tacrolimus over 31 days. Size and drug loading are adjustable for applications in small and large caliber nerves, and the wrap degrades within 120 days into biocompatible byproducts. Tacrolimus released from the nerve wrap promotes axon elongation in vitro and accelerates nerve regeneration and functional recovery in preclinical nerve repair models while off-target systemic drug exposure is reduced by 80% compared with systemic delivery. Given its surgical suitability and preclinical efficacy and safety, this system may provide a readily translatable approach to support axonal regeneration and recovery in patients undergoing nerve surgery.Item Advancing Nerve Regeneration: Translational Perspectives of Tacrolimus (FK506)(MDPI, 2023-08-14) Daeschler, Simeon C.; Feinberg, Konstantin; Harhaus, Leila; Kneser, Ulrich; Gordon, Tessa; Borschel, Gregory H.; Ophthalmology, School of MedicinePeripheral nerve injuries have far-reaching implications for individuals and society, leading to functional impairments, prolonged rehabilitation, and substantial socioeconomic burdens. Tacrolimus, a potent immunosuppressive drug known for its neuroregenerative properties, has emerged in experimental studies as a promising candidate to accelerate nerve fiber regeneration. This review investigates the therapeutic potential of tacrolimus by exploring the postulated mechanisms of action in relation to biological barriers to nerve injury recovery. By mapping both the preclinical and clinical evidence, the benefits and drawbacks of systemic tacrolimus administration and novel delivery systems for localized tacrolimus delivery after nerve injury are elucidated. Through synthesizing the current evidence, identifying practical barriers for clinical translation, and discussing potential strategies to overcome the translational gap, this review provides insights into the translational perspectives of tacrolimus as an adjunct therapy for nerve regeneration.Item Foretinib mitigates cutaneous nerve fiber loss in experimental diabetic neuropathy(Springer Nature, 2022-05-19) Daeschler, Simeon C.; Zhang, Jennifer; Gordon, Tessa; Borschel, Gregory H.; Feinberg, Konstantin; Surgery, School of MedicineDiabetes is by far, the most common cause of neuropathy, inducing neurodegeneration of terminal sensory nerve fibers associated with loss of sensation, paresthesia, and persistent pain. Foretinib prevents die-back degeneration in cultured sensory and sympathetic neurons by rescuing mitochondrial activity and has been proven safe in prospective clinical trials. Here we aimed at investigating a potential neuroprotective effect of Foretinib in experimental diabetic neuropathy. A mouse model of streptozotocin induced diabetes was used that expresses yellow fluorescent protein (YFP) in peripheral nerve fibers under the thy-1 promoter. Streptozotocin-injected mice developed a stable diabetic state (blood glucose > 270 mg/dl), with a significant reduction of intraepidermal nerve fiber density by 25% at 5 weeks compared to the non-diabetic controls. When diabetic mice were treated with Foretinib, a significantly greater volume of the cutaneous nerve fibers (67.3%) in the plantar skin was preserved compared to vehicle treated (37.8%) and non-treated (44.9%) diabetic mice while proximal nerve fiber morphology was not affected. Our results indicate a neuroprotective effect of Foretinib on cutaneous nerve fibers in experimental diabetic neuropathy. As Foretinib treated mice showed greater weight loss compared to vehicle treated controls, future studies may define more sustainable treatment regimen and thereby may allow patients to take advantage of this neuroprotective drug in chronic neurodegenerative diseases like diabetic neuropathy.Item Optical tissue clearing enables rapid, precise and comprehensive assessment of three-dimensional morphology in experimental nerve regeneration research(Wolters Kluwer, 2022) Daeschler, Simeon C.; Zhang, Jennifer; Gordon, Tessa; Borschel, Gregory H.; Surgery, School of MedicineMorphological analyses are key outcome assessments for nerve regeneration studies but are historically limited to tissue sections. Novel optical tissue clearing techniques enabling three-dimensional imaging of entire organs at a subcellular resolution have revolutionized morphological studies of the brain. To extend their applicability to experimental nerve repair studies we adapted these techniques to nerves and their motor and sensory targets in rats. The solvent-based protocols rendered harvested peripheral nerves and their target organs transparent within 24 hours while preserving tissue architecture and fluorescence. The optical clearing was compatible with conventional laboratory techniques, including retrograde labeling studies, and computational image segmentation, providing fast and precise cell quantitation. Further, optically cleared organs enabled three-dimensional morphometry at an unprecedented scale including dermatome-wide innervation studies, tracing of intramuscular nerve branches or mapping of neurovascular networks. Given their wide-ranging applicability, rapid processing times, and low costs, tissue clearing techniques are likely to be a key technology for next-generation nerve repair studies. All procedures were approved by the Hospital for Sick Children's Laboratory Animal Services Committee (49871/9) on November 9, 2019.Item QS8: The Roles of the TrkA and p75NTR NGF Receptors in Corneal Wound Healing(Wolters Kluwer, 2021-07-26) Tajdaran, Kiana; Feinberg, Konstantin; Mirmoeini, Seyed Kaveh; Zhang, Jennifer; Gordon, Tessa; Borschel, Gregory; Surgery, School of MedicinePurpose: The cornea is the window through which we see the world and is one of the most densely innervated structures in the body. Besides providing protective sensory input, corneal nerves have been postulated to stimulate limbal stem cells (LSCs), hence governing corneal epithelial maintenance and recovery. Loss of corneal innervation, through injury, diabetes, tumors, infections, and even improper contact lens use, leads to neurotrophic keratopathy (NK), a degenerative corneal disease that is characterized by corneal epithelial breakdown, scarring, and permanent vision loss1. The only non-invasive treatment option for NK is the human recombinant nerve growth factor (rhNGF), but the short half-life of exogenous neurotrophins-based therapies make this therapeutic approach less effective2. Development of the small molecule ligands for neurotrophins receptors that have better pharmacokinetics and plasma stability showed promising results in the treatment of several neurodegenerative conditions in the recent years3. In this study, we were prompted to investigate the molecular mechanism of NK and the role of NGF receptors, TrkA and p75NTR, in corneal healing. We hypothesized that TrkA inhibition would delay corneal wound healing and p75NTR inhibition accelerates corneal healing. This knowledge will lay the basis for a new non-invasive approach for NK. Methods: For this experiment, we took advantage of commercially available Ntrk1 mutant mice, which allow for pharmacological inhibition of TrkA receptor with an inhibitor known as not mammalian kinase inhibitor (1-NM-PP1)4. Ntrk1 mice (n=20) were divided into three groups, which received saline injection as a control. In one experimental group animals were received TrkA inhibitor and the other group received both TrkA and p75 inhibitor for 5 days. On day six we removed the corneal epithelium with a 0.5 mm rotating brush. To measure epithelial healing, we performed digital imaging of fluorescein staining daily for four days after injury. We then harvested the corneas for immunofluorescent and biochemical analyses. Results: Our results show a significant delay in corneal epithelial healing following TrkA inhibition and acceleration in corneal healing after p75NTR inhibition. Conclusion: A selective TrkA agonist or p75NTR inhibitors could be a new therapeutic approach for NK.Item Rapid, automated nerve histomorphometry through open-source artificial intelligence(Springer, 2022-04-08) Daeschler, Simeon Christian; Bourget, Marie-Hélène; Derakhshan, Dorsa; Sharma, Vasudev; Asenov, Stoyan Ivaylov; Gordon, Tessa; Cohen-Adad, Julien; Borschel , Gregory Howard; Surgery, School of MedicineWe aimed to develop and validate a deep learning model for automated segmentation and histomorphometry of myelinated peripheral nerve fibers from light microscopic images. A convolutional neural network integrated in the AxonDeepSeg framework was trained for automated axon/myelin segmentation using a dataset of light-microscopic cross-sectional images of osmium tetroxide-stained rat nerves including various axonal regeneration stages. In a second dataset, accuracy of automated segmentation was determined against manual axon/myelin labels. Automated morphometry results, including axon diameter, myelin sheath thickness and g-ratio were compared against manual straight-line measurements and morphometrics extracted from manual labels with AxonDeepSeg as a reference standard. The neural network achieved high pixel-wise accuracy for nerve fiber segmentations with a mean (± standard deviation) ground truth overlap of 0.93 (± 0.03) for axons and 0.99 (± 0.01) for myelin sheaths, respectively. Nerve fibers were identified with a sensitivity of 0.99 and a precision of 0.97. For each nerve fiber, the myelin thickness, axon diameter, g-ratio, solidity, eccentricity, orientation, and individual x -and y-coordinates were determined automatically. Compared to manual morphometry, automated histomorphometry showed superior agreement with the reference standard while reducing the analysis time to below 2.5% of the time needed for manual morphometry. This open-source convolutional neural network provides rapid and accurate morphometry of entire peripheral nerve cross-sections. Given its easy applicability, it could contribute to significant time savings in biomedical research while extracting unprecedented amounts of objective morphologic information from large image datasets.Item Schwann Cells Are Key Regulators of Corneal Epithelial Renewal(Association for Research in Vision and Ophthalmology (ARVO), 2023) Mirmoeini, Kaveh; Tajdaran, Kiana; Zhang, Jennifer; Gordon, Tessa; Ali, Asim; Kaplan, David R.; Feinberg, Konstantin; Borschel, Gregory H.; Surgery, School of MedicinePurpose: Corneal sensory nerves protect the cornea from injury. They are also thought to stimulate limbal stem cells (LSCs) to produce transparent epithelial cells constantly, enabling vision. In other organs, Schwann cells (SCs) associated with tissue-innervating axon terminals mediate tissue regeneration. This study defines the critical role of the corneal axon-ensheathing SCs in homeostatic and regenerative corneal epithelial cell renewal. Methods: SC localization in the cornea was determined by in situ hybridization and immunohistochemistry with SC markers. In vivo SC visualization and/or ablation were performed in mice with inducible corneal SC-specific expression of tdTomato and/or Diphtheria toxin, respectively. The relative locations of SCs and LSCs were observed with immunohistochemical analysis of harvested genetically SC-prelabeled mouse corneas with LSC-specific antibodies. The correlation between cornea-innervating axons and the appearance of SCs was ascertained using corneal denervation in rats. To determine the limbal niche cellular composition and gene expression changes associated with innervation-dependent epithelial renewal, single-cell RNA sequencing (scRNA-seq) of dissociated healthy, de-epithelized, and denervated cornea limbi was performed. Results: We observed limbal enrichment of corneal axon-associated myelinating and non-myelinating SCs. Induced local genetic ablation of SCs, although leaving corneal sensory innervation intact, markedly inhibited corneal epithelial renewal. scRNA-seq analysis (1) highlighted the transcriptional heterogenicity of cells populating the limbal niche, and (2) identified transcriptional changes associated with corneal innervation and during wound healing that model potential regulatory paracrine interactions between SCs and LSCs. Conclusions: Limbal SCs are required for innervation-dependent corneal epithelial renewal.Item Schwann Cells Are Required for Efficient Corneal Wound Healing(Wolters Kluwer, 2021) Mirmoeini, Seyed Kaveh; Feinberg, Konstantin; Tajdaran, Kiana; Zhang, Jennifer; Gordon, Tessa; Borschel, Gregory H.; Surgery, School of MedicineBACKGROUND: Corneal nerves play a crucial role in maintaining corneal health, which includes regulation of activity of limbal stem cell (LSC). Their loss leads to neurotrophic keratopathy (NK), with corneal ulceration, scarring, and ultimately, blindness. Having identified nerve-ensheathing Schwann cells (SC) in the corneal limbus, we hypothesize that SCs, via paracrine interaction with LSC, play a key role in corneal epithelial maintenance and healing. In this study we wanted to (1) Define the role of SCs in corneal healing; (2) Determine the paracrine interaction between the limbal SCs and LSC. METHODS: (1) Local corneal ablation of SCs was induced in a genetically modified mouse where the topical application of tamoxifen induced SCs apoptosis. The corneal epithelium was then removed with an Amoils brush under anesthesia and fluorescein was used to assess healing over 4 days. (2) We performed single-cell RNA expression analysis of 10,000 cells derived from dissociated rat limbus with droplet-based high throughput 10× Genomics to identify ~3000 genes. We used the data to predict possible ligand-receptor interactions between the limbal SCs and LSC. RESULTS: (1) Ablation of SCs impaired corneal wound healing in mouse cornea, suggesting the involvement of SC in innervation-dependent corneal epithelial recovery. (2) Genomic analysis suggested the presence of paracrine crosstalk between SCs and LSCs, and relevant downstream intracellular signaling events in LSCs. The latter included activation of Notch signaling and VEGF-mediated cell migration and inhibition of apoptosis. Further expression analysis comparing the limbal region of healthy and wounded corneas indicated significant changes in the expression of jag1, Pdgfa, Tgfb1, and Ptn genes by SCs. All of these genes could potentially play a role in corneal recovery. CONCLUSIONS: Our findings (i) describe the presence of a high volume of SCs at the limbus, located in close spatial vicinity to LSCs, (ii) demonstrate the importance of the limbal SCs for corneal wound healing, and (iii) suggest the presence of paracrine SC-LSC interaction that may be responsible for the limbal nerve-mediated activation of LSCs during homeostasis or the epithelial wound healing after injury. These findings suggest new therapeutic targets for treating NK.Item Sustained Release of Tacrolimus From a Topical Drug Delivery System Promotes Corneal Reinnervation(Association for Research in Vision and Ophthalmology (ARVO), 2022) Daeschler, Simeon C.; Mirmoeini, Kaveh; Gordon, Tessa; Chan, Katelyn; Zhang, Jennifer; Ali, Asim; Feinberg, Konstantin; Borschel, Gregory H.; Surgery, School of MedicinePurpose: Corneal nerve fibers provide sensation and maintain the epithelial renewal process. Insufficient corneal innervation can cause neurotrophic keratopathy. Here, topically delivered tacrolimus is evaluated for its therapeutic potential to promote corneal reinnervation in rats. Methods: A compartmentalized neuronal cell culture was used to determine the effect of locally delivered tacrolimus on sensory axon regeneration in vitro. The regenerating axons but not the cell bodies were exposed to tacrolimus (50 ng/mL), nerve growth factor (50 ng/mL), or a vehicle control. Axon area and length were measured after 48 hours. Then, a biodegradable nanofiber drug delivery system was fabricated via electrospinning of a tacrolimus-loaded polycarbonate-urethane polymer. Biocompatibility, degradation, drug biodistribution, and therapeutic effectiveness were tested in a rat model of neurotrophic keratopathy induced by stereotactic trigeminal nerve ablation. Results: Sensory neurons whose axons were exposed to tacrolimus regenerated significantly more and longer axons compared to vehicle-treated cultures. Trigeminal nerve ablation in rats reliably induced corneal denervation. Four weeks after denervation, rats that had received tacrolimus topically showed similar limbal innervation but a significantly higher nerve fiber density in the center of the cornea compared to the non-treated control. Topically applied tacrolimus was detectable in the ipsilateral vitreal body, the plasma, and the ipsilateral trigeminal ganglion but not in their contralateral counterparts and vital organs after 4 weeks of topical release. Conclusions: Locally delivered tacrolimus promotes axonal regeneration in vitro and corneal reinnervation in vivo with minimal systemic drug exposure. Translational relevance: Topically applied tacrolimus may provide a readily translatable approach to promote corneal reinnervation.