Multicenter exploration of tenecteplase transition factors: A quantitative analysis.

BACKGROUND: Tenecteplase (TNK) is gaining recognition as a novel therapy for acute ischemic stroke (AIS). Despite TNK offering a longer half-life, time and cost saving benefits and comparable treatment and safety profiles to Alteplase (ALT), the adoption of TNK as a treatment for AIS presents challenges for hospital systems. OBJECTIVE: Identify barriers and facilitators of TNK implementation at acute care hospitals in Texas. METHODS: This prospective survey used open-ended questions and Likert statements generated from content experts and informed by qualitative research. Stroke clinicians and nurses working at 40 different hospitals in Texas were surveyed using a virtual platform. RESULTS: The 40 hospitals had a median of 34 (IQR 24.5-49) emergency department beds and 42.5 (IQR 23.5-64.5) inpatient stroke beds with 506.5 (IQR 350-797.5) annual stroke admissions. Fifty percent of the hospitals were Comprehensive Stroke Centers, and 18 (45 %) were solely using ALT for treatment of eligible AIS patients. Primary facilitators to TNK transition were team buy-in and a willingness of stroke physicians, nurses, and pharmacists to adopt TNK. Leading barriers were lack of clinical evidence supporting TNK safety profile inadequate evidence supporting TNK use and a lack of American Heart Association guidelines support for TNK administration in all AIS cases. CONCLUSION: Understanding common barriers and facilitators to TNK adoption can assist acute care hospitals deciding to implement TNK as a treatment for AIS. These findings will be used to design a TNK adoption Toolkit, utilizing implementation science techniques, to address identified obstacles and to leverage facilitators.

A qualitative study of barriers and facilitators to using tenecteplase to treat acute ischemic stroke.

BACKGROUND: Tenecteplase (TNK) is emerging as an alternative to alteplase (ALT) for thrombolytic treatment of acute ischemic stroke (AIS). Compared to ALT, TNK has a longer half-life, shorter administration time, lower cost, and similarly high efficacy in treating large vessel occlusion. Nevertheless, there are barriers to adopting TNK as a treatment for AIS. This study aimed to identify thematic barriers and facilitators to adopting TNK as an alternative to ALT as a thrombolytic for eligible AIS patients. METHODS: Qualitative research methodology using hermeneutic cycling and purposive sampling was used to interview four stroke clinicians in Texas. Interviews were recorded and transcribed verbatim. Enrollment was complete when saturation was reached. All members of the research team participated in content analysis during each cycle and in thematic analysis after saturation. RESULTS: Interviews were conducted between November 2022 and February 2023 with stroke center representatives from centers that either had successfully adopted TNK, or had not yet adopted TNK. Three themes and eight sub-themes were identified. The theme "Evidence" had three sub-themes: Pro-Con Balance, Fundamental Knowledge, and Pharmacotherapeutics. The theme "Process Flow" had four subthemes: Proactive, Reflective self-doubt, Change Process Barriers, and Parameter Barriers. The theme "Consensus" had one sub-theme: Getting Buy-In. CONCLUSION: Clinicians experience remarkably similar barriers and facilitators to adopting TNK. The results lead to a hypothesis that providing evidence to support a practice change, and identifying key change processes, will help clinicians achieve consensus across teams that need to 'buy in' to adopting TNK for AIS treatment.

Using Operant Reach Chambers to Assess Mouse Skilled Forelimb Use After Stroke.

Skilled forelimb reaching and grasping are important components of rodent motor performance. The isometric pull task can serve as a tool for quantifying forelimb function following stroke or other CNS injury as well as in forelimb rehabilitation. This task has been extensively developed for use in rats. Here, we describe methods of setup and training of an operant reach chamber for mice. Using a reward of peanut oil, mice are adaptively trained to pull a handle positioned slightly outside of an operant chamber, with automated recording of the number of attempts, force generated, success rate, and latency to maximal force.

LZK-dependent stimulation of astrocyte reactivity promotes corticospinal axon sprouting.

Injury to the adult mammalian central nervous system induces compensatory plasticity of spared axons-referred to as collateral axon sprouting-that can facilitate neural recovery. The contribution of reactive astrocytes to axon sprouting remains elusive. Here, we sought to investigate the role of axon degeneration-reactive astrocytes in the regulation of collateral axon sprouting that occurs in the mouse spinal cord after unilateral photothrombotic stroke of the primary motor cortex. We identified astrocytic leucine zipper-bearing kinase (LZK) as a positive regulator of astrocyte reactivity to corticospinal axon degeneration. Remarkably, genetic stimulation of astrocyte reactivity, via LZK overexpression in adult astrocytes, enhanced corticospinal axon sprouting. LZK promoted the production of astrocyte-derived ciliary neurotrophic factor (CNTF) that likely enhanced axon growth in mice with astrocytic LZK overexpression after injury. Our finding that LZK-dependent stimulation of astrocyte reactivity promotes corticospinal axon sprouting highlights the potential of engineering astrocytes to support injury-induced axon plasticity for neural repair.

Outcomes From a Nursing-Driven Acute Stroke Care Protocol for Telehealth Encounters.

INTRODUCTION: Nursing care is widely recognized to be a vital element in stroke care delivery. However, no publications examining clinical education and optimal workflow practices as predictors of acute ischemic stroke care metrics exist. This study aimed to explore the impact of a nurse-led workflow to improve patient care that included telestroke encounters in the emergency department. METHODS: A nonrandomized prospective pre- and postintervention unit-level feasibility study design was used to explore how implementing nurse-driven acute stroke care affects the efficiency and quality of telestroke encounters in the emergency department. Nurses and providers in the emergency department received education/training, and then the Nursing-Driven Acute Ischemic Stroke Care protocol was implemented. RESULTS: There were 180 acute ischemic stroke encounters (40.3%) in the control phase and 267 (59.7%) in the postintervention phase with similar demographic characteristics. Comparing the control with intervention times directly affected by the nurse-driven protocol, there was a significant reduction in median door-to-provider times (5 [interquartile range 12] vs 2 [interquartile range 9] minutes, P < .001) and in median door-to-computed tomography scan times (9 [interquartile range 18] vs 5 [interquartile range 11] minutes, P < .001); however, the metrics potentially affected by extraneous variables outside of the nurse-driven protocol demonstrated longer median door-to-ready times (21 [interquartile range 24] vs 25 [interquartile range 25] minutes, P < .001). Door-to-specialist and door-to-needle times were not significantly different. DISCUSSION: In this sample, implementation of the nurse-driven acute stroke care protocol is associated with improved nurse-sensitive stroke time metrics but did not translate to faster delivery of thrombolytic agents for acute ischemic stroke, emphasizing the importance of well-outlined workflows and standardized stroke code protocols at every point in acute ischemic stroke care.

Delayed diapedesis of CD8 T cells contributes to long-term pathology after ischemic stroke in male mice.

OBJECTIVE: Stroke is a debilitating disorder with significant annual mortality and morbidity rates worldwide. Immune cells are recruited to the injured brain within hours after stroke onset and can exhibit either protective or detrimental effects on recovery. However, immune cells, including CD8 T cells, persist in the injured brain for weeks, suggesting a longer-term role for the adaptive immune system during functional recovery. The aim of this study was to determine if the delayed secondary diapedesis of CD8 T cells into the ischemic brain negatively impacts functional recovery after transient ischemic stroke in male mice. RESULTS: Mice exhibited an increased number of leukocytes in the ipsilesional hemispheres at 14 days (3-fold; p < 0.001) and 30 days (2.2-fold; p = 0.02) after transient middle cerebral artery occlusion (tMCAo) compared to 8 days post-tMCAo, at which time acute neuroinflammation predominantly resolves. Moreover, mice with higher ipsilesional CD8 T cells at 30 days (R2 = 0.52, p < 0.01) exhibited worse functional recovery. To confirm a detrimental role of chronic CD8 T cell diapedesis on recovery, peripheral CD8 T cells were depleted beginning 10 days post-tMCAo. Delayed CD8 T cell depletion improved motor recovery on the rotarod (F(1,28) = 4.264; p = 0.048) compared to isotype control-treated mice. CD8 T cell-depleted mice also exhibited 2-fold (p < 0.001) reduced leukocyte infiltration at 30 days post-tMCAo. Specifically, macrophage, neutrophil, and CD4 T cell numbers were reduced in the ipsilesional hemisphere of the CD8 T cell-depleted mice independent of inflammatory status of the post-stroke CNS (e.g. microglial phenotype and cytokine production). RNAseq identified a unique profile for brain infiltrating CD8 T cells at 30 days post-tMCAo, with 46 genes differentially expressed relative to CD8 T cells at 3 days post-tMCAo. CONCLUSION: Our data reveal a role for CD8 T cells in the chronic phase post-stroke that can be therapeutically targeted. We demonstrate long-term CD8 T cell recruitment into the ipsilesional hemisphere that affects both immune cell numbers present in the injured brain and functional recovery through one month after stroke onset.

IAT-TiMeS: Intra-Arterial Thrombectomy Transfer Metric Study in Texas.

OBJECTIVE: We aim to report intra-arterial thrombectomy transfer metrics for ischemic stroke patients that were transferred to hub hospitals for possible intra-arterial thrombectomy in multiple geographic regions throughout the state of Texas and to identify potential barriers and delays in the intra-arterial thrombectomy transfer process. METHOD: We prospectively collected data from 8 participating Texas comprehensive stroke/thrombectomy capable centers from 7 major regions in the State of Texas. We collected baseline clinical and imaging data related to the pre-transfer evaluation, transfer metrics, and post-transfer clinical and imaging data. RESULTS: A total of 103 acute ischemic stroke patients suspected/confirmed to have large vessel occlusions between December 2016 to May 2019 that were transferred to hubs as possible intra-arterial thrombectomy candidates were enrolled. A total of 56 (54%) patients were sent from the spoke to the hub via ground ambulance with 47 (46%) patients traveling via air ambulance. The median spoke arrival to hub arrival time was 174 min, median spoke arrival to departure from spoke was 131 min, and median travel time was 39 min. The spoke arrival time to transfer initiation was 68 min. CT-perfusion obtained at the spoke and earlier initiation of transfer were statistically associated with shorter transfer times. CONCLUSION: Transfer of intra-arterial thrombectomy patients in Texas may take over 4 h from spoke arrival to hub arrival. This time may be shortened by earlier transfer initiation and acceptance.

Forelimb Cortical Stroke Reduces Precision of Motor Control in Mice.

Background and Objective. Rodent models of stroke impairment should capture translatable features of behavioral injury. This study characterized poststroke impairment of motor precision separately from strength in an automated behavioral assay. Methods. We measured skilled distal forelimb reach-and-grasp motions within a target force range requiring moderate-strength. We assessed whether deficits reflected an increase in errors on only one or both sides of the target force range after photothrombotic cortical stroke. Results. Pull accuracy was impaired for 6 weeks after stroke, with errors redistributing to both sides of the target range. No decrease in maximum force was measured. Conclusions. This automated reach task measures sustained loss of motor precision following cortical stroke in mice.

B cells migrate into remote brain areas and support neurogenesis and functional recovery after focal stroke in mice.

Lymphocytes infiltrate the stroke core and penumbra and often exacerbate cellular injury. B cells, however, are lymphocytes that do not contribute to acute pathology but can support recovery. B cell adoptive transfer to mice reduced infarct volumes 3 and 7 d after transient middle cerebral artery occlusion (tMCAo), independent of changing immune populations in recipient mice. Testing a direct neurotrophic effect, B cells cocultured with mixed cortical cells protected neurons and maintained dendritic arborization after oxygen-glucose deprivation. Whole-brain volumetric serial two-photon tomography (STPT) and a custom-developed image analysis pipeline visualized and quantified poststroke B cell diapedesis throughout the brain, including remote areas supporting functional recovery. Stroke induced significant bilateral B cell diapedesis into remote brain regions regulating motor and cognitive functions and neurogenesis (e.g., dentate gyrus, hypothalamus, olfactory areas, cerebellum) in the whole-brain datasets. To confirm a mechanistic role for B cells in functional recovery, rituximab was given to human CD20+ (hCD20+) transgenic mice to continuously deplete hCD20+-expressing B cells following tMCAo. These mice experienced delayed motor recovery, impaired spatial memory, and increased anxiety through 8 wk poststroke compared to wild type (WT) littermates also receiving rituximab. B cell depletion reduced stroke-induced hippocampal neurogenesis and cell survival. Thus, B cell diapedesis occurred in areas remote to the infarct that mediated motor and cognitive recovery. Understanding the role of B cells in neuronal health and disease-based plasticity is critical for developing effective immune-based therapies for protection against diseases that involve recruitment of peripheral immune cells into the injured brain.

Short-Chain Fatty Acids Improve Poststroke Recovery via Immunological Mechanisms.

Recovery after stroke is a multicellular process encompassing neurons, resident immune cells, and brain-invading cells. Stroke alters the gut microbiome, which in turn has considerable impact on stroke outcome. However, the mechanisms underlying gut-brain interaction and implications for long-term recovery are largely elusive. Here, we tested the hypothesis that short-chain fatty acids (SCFAs), key bioactive microbial metabolites, are the missing link along the gut-brain axis and might be able to modulate recovery after experimental stroke. SCFA supplementation in the drinking water of male mice significantly improved recovery of affected limb motor function. Using in vivo wide-field calcium imaging, we observed that SCFAs induced altered contralesional cortex connectivity. This was associated with SCFA-dependent changes in spine and synapse densities. RNA sequencing of the forebrain cortex indicated a potential involvement of microglial cells in contributing to the structural and functional remodeling. Further analyses confirmed a substantial impact of SCFAs on microglial activation, which depended on the recruitment of T cells to the infarcted brain. Our findings identified that microbiota-derived SCFAs modulate poststroke recovery via effects on systemic and brain resident immune cells.SIGNIFICANCE STATEMENT Previous studies have shown a bidirectional communication along the gut-brain axis after stroke. Stroke alters the gut microbiota composition, and in turn, microbiota dysbiosis has a substantial impact on stroke outcome by modulating the immune response. However, until now, the mediators derived from the gut microbiome affecting the gut-immune-brain axis and the molecular mechanisms involved in this process were unknown. Here, we demonstrate that short-chain fatty acids, fermentation products of the gut microbiome, are potent and proregenerative modulators of poststroke neuronal plasticity at various structural levels. We identified that this effect was mediated via circulating lymphocytes on microglial activation. These results identify short-chain fatty acids as a missing link along the gut-brain axis and as a potential therapeutic to improve recovery after stroke.

Visualization and Quantification of Post-stroke Neural Connectivity and Neuroinflammation Using Serial Two-Photon Tomography in the Whole Mouse Brain.

Whole-brain volumetric microscopy techniques such as serial two-photon tomography (STPT) can provide detailed information on the roles of neuroinflammation and neuroplasticity throughout the whole brain post-stroke. STPT automatically generates high-resolution images of coronal sections of the entire mouse brain that can be readily visualized in three dimensions. We developed a pipeline for whole brain image analysis that includes supervised machine learning (pixel-wise random forest models via the "ilastik" software package) followed by registration to a standardized 3-D atlas of the adult mouse brain (Common Coordinate Framework v3.0; Allen Institute for Brain Science). These procedures allow the detection of cellular fluorescent signals throughout the brain in an unbiased manner. To illustrate our imaging techniques and automated image quantification, we examined long-term post-stroke motor circuit connectivity in mice that received a motor cortex photothrombotic stroke. Two weeks post-stroke, mice received intramuscular injections of pseudorabies virus (PRV-152), a trans-synaptic retrograde herpes virus driving expression of green fluorescent protein (GFP), into the affected contralesional forelimb to label neurons in descending tracts to the forelimb musculature. Mice were sacrificed 3 weeks post-stroke. We also quantified sub-acute neuroinflammation in the post-stroke brain in a separate cohort of mice following a 60 min transient middle cerebral artery occlusion (tMCAo). Naive e450+-labeled splenic CD8+ cytotoxic T cells were intravenously injected at 7, 24, 48, and 72 h post-tMCAo. Mice were sacrificed 4 days after stroke. Detailed quantification of post-stroke neural connectivity and neuroinflammation indicates a role for remote brain regions in stroke pathology and recovery. The workflow described herein, incorporating STPT and automated quantification of fluorescently labeled features of interest, provides a framework by which one can objectively evaluate labeled neuronal or lymphocyte populations in healthy and injured brains. The results provide region-specific quantification of neural connectivity and neuroinflammation, which could be a critical tool for investigating mechanisms of not only stroke recovery, but also a wide variety of brain injuries or diseases.

Closed-loop neuromodulation restores network connectivity and motor control after spinal cord injury.

Recovery from serious neurological injury requires substantial rewiring of neural circuits. Precisely-timed electrical stimulation could be used to restore corrective feedback mechanisms and promote adaptive plasticity after neurological insult, such as spinal cord injury (SCI) or stroke. This study provides the first evidence that closed-loop vagus nerve stimulation (CLV) based on the synaptic eligibility trace leads to dramatic recovery from the most common forms of SCI. The addition of CLV to rehabilitation promoted substantially more recovery of forelimb function compared to rehabilitation alone following chronic unilateral or bilateral cervical SCI in a rat model. Triggering stimulation on the most successful movements is critical to maximize recovery. CLV enhances recovery by strengthening synaptic connectivity from remaining motor networks to the grasping muscles in the forelimb. The benefits of CLV persist long after the end of stimulation because connectivity in critical neural circuits has been restored.

Leucine Zipper-Bearing Kinase Is a Critical Regulator of Astrocyte Reactivity in the Adult Mammalian CNS.

Reactive astrocytes influence post-injury recovery, repair, and pathogenesis of the mammalian CNS. Much of the regulation of astrocyte reactivity, however, remains to be understood. Using genetic loss and gain-of-function analyses in vivo, we show that the conserved MAP3K13 (also known as leucine zipper-bearing kinase [LZK]) promotes astrocyte reactivity and glial scar formation after CNS injury. Inducible LZK gene deletion in astrocytes of adult mice reduced astrogliosis and impaired glial scar formation, resulting in increased lesion size after spinal cord injury. Conversely, LZK overexpression in astrocytes enhanced astrogliosis and reduced lesion size. Remarkably, in the absence of injury, LZK overexpression alone induced widespread astrogliosis in the CNS and upregulated astrogliosis activators pSTAT3 and SOX9. The identification of LZK as a critical cell-intrinsic regulator of astrocyte reactivity expands our understanding of the multicellular response to CNS injury and disease, with broad translational implications for neural repair.

Piloting a Sex-Specific, Technology-Enhanced, Active Learning Intervention for Stroke Prevention in Women.

BACKGROUND: Recent studies reveal deficiencies in stroke awareness and knowledge of risk factors among women. Existing stroke education interventions may not address common and sex-specific risk factors in the population with the highest stroke-related rate of mortality. OBJECTIVE: This pilot study assessed the efficacy of a technology-enhanced, sex-specific educational program ("SISTERS") for women's knowledge of stroke. METHODS: This was an experimental pretest-posttest design. The sample consisted of 150 women (mean age, 55 years) with at least 1 stroke risk factor. Participants were randomized to either the intervention (n = 75) or control (n = 75) group. Data were collected at baseline and at a 2-week posttest. RESULTS: There was no statistically significant difference in mean knowledge score (P = .67), mean confidence score (P = .77), or mean accuracy score (P = .75) between the intervention and control groups at posttest. Regression analysis revealed that older age was associated with lower knowledge scores (P < .001) and lower confidence scores (P < .001). After controlling for age, the SISTERS program was associated with a statistically significant difference in knowledge (P < .001) and confidence (P < .001). CONCLUSIONS: Although no change occurred overall, after controlling for age, there was a statistically significant benefit. Older women may have less comfort with technology and require consideration for cognitive differences.

Cystic Lesions as a Rare Complication of Deep Brain Stimulation.

DBS is a typically well-tolerated operation for treatment of Parkinson's disease, dystonia, and essential tremor (ET). Complications related to the surgical procedure and implanted hardware may occur. More commonly reported complications include hemorrhage, seizure, confusion, and infection. In this article, we report on a rare, but important, complication of DBS surgery, a brain cyst formation at the tip of the implanted ventralis intermedius nucleus (VIM) DBS lead in 2 patients who underwent the procedure at 2 different centers. The indication for surgery was debilitating ET, and in both cases, there was development of a delayed-onset neurological deficit associated with an internal capsule/thalamic cystic lesion formation located at the tip of the DBS lead. Case 1 presented within a few months post-DBS, whereas case 2 had a 10-mo delay to onset of symptoms. No clinical and radiological signs of infection were observed and both DBS systems were explanted with uneventful recovery.

An automated task for the training and assessment of distal forelimb function in a mouse model of ischemic stroke.

BACKGROUND: Behavioral models relevant to stroke research seek to capture important aspects of motor skills typically impaired in human patients, such as coordination of distal musculature. Such models may focus on mice since many genetic tools are available for use only in that species and since the training and behavioral demands of mice can differ from rats even for superficially similar behavioral readouts. However, current mouse assays are time consuming to train and score, especially in a manner producing continuous quantification. An automated assay of mouse forelimb function may provide advantages for quantification and speed, and may be useful for many applications including stroke research. NEW METHOD: We present an automated assay of distal forelimb function. In this task, mice reach forward, grip and pull an isometric handle with a prescribed force. The apparatus partially automates the training process so that mice can be trained quickly and simultaneously. RESULTS: Using this apparatus, it is possible to measure long-lasting impairment in success rate, force pulled, latency to pull, and latency to success up to 22 weeks following photothrombotic cortical strokes in mice. COMPARISON WITH EXISTING METHOD(S): This assessment measures forelimb function as do pellet reach tasks, but it utilizes a different motion and provides automatic measures that can ease and augment the research process. CONCLUSIONS: This high-throughput behavioral assay can detect long-lasting motor impairments, eliminates the need for subjective scoring, and produces a rich, continuous data set from which many aspects of the reach and grasp motion can be automatically extracted.

Erythropoietin-mediated neuroprotection in a pediatric mouse model of chronic hypoxia.

Chronic hypoxia (CH), a disease state that accounts for significant morbidity and mortality in pediatrics, occurs in many children during critical periods of hippocampal development and cortical myelination. Hippocampal neurogenesis occurs throughout postnatal life and is important for normal development, thus impairment results in long-term cognitive deficits. Erythropoietin (EPO), a drug commonly known for its role in erythrogenesis, has recently been evaluated in neuroprotection in neonatal injury models and preterm brain injury. However, the effects of EPO therapy on hippocampal neurogenesis and myelination in pediatric CH are unknown. We show that CH decreases hippocampal neurogenesis in a pediatric mouse model. This decrease in early and late progenitors, and actively dividing cells is rescued with EPO treatment. Furthermore, we show that CH during this critical time decreases oligodendrocyte progenitor (OPC) populations in the cortex, leading to defective myelination. However, EPO therapy is only able to rescue the OPC but not the loss of mature myelin. Overall, our findings demonstrate that CH in developing mice has significant effects on hippocampal neurogenesis and OPCs, which can be rescued with EPO treatment. Future studies should confirm the role of this FDA-approved therapy in neuroprotection in at-risk pediatric populations.

Synthesis and properties of Asante Calcium Red--a novel family of long excitation wavelength calcium indicators.

Although many synthetic calcium indicators are available, a search for compounds with improved characteristics continues. Here, we describe the synthesis and properties of Asante Calcium Red-1 (ACR-1) and its low affinity derivative (ACR-1-LA) created by linking BAPTA to seminaphthofluorescein. The indicators combine a visible light (450-540 nm) excitation with deep-red fluorescence (640 nm). Upon Ca2+ binding, the indicators raise their fluorescence with longer excitation wavelengths producing higher responses. Although the changes occur without any spectral shifts, it is possible to ratio Ca(2+)-dependent (640 nm) and quasi-independent (530 nm) emission when using visible (< 490 nm) or multiphoton (∼780 nm) excitation. Therefore, both probes can be used as single wavelength or, less dynamic, ratiometric indicators. Long indicator emission might allow easy [Ca2+]i measurement in GFP expressing cells. The indicators bind Ca2+ with either high (Kd = 0.49 ± 0.07 µM; ACR-1) or low affinity (Kd = 6.65 ± 0.13 µM; ACR-1-LA). Chelating Zn2+ (Kd = 0.38 ± 0.02 nM) or Mg2+ (Kd∼5mM) slightly raises and binding Co2+ quenches dye fluorescence. New indicators are somewhat pH-sensitive (pKa = 6.31 ± 0.07), but fairly resistant to bleaching. The probes are rather dim, which combined with low AM ester loading efficiency, might complicate in situ imaging. Despite potential drawbacks, ACR-1 and ACR-1-LA are promising new calcium indicators.

Maintaining energy homeostasis is an essential component of Wld(S)-mediated axon protection.

Wld(S) mutation protects axons from degeneration in diverse experimental models of neurological disorders, suggesting that the mutation might act on a key step shared by different axon degeneration pathways. Here we test the hypothesis that Wld(S) protects axons by preventing energy deficiency commonly encountered in many diseases. We subjected compartmentally cultured, mouse cortical axons to energy deprivation with 6mM azide and zero glucose. In wild-type (WT) culture, the treatment, which reduced axon ATP level ([ATP]axon) by 65%, caused immediate axon depolarization followed by gradual free calcium accumulation and subsequent irreversible axon damage. The calcium accumulation resulted from calcium influx partially via L-type voltage-gated calcium channel (L-VGCC). Blocking L-VGCC with nimodipine reduced calcium accumulation and protected axons. Without altering baseline [ATP]axon, the presence of Wld(S) mutation significantly reduced the axon ATP loss and depolarization, restrained the subsequent calcium accumulation, and protected axons against energy deprivation. Wld(S) neurons possessed higher than normal nicotinamide mononucleotide adenylyltransferase (NMNAT) activity. The intrinsic Wld(S) NMNAT activity was required for the Wld(S)-mediated energy preservation and axon protection during but not prior to energy deprivation. NMNAT catalyzes the reversible reaction that produces nicotinamide adenine dinucleotide (NAD) from nicotinamide mononucleotide (NMN). Interestingly, preventing the production of NAD from NMN with FK866 increased [ATP]axon and protected axons from energy deprivation. These results indicate that the Wld(S) mutation depends on its intrinsic Wld(S) NMNAT activity and the subsequent increase in axon ATP but not NAD to protect axons, implicating a novel role of Wld(S) NMNAT in axon bioenergetics and protection.