Axonal Viability Research Articles

NAD+, Axonal Maintenance, and Neurological Disease.

Significance: The remarkable geometry of the axon exposes it to unique challenges for survival and maintenance. Axonal degeneration is a feature of peripheral neuropathies, glaucoma, and traumatic brain injury, and an early event in neurodegenerative diseases. Since the discovery of Wallerian degeneration (WD), a molecular program that hijacks nicotinamide adenine dinucleotide (NAD+) metabolism for axonal self-destruction, the complex roles of NAD+ in axonal viability and disease have become research priority. Recent Advances: The discoveries of the protective Wallerian degeneration slow (WldS) and of sterile alpha and TIR motif containing 1 (SARM1) activation as the main instructive signal for WD have shed new light on the regulatory role of NAD+ in axonal degeneration in a growing number of neurological diseases. SARM1 has been characterized as a NAD+ hydrolase and sensor of NAD+ metabolism. The discovery of regulators of nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2) proteostasis in axons, the allosteric regulation of SARM1 by NAD+ and NMN, and the existence of clinically relevant windows of action of these signals has opened new opportunities for therapeutic interventions, including SARM1 inhibitors and modulators of NAD+ metabolism. Critical Issues: Events upstream and downstream of SARM1 remain unclear. Furthermore, manipulating NAD+ metabolism, an overdetermined process crucial in cell survival, for preventing the degeneration of the injured axon may be difficult and potentially toxic. Future Directions: There is a need for clarification of the distinct roles of NAD+ metabolism in axonal maintenance as contrasted to WD. There is also a need to better understand the role of NAD+ metabolism in axonal endangerment in neuropathies, diseases of the white matter, and the early stages of neurodegenerative diseases of the central nervous system. Antioxid. Redox Signal. 39, 1167-1184.

Survival of rat sciatic nerve segments preserved in storage solutions ex vivo assessed by novel electrophysiological and morphological criteria.

Most organ or tissue allografts with viable cells are stored in solutions ex vivo for hours to several days. Most allografts then require rapid host revascularization upon transplantation to maintain donor-cell functions (e.g., cardiac muscle contractions, hepatic secretions). In contrast, peripheral nerve allografts stored ex vivo do not require revascularization to act as scaffolds to guide outgrowth by host axons at 1-2 mm/d, likely aided by viable donor Schwann cells. Using current storage solutions and protocols, axons in all these donor organ/tissue/nerve transplants are expected to rapidly become non-viable due to Wallerian degeneration within days. Therefore, ex vivo storage solutions have not been assessed for preserving normal axonal functions, i.e., conducting action potentials or maintaining myelin sheaths. We hypothesized that most or all organ storage solutions would maintain axonal viability. We examined several common organ/tissue storage solutions (University of Wisconsin Cold Storage Solution, Normosol-R, Normal Saline, and Lactated Ringers) for axonal viability in rat sciatic nerves ex vivo as assessed by maintaining: (1) conduction of artificially-induced compound action potentials; and (2) axonal and myelin morphology in a novel assay method. The ten different storage solution conditions for peripheral nerves with viable axons (PNVAs) differed in their solution composition, osmolarity (250-318 mOsm), temperature (4°C vs. 25°C), and presence of calcium. Compound action potentials and axonal morphology in PNVAs were best maintained for up to 9 days ex vivo in calcium-free hypotonic diluted (250 mOsm) Normosol-R (dNR) at 4°C. Surprisingly, compound action potentials were maintained for only 1-2 days in UW and NS at 4°C, a much shorter duration than PNVAs maintained in 4°C dNR (9 days) or even in 25°C dNR (5 days). Viable axons in peripheral nerve allografts are critical for successful polyethylene glycol (PEG)-fusion of viable proximal and distal ends of host axons with viable donor axons to repair segmental-loss peripheral nerve injuries. PEG-fusion repair using PNVAs prevents Wallerian degeneration of many axons within and distal to the graft and results in excellent recovery of sensory/motor functions and voluntary behaviors within weeks. Such PEG-fused PNVAs, unlike all other types of conventional donor transplants, are immune-tolerated without tissue matching or immune suppression. Preserving axonal viability in stored PNVAs would enable the establishment of PNVA tissue banks to address the current shortage of transplantable nerve grafts and the use of stored PEG-fused PNVAs to repair segmental-loss peripheral nerve injuries. Furthermore, PNVA storage solutions may enable the optimization of ex vivo storage solutions to maintain axons in other types of organ/tissue transplants.

Predictors of Co-activation in Erb's Palsy: A Retrospective Study.

Three per thousand births have Erb's palsy. Spontaneous recovery is 50%. Co-activation yields poor outcomes. There are no objective indicators of its emergence. Analyze if 1 month Axon Viability Index (AVI) of the axillary nerve and which active movement score (AMS) measures can predict co-activation. Tertiary level rehabilitation center, retrospective design. The electronic medical record (EMR) was reviewed for patients with Erb's palsy with Narakas grade 2 lesions, as having co-activation or not. The one-month Axillary AVI was used with monthly AMS scores. The inclusion criteria were an AVI greater than ten percent. Exclusion criteria were bi-brachial palsy, congenital anomalies, concomitant or subsequent neurological injuries, and orthopedic injuries. Descriptive statistics were used to calculate the median and interquartile values for AMS scores at each respective time point. Statistical significance for each time point was determined using a student's t-test. Regarding the t-test on the AVI data, a significant P value of 0.001 was found favoring the co-activation group. AVI of the Axillary nerve between 0.1 and 0.5 at 1 month is a reliable indicator of future development of co-activation. The following were strong indicators of the emergence of co-activation respectively: month three Wrist Extension in sitting, Shoulder Abduction in supine, Shoulder Abduction in sitting, Elbow Flexion in sitting, month six Elbow Flexion in sitting, month seven Elbow Flexion in sitting. The axillary AVI at one month is a good predictor of future development of co-activation. The mentioned AMS items are the earliest indicators of co-activation.

Brain metabolite alterations related to alcohol use: a meta-analysis of proton magnetic resonance spectroscopy studies.

Alcohol misuse and alcohol use disorder (AlUD) have neurobiological consequences. This meta-analysis of proton magnetic resonance spectroscopy (MRS) studies aimed to assess the differences in brain metabolite levels in alcohol misuse and AUD relative to controls (PROSPERO registration: CRD42020209890). Hedge's g with random-effects modeling was used. Sub-group and meta-regression techniques explored potential sources of demographic and MRS parameter heterogeneity. A comprehensive literature review identified 43 studies, resulting in 69 models across gray and white matter (GM, WM). Lower N-acetylaspartate levels were found in frontal, anterior cingulate cortex (ACC), hippocampal, and cerebellar GM, and frontal and parietal WM, suggesting decreased neuronal and axonal viability. Lower choline-containing metabolite levels (all metabolites contributing to choline peak) were found in frontal, temporal, thalamic, and cerebellar GM, and frontal and parietal WM, suggesting membrane alterations related to alcohol misuse. Lower creatine-containing metabolite levels (Cr; all metabolites contributing to Cr peak) were found in temporal and occipital cortical GM, while higher levels were noted in midbrain/brainstem GM; this finding may have implications for using Cr as an internal reference. The lack of significant group differences in glutamate-related levels is possibly related to biological and methodological complexities. The few studies reporting on GABA found lower levels restricted to the ACC. Confounding variables were age, abstinence duration, treatment status, and MRS parameters (echo time, quantification type, data quality). This first meta-analysis of proton MRS studies consolidates the numerous individual studies to identify neurometabolite alterations within alcohol misuse and AUD. Future studies can leverage this new formalized information to investigate treatments that might effectively target the observed disturbances.

QS1: Pre-implanted Nerve Grafts (PING) to Improve Functional Outcomes in VCA

Purpose:Improved graft reinnervation and functional recovery would greatly enhance the utility of vascularized composite allotransplantation (VCA). Pre-implantation of nerve grafts in a limb transplant recipient prior to transplantation to increase the length of the severed nerves in the amputation stump offers a novel, readily translatable approach to this problem. At the time of transplantation a pre-implanted nerve graft (PING) would be elevated from its wound bed in the amputated stump and unfurled distally into the transplanted limb, allowing for a more distal nerve coaptation.Methods:We used a reversed sciatic-tibial nerve isograft coapted end-to-end to a median nerve in a rat to: (1) determine viability of distal axons after elevating PINGs; and (2) assess for ischemic injury to axons in the distal PING following elevation from the wound bed. For axonal viability, grafts of varying lengths (2cm,3cm,4cm, n≥5/group) were left to regenerate for 8 weeks. The distal ends were then biopsied prior to elevation, and again one week after elevation. For ischemia assessment, blood flow along 4cm long PINGs was measured with laser doppler at multiple timepoints: baseline (at time of grafting), 8 weeks after grafting (before elevation), immediately after elevation, and 3 and 7 days post-elevation (n=8/timepoint). Means were compared with t-tests and ANOVA. A pig forelimb model was used to assess the functional impact of PINGs. A 6cm long reversed ulnar-PING was coapted to the proximally transected ulnar nerve. After 12 weeks of regeneration, the distal PING was transferred end-to-end to the median nerve just proximal to the elbow. A control group underwent ulnar-to-median nerve transfer 6cm proximal to the elbow (n=4 limbs/group). Hoof flexion force was measured every three weeks.Results Axonal viability:after 8 weeks, axons had regenerated into the distal grafts in all lengths: mean myelinated axon counts (±SD): 7043 (±790), 6066 (±1838), 6491 (±983) in 2cm,3cm,4cm grafts, with no differences between graft lengths (p=0.48). One week after elevation and unfurling, the number of myelinated axons was significantly decreased in all groups (p<0.006): 3821 (±567), 2953 (±1107), 2894 (±1761) in 2cm,3cm,4cm grafts, with no differences between lengths (p=0.91). Ischemic injury: immediately after elevating a PING, blood flow within the distal half of the graft fell to baseline levels (i.e. no blood flow). 3 days after elevation, blood flow throughout the graft was significantly above baseline (p≤0.04) and matched/exceeded flow measured immediately prior to elevation. Functional impact: preliminary results suggest PINGs can deliver rapid functional recovery.Conclusions:Axons remain present in clinically relevant numbers at the distal ends of PINGs after elevation and unfurling. A decrease in axon counts after elevation may relate to ischemia in the distal PING immediately after elevation. Preliminary functional results show PINGs are capable of delivering early functional recovery.

Neurotoxic Effect of Fipronil in Neuroblastoma SH-SY5Y Cell Line.

Fipronil is a broad-spectrum insecticide belonging to the phenyl pyrazole chemical family. Fipronil disrupts gamma-aminobutyric acid (GABA) receptors in the central nervous system, thereby blocking GABA-gated chloride channels. Neurotoxic symptoms of fipronil poisoning in humans are typically associated with the antagonism of central GABA receptors. In this study, the cytotoxic effects of fipronil in SH-SY5Y neuroblastoma cell line, the human neural cell line, were investigated. SH-SY5Y cells were exposed to 125, 250, and 500μM fipronil doses and incubated for 24 and 48h, and its neurotoxic effect was examined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide) test, lactate dehydrogenase (LDH) test, trypan blue dye exclusion test, and neutral red uptake assay. Acridine orange-propidium iodide and DAPI (4',6-diamidino-2-phenylindole dihydrochloride) staining methods were also used to determine the type of cell death. In addition, neurodegenerative changes were also examined by immunofluorescence staining of the cytoskeleton. The amount of neurofilament heavy chain and the degree of phosphorylation of neurofilament subunits were investigated to determine the effect of fipronil on the axonal viability, and we observed a reduction in the amount of neurofilament heavy chain, speculating disruption of the axonal transport leading to cell death. According to our results, we may suggest that fipronil caused cytotoxicity in SH-SY5Y cells.

Neuron-specific PERK inactivation exacerbates neurodegeneration during experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), are chronic inflammatory demyelinating and neurodegenerative diseases of the CNS. Although neurodegeneration is the major contributor to chronic disability in MS, mechanisms governing the viability of axons and neurons in MS and EAE remain elusive. Data indicate that activation of pancreatic endoplasmic reticulum kinase (PERK) influences, positively or negatively, neuron and axon viability in various neurodegenerative diseases through induction of ATF4. In this study, we demonstrate that the PERK pathway was activated in neurons during EAE. We found that neuron-specific PERK inactivation impaired EAE resolution and exacerbated EAE-induced axon degeneration, neuron loss, and demyelination. Surprisingly, neuron-specific ATF4 inactivation did not alter EAE disease course or EAE-induced axon degeneration, neuron loss, and demyelination. These results suggest that PERK activation in neurons protects axons and neurons against inflammation in MS and EAE through ATF4-independent mechanisms.

Late Endosomes Act as mRNA Translation Platforms and Sustain Mitochondria in Axons

SummaryLocal translation regulates the axonal proteome, playing an important role in neuronal wiring and axon maintenance. How axonal mRNAs are localized to specific subcellular sites for translation, however, is not understood. Here we report that RNA granules associate with endosomes along the axons of retinal ganglion cells. RNA-bearing Rab7a late endosomes also associate with ribosomes, and real-time translation imaging reveals that they are sites of local protein synthesis. We show that RNA-bearing late endosomes often pause on mitochondria and that mRNAs encoding proteins for mitochondrial function are translated on Rab7a endosomes. Disruption of Rab7a function with Rab7a mutants, including those associated with Charcot-Marie-Tooth type 2B neuropathy, markedly decreases axonal protein synthesis, impairs mitochondrial function, and compromises axonal viability. Our findings thus reveal that late endosomes interact with RNA granules, translation machinery, and mitochondria and suggest that they serve as sites for regulating the supply of nascent pro-survival proteins in axons.

Caspase-6 is a Dispensable Enabler of Adult Mammalian Axonal Degeneration

The progress of axonal degeneration (AxD) following injury or insult impacts both recovery from axonal transection and protection of axons from diverse insults, or axonopathy. Here we provide evidence that increases in capase-6 (Casp6) expression and action contribute to the progression of AxD. The expression of Casp6 protein and mRNA in distal branches of sensory axons undergoing AxD was confirmed. We developed and utilized a new model of axonopathy in live mice by serially visualizing the viability of cutaneous axons in the ear pinna that expressed an axonal YFP transgene, in response to capasaicin-induced AxD. Both specific pharmacological inhibition of caspase-6 and local knockdown offered early but subtle and mild attenuation of axonopathy. To evaluate an axon autonomous role of Casp6, we examined axon integrity following transection ex vivo, and analyzed the serial morphological fragmentation of neurofilament expression as a structural index of AxD. Adding a specific Casp6 inhibitor to the preparation delayed neurofilament fragmentation. Intact motor axons of Casp6 null mice had normal electrophysiological properties but, as tested serially during AxD, there was attenuated loss of excitability. Following transection, morphological features of AxD were evident in both wild type and Casp6−/− mice but the latter had evidence of slowed progression. Taken together, our findings suggest a subtle but dispensable enabling role of local Casp6 expression in axons undergoing AxD. Serial analysis of cutaneous ear pinna axons in live mice provides a useful and novel model of axonal integrity.

NTE/PNPLA6 is expressed in mature Schwann cells and is required for glial ensheathment of Remak fibers.

Neuropathy target esterase (NTE) or patatin-like phospholipase domain containing 6 (PNPLA6) was first linked with a neuropathy occurring after organophosphate poisoning and was later also found to cause complex syndromes when mutated, which can include mental retardation, spastic paraplegia, ataxia, and blindness. NTE/PNPLA6 is widely expressed in neurons but experiments with its Drosophila orthologue Swiss-cheese (SWS) suggested that it may also have glial functions. Investigating whether NTE/PNPLA6 is expressed in glia, we found that NTE/PNPLA6 is expressed by Schwann cells in the sciatic nerve of adult mice with the most prominent expression in nonmyelinating Schwann cells. Within Schwann cells, NTE/PNPLA6 is enriched at the Schmidt-Lanterman incisures and around the nucleus. When analyzing postnatal expression patterns, we did not detect NTE/PNPLA6 in promyelinating Schwann cells, while weak expression was detectable at postnatal day 5 in Schwann cells and increased with their maturation. Interestingly, NTE/PNPLA6 levels were upregulated after nerve crush and localized to ovoids forming along the nerve fibers. Using a GFAP-based knock-out of NTE/PNPLA6, we detected an incomplete ensheathment of Remak fibers whereas myelination did not appear to be affected. These results suggest that NTE/PNPLA6 is involved in the maturation of nonmyelinating Schwann cells during development and de-/remyelination after neuronal injury. Since Schwann cells play an important role in maintaining axonal viability and function, it is therefore likely that changes in Schwann cells contribute to the locomotory deficits and neuropathy observed in patients carrying mutations in NTE.

Neuroprotective effects of vascular endothelial growth factor A in the experimental autoimmune encephalomyelitis model of multiple sclerosis

Neuroprotective effects of vascular endothelial growth factor A in the experimental autoimmune encephalomyelitis model of multiple sclerosis

Axonal motor protein KIF5A and associated cargo deficits in multiple sclerosis lesional and normal-appearing white matter.

Understanding the causes of axonal pathology remains a key goal in the pursuit of new therapies to target disease progression in multiple sclerosis (MS). Anterograde axonal transport of many proteins vital for axonal viability is mediated by the motor protein KIF5A, which has been linked to several neurological diseases. This study aimed to investigate the expression of KIF5A protein and its associated cargoes: amyloid precursor protein (APP) and neurofilament (NF) in post mortem MS and control white matter (WM) and to determine if KIF5A expression is influenced by the presence of MS risk single nucleotide polymorphisms (SNPs) identified in the region of the KIF5A gene. Using immunoblotting assays we analysed the expression of KIF5A, APP and NF phospho-isoforms in 23 MS cases and 12 controls. We found a significant reduction in KIF5A and associated cargoes in MS WM and an inverse correlation between KIF5A and APP/NF protein levels. Furthermore, homozygous carriers of MS risk gene SNPs show significantly lower levels of KIF5A protein compared to MS patients with no copies of the risk SNPs. We conclude that reduced expression of axonal motor KIF5A may have important implications in determining axonal transport deficits and ongoing neurodegeneration in MS.

The RNA-binding protein SFPQ orchestrates an RNA regulon to promote axon viability.

To achieve accurate spatiotemporal patterns of gene expression, RNA-binding proteins (RBPs) guide nuclear processing, intracellular trafficking, and local translation of target mRNAs. In neurons, RBPs direct transport of target mRNAs to sites of translation in remote axons and dendrites. However, it is not known whether an individual RBP coordinately regulates multiple mRNAs within these morphologically complex cells. Here we identify SFPQ (Splicing factor, proline-glutamine rich) as an RBP that binds and regulates multiple mRNAs in dorsal root ganglion (DRG) sensory neurons and thereby promotes neurotrophin-dependent axonal viability. SFPQ acts within nuclei, cytoplasm and axons to regulate multiple functionally related mRNAs essential for axon survival. Notably SFPQ is required for co-assembly of laminb2 and bclw within RNA granules and for axonal trafficking of these mRNAs. Together these data demonstrate that SFPQ orchestrates spatial gene expression of a newly identified RNA regulon essential for axonal viability.

Functional consequences of ethidium bromide demyelination of the mouse ventral spinal cord

Ethidium bromide (EB) has been extensively used in the rat as a model of spinal cord demyelination. However, this lesion has not been addressed in the adult mouse, a model with unlimited genetic potential. Here we characterize behavioral function, inflammation, myelin status and axonal viability following bilateral injection of 0.20mg/mL ethidium bromide or saline into the ventral white matter (VWM) of female C57Bl/6 mice. EB-induced VWM demyelination significantly reduced spared VWM and Basso Mouse Scale (BMS) scores persisting out to 2months. Chronic hindlimb dysfunction was accompanied by a persistent inflammatory response (demonstrated by CD45+ immunofluorescence) and axonal loss (demonstrated by NF-M immunofluorescence and electron microscopy; EM). These cellular responses differ from the rat where inflammation resolves by 3–4weeks and axon loss is minimal following EB demyelination. As these data suggest that EB-injection in the mouse spinal cord is a non-remyelinating lesion, we sought to ask whether wheel running could promote recovery by enhancing plasticity of local lumbar circuitry independent of remyelination. This did not occur as BMS and Treadscan® assessment revealed no significant effect of wheel running on recovery. However, this study defines the importance of descending ventral motor pathways to locomotor function in the mouse as VWM loss results in a chronic hindlimb deficit.

Proton Magnetic Resonance Spectroscopy

Magnetic resonance spectroscopy (MRS) provides information on neuronal and axonal viability, energetics of cellular structures, and status of cellular membranes. Proton MRS appeals to clinicians and scientists because its application in the clinical setting can increase the specificity of MR imaging. The objective of this article is to provide descriptive concepts of the technique and its application in vivo for a variety of patient populations. When appropriately incorporating MRS into the neuroradiologic evaluation, this technique produces relevant information to radiologists and clinicians for their understanding of adult and pediatric neurologically based disease processes.

Letter to the Editor Involvement of the corpus callosum splenium in a case with SSPE: magnetic resonance spectroscopy findings

Subacute sclerosing panencephalitis (SSPE) is a fatal, incurable, inflammatory, and neurodegenerative disease. The parieto-occipital lobes are the most frequently and severely affected. The basal ganglia, cerebellum, spinal cord and corpus callosum are less frequently involved [1–4]. Because magnetic resonance spectroscopy (MRS) provides information regarding in vivo brain metabolism and neuronal function, it is a promising method that may illustrate the pathophysiological features of SSPE [5–8]. To our knowledge, this is the first report of MRS findings of corpus callosum in a case with SSPE. A 15-year-old, previously healthy boy was hospitalized because of 4-week history of muscle weakness, impaired vision, and gait imbalance. He had not been vaccinated. A neurological examination revealed symmetric muscular weakness, ataxia, sequential slow movement, decreased motor activity and deep tendon reflexes, and negative Babinski reflex. He had exhibited myoclonic jerks, intellectual deterioration and behavioral changes. The characteristic burst suppression pattern was seen on EEG. Cerebrospinal fluid investigation revealed oligoclonal IgG bands and elevated titers of the measles antibody. He was diagnosed as clinical stage II according to the Jabbour classification. Magnetic resonance imaging (MRI) demonstrated diffuse hyperintensity in the cortical gray and subcortical, deep and periventricular white matter of the parieto-occipital region with mass effect observed as sulcal effacement and hyperintensity in the splenium of the corpus callosum on T2 and FLAIR weighted images. MRS was performed (TE: 135 ms). Voxels were placed in the right parietooccipital region and splenium of the corpus callosum. MR spectrum obtained from right parietooccipital subcortical and deep white matter and splenium of the corpus callosum showed a significant decrease in N-acetylaspartate (NAA)/creatine (Cr) and increase in the ratio of choline (Cho)/Cr. Additionally, there were also inverted prominent lactate peaks (Figures 1 and ​and22). Figure 1 Sagittal T2-weighted images show widespread hyperintensities in cortical-subcortical and deep periventricular white matter of right parietooccipital region and in splenium of corpus callosum Figure 2 Multivoxel MRS (TE: 135 ms) obtained from splenium of corpus callosum reveal prominent decreased NAA/Cr, increased Cho/Cr and presence of lactate at 1.33 ppm (inverted double) Subacute sclerosing panencephalitis is persistent and chronic encephalitis secondary to measles virus infection that causes widespread demyelination of the central nervous system. Since clinical profiles of the disease lead to various presentations, early diagnosis and true clinical staging are not always easy. Usually, MRI findings and the clinical stage of SSPE are not correlated. It is not possible to define these findings as improvement or progression based on the MRI findings alone. Despite the progress of SSPE, improvement of MRI findings may be seen [2–5]. It has been reported that histopathological examinations show evidence of neuronal loss, astrogliosis, demyelination, and infiltration of inflammatory cells [5]. Magnetic resonance spectroscopy can provide information on neuronal and axonal viability, cellular energetics, and membrane status. In our case, MRS showed significantly decreased NAA/Cr ratios in the right parietooccipital subcortical white matter and splenium of the corpus callosum. The MR spectrum of the corpus callosal splenial lesion revealed increased Cho/Cr and presence of lactate. These findings might indicate neuronal loss, demyelination, active inflammation and/or anaerobic metabolism. Magnetic resonance spectroscopy findings were consistent with stage III SSPE [5]. Although to date SSPE is not completely curable, with early diagnosis it is possible to slow the progression of the disease. Although MRS is not specific or diagnostic, it can show metabolic changes in the early-late stage [7]. For this reason, MRS may be useful to reveal the extent and progression of SSPE.

Computational Modeling of Axonal Microtubule Bundles under Tension

Microtubule bundles cross-linked by tau protein serve a variety of neurological functions including maintaining mechanical integrity of the axon, promoting axonal growth, and facilitating cargo transport. It has been observed that axonal damage in traumatic brain injury leads to bundle disorientation, loss of axonal viability, and cognitive impairment. This study investigates the initial mechanical response of axonal microtubule bundles under uniaxial tension using a discrete bead-spring representation. Mechanisms of failure due to traumatic stretch loading and their impact on the mechanical response and stability are also characterized. This study indicates that cross-linked axonal microtubule bundles in tension display stiffening behavior similar to a power-law relationship from nonaffine network deformations. Stretching of cross-links and microtubule bending were the primary deformation modes at low stresses. Microtubule stretch was negligible up to tensile stresses of ∼1 MPa. Bundle failure occurred by failure of cross-links leading to pull-out of microtubules and loss of bundle integrity. This may explain the elongation, undulation, and delayed elasticity of axons following traumatic stretch loading. More extensively cross-linked bundles withstood higher tensile stresses before failing. The bundle mechanical behavior uncovered by these computational techniques should guide future experiments on stretch-injured axons.

Neurofilament dot blot assays: Novel means of assessing axon viability in culture

Axonal structure and integrity are vital to overall neuronal maintenance and action potential propagation. Neurofilaments (NFs) are one of the main cytoskeletal components of axons and phosphorylation of NF subunits regulates speed of NF transport through axons and determines optimal axonal calibre required for signal propagation. Many previous studies of neuroprotective agents have focussed on neuronal viability in models of neurodegenerative disease, without specifically considering axon function as an indicator of neuronal damage. In this study, we have focused on developing novel assays for determining axon viability by measuring levels of neurofilament phosphorylation in cultured cortical neurons. The nitric oxide donor DETANONOate (NO) was used as an inflammatory insult and glial cell line-derived neurotrophic factor (GDNF) and superoxide dismutase (SOD) were tested as potential axonal protective agents. Using ‘dot blot’ methodologies, we show a decrease in NF phosphorylation in cortical neurons exposed to NO-mediated cell toxicity and an attenuation of NO-mediated changes in NF phosphorylation associated with GDNF and SOD treatment. These results correlated well with immunocytochemical counts. We propose therefore that the dot blot assay is a novel method for assessing axonal integrity in vitro and may play a useful role in the future for testing the effects of agents on axonal viability, providing a reliable and reproducible screening method for potential therapeutics for neurodegenerative diseases.

Motor Nerve-Conduction Studies in Obstetric Brachial Plexopathy for a Selection of Patients with a Poor Outcome

The criteria and timing for nerve surgery in infants with obstetric brachial plexopathy remain controversial. Our aim was to develop a new method for early prognostic assessment to assist this decision process. Fifty-four patients with unilateral obstetric brachial plexopathy who were ten to sixty days old underwent bilateral motor-nerve-conduction studies of the axillary, musculocutaneous, proximal radial, distal radial, median, and ulnar nerves. The ratio between the amplitude of the compound muscle action potential of the affected limb and that of the healthy side was called the axonal viability index. The patients were followed and classified in three groups according to the clinical outcome. We analyzed the receiver operating characteristic curve of each index to define the best cutoff point to detect patients with a poor recovery. The best cutoff points on the axonal viability index for each nerve (and its sensitivity and specificity) were <10% (88% and 89%, respectively) for the axillary nerve, 0% (88% and 73%) for the musculocutaneous nerve, <20% (82% and 97%) for the proximal radial nerve, <50% (82% and 97%) for the distal radial nerve, and <50% (59% and 97%) for the ulnar nerve. The indices from the proximal radial, distal radial, and ulnar nerves had better specificities compared with the most frequently used clinical criterion: absence of biceps function at three months of age. The axonal viability index yields an earlier and more specific prognostic estimation of obstetric brachial plexopathy than does the clinical criterion of biceps function, and we believe it may be useful in determining surgical indications in these patients.

Tau inclusions in retinal ganglion cells of human P301S tau transgenic mice: Effects on axonal viability

Tau inclusions play a key role in the pathogenesis of tauopathies. Altered tau levels have been detected in retina and optic nerve of patients with glaucoma, suggesting the possibility of shared pathogenic mechanisms with tauopathies. Here we report that hyperphosphorylated transgenic tau accumulates in the nerve fibre layer and, from 2 months of age, aggregates into filamentous inclusions in retinal ganglion cells of human P301S tau transgenic mice. Axonopathy and accumulation of hyperphosphorylated tau in the nerve fibre layer preceded inclusion formation. Hyperphosphorylated tau and tau inclusions were also detected in cultured retinal explants from 5-month-old transgenic mice. Axonal outgrowth was similar in transgenic and wild-type retinal explants under basal conditions. However, when exposed to growth-promoting stimuli, axon elongation was enhanced in explants from wild-type but not transgenic mice, indicating that the presence of abnormal tau can impair stimulated axonal outgrowth. These findings suggest that the retina is a good model system for investigating tau-driven neurodegeneration and for assessing potential pharmacological modifiers for tauopathies.