Illuminating Corneal Nerve Injury: Analysis of Neuronal Phenotypes Following Acute and Chronic Corneal Nerve Injury

The cornea is uniquely avascular, transparent, and densely innervated with a highly complex and rapidly adapting sensory system. Combined with the tear film coating its surface, it is the first line of protection for the visual system. Clear vision provides tremendous survival advantage and thus uses long evolved and highly conserved mechanisms within the corneal protective barrier to preserve its function. Corneal nerves have a critical role in maintaining corneal homeostasis, initiating blinking, and tearing circuits to block potentially damaging environmental insults, and for engaging rapid-response regeneration programs. Many of these critical molecular mechanisms have been broadly characterized over the past century. However, with new advances in transgenic models and precision medicine, current studies focus on specific molecular factors that can be targeted in translational research and treatments. Sry-Box Transcription Factor 11 (SOX11) is a known upstream regulator of the injury response regeneration associated gene network and is highly expressed in primary afferent neurons following injury. Currently, little is known about the role of SOX11 in nerve regeneration following corneal injury. The aim of this study was to examine corneal injury-induced pathology in direct and indirect corneal injury models, which include trephine-only (TO), corneal abrasion (CA), and lacrimal gland excision (LGE). Both wild-type C57B6/J, Nav1.8Cre-tdTomato and Nav1.8Cre-Sox11-tdTomato mice were used to characterize corneal pathology following these injuries. LGE-induced dry eye reduces the aqueous component of tears, resulting in persistent corneal epithelial cell damage and retraction of corneal afferent nerve terminals. TO ligates axon terminals to the subbasal plexus in the corneal epithelium. CA first uses TO injury directly followed by mechanically removing the corneal epithelium and axon terminals from the central cornea. Before and after injury, assays were performed to evaluate tearing, mechanical sensitivity, ocular discomfort, and the corneal epithelium. Tissue was collected at terminal timepoints, and whole-mount corneas were imaged, and afferent terminals were analyzed. Before injury, corneal cell bodies were labeled using retrograde tracer, and somal phenotype was evaluated using immunohistochemistry and in situ hybridization. Additionally, rt-qPCR was performed on both corneas and trigeminal ganglia in some experiments. The results show that while corneal innervation density decreased between 1-2 weeks following LGE in control animals, nerves regenerated to near normal levels by four weeks, albeit in a disorganized manner. In Nav1.8Cre-tdTomato-Sox11f/f (Sox11fl/fl) animals, innervation density was significantly reduced at the 4-week time point compared to control animals. As determined with fluorescein staining, corneal epithelial cell damage was similar between Sox11fl/fl and control animals over the 4 -weeks after LGE. Directly following CA-induced injury, both control and Sox11fl/fl animals showed significant decreases in innervation density at 24 hours. By 48 hours after injury, Sox11fl/fl animals showed a small yet significant increase in nerve growth. Both control and Sox11fl/fl animals demonstrated comparable reductions in corneal epithelial cell damage 48 hours after injury. Taken together, these results provide support for the critical role of SOX11 in nerve regeneration and healing following corneal injury

Psr1p interacts with SUN/sad1p and EB1/mal3p to establish the bipolar spindle

Regular Abstracts - Sunday Poster Presentations: no. 382During mitosis, interpolar microtubules from two spindle pole bodies (SPBs) interdigitate to create an antiparallel microtubule array for accommodating numerous regulatory proteins. Among these proteins, the kinesin-5 cut7p/Eg5 is the key player responsible for sliding apart antiparallel microtubules and thus helps in establishing the bipolar spindle. At the onset of mitosis, two SPBs are adjacent to one another with most microtubules running nearly parallel toward the nuclear envelope, creating an unfavorable microtubule configuration for the kinesin-5 kinesins. Therefore, how the cell organizes the antiparallel microtubule array in the first place at mitotic onset remains enigmatic. Here, we show that a novel protein psrp1p localizes to the SPB and plays a key role in organizing the antiparallel microtubule array. The absence of psr1+ leads to a transient monopolar spindle and massive chromosome loss. Further functional characterization demonstrates that psr1p is recruited to the SPB through interaction with the conserved SUN protein sad1p and that psr1p physically interacts with the conserved microtubule plus tip protein mal3p/EB1. These results suggest a model that psr1p serves as a linking protein between sad1p/SUN and mal3p/EB1 to allow microtubule plus ends to be coupled to the SPBs for organization of an antiparallel microtubule array. Thus, we conclude that psr1p is involved in organizing the antiparallel microtubule array in the first place at mitosis onset by interaction with SUN/sad1p and EB1/mal3p, thereby establishing the bipolar spindle.postprin

Removal of antagonistic spindle forces can rescue metaphase spindle length and reduce chromosome segregation defects

Regular Abstracts - Tuesday Poster Presentations: no. 1925Metaphase describes a phase of mitosis where chromosomes are attached and oriented on the bipolar spindle for subsequent segregation at anaphase. In diverse cell types, the metaphase spindle is maintained at a relatively constant length. Metaphase spindle length is proposed to be regulated by a balance of pushing and pulling forces generated by distinct sets of spindle microtubules and their interactions with motors and microtubule-associated proteins (MAPs). Spindle length appears important for chromosome segregation fidelity, as cells with shorter or longer than normal metaphase spindles, generated through deletion or inhibition of individual mitotic motors or MAPs, showed chromosome segregation defects. To test the force balance model of spindle length control and its effect on chromosome segregation, we applied fast microfluidic temperature-control with live-cell imaging to monitor the effect of switching off different combinations of antagonistic forces in the fission yeast metaphase spindle. We show that spindle midzone proteins kinesin-5 cut7p and microtubule bundler ase1p contribute to outward pushing forces, and spindle kinetochore proteins kinesin-8 klp5/6p and dam1p contribute to inward pulling forces. Removing these proteins individually led to aberrant metaphase spindle length and chromosome segregation defects. Removing these proteins in antagonistic combination rescued the defective spindle length and, in some combinations, also partially rescued chromosome segregation defects. Our results stress the importance of proper chromosome-to-microtubule attachment over spindle length regulation for proper chromosome segregation.postprin

Biomarkers of neurological tissue injury and inflammation in paediatric tuberculous meningitis

Includes bibliographical references.[Background] Tuberculous meningitis (TBM) in children has high mortality and neurological morbidity rates. The assessment of disease severity and prognostication are difficult because several factors influence initial presentation, and advanced tools for these are lacking. Biomarkers of neurological injury could help to assess severity and to prognosticate, but have not been assessed in paediatric TBM. This study examined serum and cerebrospinal fluid (CSF) biomarkers of neurological injury in paediatric TBM in association with clinical and physiological data, radiology, inflammatory markers, and outcome. [ Methods ] Serum and CSF (ventricular and lumbar) samples were taken on admission and over 3 weeks in children with probable TBM and hydrocephalus. These were analysed with ELISA for neuromarkers S100B, neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP), and with Luminex multianalyte array assay for a panel of inflammatory markers. Results were compared with 2 controls groups. Computerized tomography was done on admission and magnetic resonance imaging (brain, spine and magnetic resonance angiography) at 3 weeks. Brain oxygenation was monitored invasively and non-invasively in selected patients. Clinical and neurodevelopmental outcomes were assessed at 6 months. Data were analysed with various statistical tools, including principal component analysis. [ Results ] Data were collected from 44 children. Of these, 16% died and 36% had disability (25% mildmoderate, 11% severe). S100B, NSE, GFAP and inflammatory markers were elevated in CSF on admission and for up to 3 weeks, but not in serum. Elevated neuromarkers were significantly associated with poor outcome and increased over time in patients who died, although combined inflammatory biomarkers decreased. Cerebral infarcts occurred in 66% of patients and were associated with neuromarker elevation. Novel findings on spinal MRI were the high frequency of asymptomatic disease. Cerebral vascular pathology was documented frequently on imaging but did not predict infarcts. Low brain oxygenation was common and in keeping with physiological events and outcome. [ Conclusion ] CSF neuro- and inflammatory markers are elevated in TBM. Neuromarkers were prognostic of clinical and radiological outcome and an increasing trend suggested ongoing injury. This does not appear to be related to ongoing inflammation as measured by cytokines but may reflect the ongoing secondary injury processes initiated by inflammation

Chemokine modulation of hippocampal function across aging

The hippocampus undergoes several structural, cellular, and functional changes during normal aging which is directly associated with cognitive decline and the pathogenesis of neurodegenerative disease. Dysregulation of innate immune function in the peripheral and central nervous system during aging is thought to contribute to reduced cognition and the pathophysiological events leading to neurodegeneration. Most studies of aging compare young individuals with elderly, however there is limited investigation into whether hippocampal function also declines in early aging. In this thesis I used in vitro electrophysiology on mouse hippocampal slices to examine hippocampal excitability along the perforant path in an early aging model, comparing neuronal activity between juvenile (9-15 weeks old) and adult (25-35 weeks old) C57BL/6J mice. I also examined if the Duffy antigen receptor for chemokines (DARC), which mediates circulating pro-inflammatory chemokine concentration, was affecting hippocampal excitability using homozygous DARC knockout mice. Extracellular measurements of dentate gyrus neurons during perforant pathway stimulation showed that hippocampal excitability increases in early aging. Intracellular measurements of intrinsic membrane properties indicated that dentate gyrus granule neurons become hyperpolarised and have increased membrane resistance during early aging, which occurred only in the supra-pyramidal subregion. Bath application of chemokine CCL2 increased hippocampal excitability via its cognate receptor CCR2, however not in an age-dependent manner, indicating that CCL2 does not regulate early age-dependent excitability. Measurements of population excitability and granule cell intrinsic properties in DARC knockout mice indicated that DARC regulates neuronal excitability in early aging and resting membrane potential of supra-pyramidal granule neurons. Adult DARC knockout mice also had increased hippocampal microglia proliferation. These are the first reported effects of DARC deficiency on brain cells. These implications of these findings, for our overall understanding of how hippocampal function changes in early aging and how chemokines and chemokine receptors alter hippocampal excitability, are discussed

MOLECULAR ENGINEERING STRATEGIES FOR THE DEVELOPMENT OF ENERGY-TRANSPORTING CONJUGATED SYSTEMS TOWARDS BIOELECTRONICS

Peptidic nanostructures appended with π-electron units present a powerful class of biomaterials that merges the chemical versatility of self-assembling peptides and the optoelectronic function of organic π-electron units. This dissertation spans research efforts from understanding some rational design concepts to the bioelectronic utility of π-conjugated peptides. Chapter 1 describes the progress in the field of peptidic nanomaterials bearing π-electron systems and how this relates to advances in bioelectronics. In Chapters 2 and 3, key properties and aspects of molecular design that can be utilized to rationally tune optoelectronic and mechanical properties of oligothiophene-peptide hybrid hydrogelators are discussed. Chapter 2 focuses on the effect of varied amino acid size and hydrophobicity on material properties at different length scales. Chapter 3 investigates the photophysical effects of confining individual π-units within nanostructures and how the coassembly behavior is affected by local fields imparted by the peptide moieties. The next two chapters introduce a multicomponent strategy to create and characterize the nanostructure of different functional materials, either with the co-incorporation of bioactive epitopes within peptide nanostructures or showing energy and/or electron transfer events among π-electron systems that are spatially engineered within peptidic constructs. Chapter 4 presents energy-transporting nanomaterials comprised of donor-acceptor peptide pairs existing in either solution or hydrogel phase. Chapter 5 aims to shed light on the local structure formed upon supramolecular coassembly in both solution and hydrogel phases using solid state NMR and small-angle neutron scattering techniques. Finally, Chapter 6 presents the developmental efforts towards creating biological scaffolds out of these peptidic nanostructures with tunable physicochemical properties that can potentially facilitate nanostructural energy transport upon electrical or light stimulation. These peptide nanomaterials offer a platform for scaffolds that can mimic the natural environment of electrosensitive tissues such as nerves. Much of the progress accomplished towards this application is to establish a stable system against material degradation during long periods under cell culture conditions. We show that aligned constructs that are pre-assembled by an external trigger followed by covalent crosslinking were successful in imparting topographical guidance to human neural stem cells or dorsal root ganglion neuron explants. The latter part of this Chapter reports the extension of these efforts towards developing known electroactive organic polymers that can be processed as aligned soft materials for neural stem cells and neonatal rat ventricular cardiomyocytes. This dissertation aims to bridge the understanding of chemical design and self-assembly principles with the biomaterial applications of peptide-based optoelectronic assemblies and related conjugated polymers