Gria1, an ionotropic glutamate receptor, was upregulated in DCN of control mice but failed to be upregulated in ataxia mice after sciatic nerve crush. AAV-mediated overexpression of Gria1 in DCN rescued motor deficits of ataxia mice after PNI. Finally, we found a correlative decrease in human GRIA1 mRNA expression in the cerebellum of patients with ataxia-telangiectasia and spinocerebellar ataxia type 6 patient iPSC-derived Purkinje cells, pointing to the clinical relevance of glutamatergic system. By conducting a large-scale analysis of 9,655,320 patients with ataxia, they failed to recover from carpal tunnel decompression surgery and tibial neuropathy, while aged-match non-ataxia patients fully recovered. Our results provide insight into cerebellar disorders and motor deficits after PNI.Peripheral nerve injury (PNI) often results in spontaneous motor recovery; however, how disrupted cerebellar circuitry affects PNI-associated motor recovery is unknown. Here, we demonstrated disrupted cerebellar circuitry and poor motor recovery in ataxia mice after PNI. This effect was mimicked by deep cerebellar nuclei (DCN) lesion, but not by damaging non-motor area hippocampus. By restoring cerebellar circuitry through DCN stimulation, and reversal of neurotransmitter imbalance using baclofen, ataxia mice achieve full motor recovery after PNI. Mechanistically, elevated glutamate-glutamine level was detected in DCN of ataxia mice by magnetic resonance spectroscopy. Transcriptomic study revealed that  Damage to the peripheral nerve is followed by a slow rate of axon regeneration (1–2 mm/day) and is usually associated with a sub-optimal function recovery7. For instance, proximal nerve injuries that involve partial or complete transection of peripheral nerves require long-distance axon regeneration to reinnervate their target muscles, generally resulting in the unsatisfactory recovery of motor function8 after prolonged muscle denervation7. The success of function recovery depends on the reinnervation of denervated target muscle by regenerating axons and collateral sprouting of uninjured axons, and the reorganization of the nervous system circuitry. Over the past decade, there are no significant nerve repair therapeutic advances have been made to improve function recovery, especially for severe nerve injuries. Therefore, immediate attention is required for strengthening our understanding on the causal relationship between the remodeling of central networks and function recovery after PNIs15.Injury to the peripheral nervous system (PNS) occurs when a nerve is crushed, transected or the proper communication between the PNS and the central nervous system (CNS) is disrupted 17. Dysfunction of Purkinje cells causes ataxia, a neurological disease with clinical features of tremors and loss of motor coordination20. Clinical and animal studies showed that changes in the somatosensory cortex activities detected by functional magnetic resonance imaging (MRI) were observed following amputation9, spinal cord injury10, and PNI even 1 year after nerve transection and surgical repair11. These cortical changes are generally attributed to the basis of post-injury sensory and motor function deficits. Cortical plasticity  can be either adaptive—compensating the reduction of peripheral sensory input21 or maladaptive—potentiating neuropathic pain (i.e. phantom limb pain) and dystonia22. Although peripheral nerves could regenerate after lesions7, little is known about how exactly cortical plasticity contributes to the adaptive  or maladaptive responses  in the PNS after nerve injuries7.The cytoarchitecture and neural circuitry system of the cerebellum are highly organized and uniform. Cerebellum integrates the motor and sensory inputs from the PNS to produce a feedback output to the descending motor pathways that control movements. This motor output is precisely modulated by the inhibitory input to the deep cerebellar nuclei (DCN) from Purkinje cells, and the inhibitory Purkinje cell output results in a reduction of excitatory output from DCN to the motor cortex that leads to modification of motor controlLhx1 and Lhx5 conditional double knockout (ataxia) mouse with thinner Purkinje cell dendrites and abnormal dendritic spines (arborization)23. By using a combination of animal behavioral, electrophysiological, pharmacological, and MRI approaches, we reveal that cerebellar circuitry disruption and neurotransmitter imbalance delay spontaneous motor recovery after PNIs. Correction of defective cerebellar circuitry by electrical stimulation of DCN or by pharmacological neuromodulation recovered motor function after PNIs. Furthermore, magnetic resonance spectroscopy (MRS) study revealed the imbalance of glutamate-glutamine (Glx) level in the cerebellum of ataxia mice, suggesting the involvement of glutamate receptors. Transcriptomic analysis of ataxic cerebellum revealed the involvement of an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) subtype of ionotropic glutamate receptor Gria1 in spontaneous motor recovery after PNI. In AMAP receptors, there are four subunits, including GluA1–4 (or GluR1–4), which form functionally different tetramers24. GluA1 gene deletion impaired short-term spatial memory and enhanced long-term spatial memory, and these mice have been reported to show normal overall locomotor activity27. Gria1, the gene encoding AMPA receptor subunit glutamate A1 (GLUA1), was originally identified as a putative risk gene for schizophrenia. Decreased GRIA1 expression has been found in the hippocampus of schizophrenia patients in three independent studies30. GluA1 receptor and GluA1 knockout mice have been studied extensively in many aspects of neurological diseases and have been implicated in long-term potentiation, Alzheimer’s disease, schizophrenia, depression, sleep and wakefulness, epilepsy, and neuropathic pain36. However, the roles of cerebellar Gria1 in spontaneous motor recovery after PNI is largely unknown. To address the question of whether Gria1 acts as a key gene for cortical plasticity and PNS regeneration, we showed that intracerebroventricular administration of an AMPA receptor blocker, 6,7-dinitroquinoxaline-2,3-dione (DNQX), impaired functional recovery mimicking the inhibitory effect of ataxia mice on motor recovery following PNI. Gria1 overexpression via intracerebroventricular delivery of adeno-associated virus (AAV) rescued motor deficits in ataxia mice. Finally, we extended these findings to clinical studies of human GRIA1 expression and motor recovery in patients with ataxic manifestations. Downregulation of human GRIA1 expression in patients with ataxic manifestations was observed across several human Gene Expression Omnibus (GEO) datasets. Our clinical data showed that ataxia patients were unable to recover motor function after carpal tunnel decompression and tibial nerve neuropathy, while non-ataxia patients showed complete motor recovery. These findings reveal cerebellar control of motor recovery that provides the basis for the unexplored link between other cerebellar disorders and poor clinical outcomes following PNIs.To address these key questions, we manipulated and directly observed interactions between cerebellar circuitry integrity and functional recovery after PNIs using a Purkinje cell-specific LIM-homeodomain transcription factors Lhx1/5 double knockout mice (ataxia mice) by crossing Purkinje cell protein (Pcp)2-Cre mice with Lhx1 floxed (fl) and Lhx5 fl mice, to obtain adult littermates (Pcp2-Cre−/−/Lhx1fl/fl/Lhx5fl/fl) and ataxia mice (Pcp2-Cre+/−/Lhx1fl/fl/Lhx5fl/fl) for subsequent experiments23  in proximal (gastrocnemius) and distal (interosseous) plantar muscles of ataxia mice at weeks 2, 3 and 4 after injury. The EMG activities of ataxia mice dropped to 77.7% (gastrocnemius) and 69.5% (interosseous) of the baseline values, and the controls were fully recovered in both proximal  Fig. . We genes23 Fig. . Ataxia s23 Fig.  and was s23 Fig. . As expes23 Fig. . Motor fmal Fig.  in aboutmal Fig.  and scia7. We have developed a mouse model to study critical periods by performing repeated SNCs to delay regenerating axons from reaching distal muscle for a specific period of time7. We start with 16 days of muscle denervation by crushing the sciatic nerve every 2 days as it takes 10 days for the regenerating axons to reach the distal target muscle1  and regain full motor function recovery only if regenerating axons reach distal muscle within 37 days38, however; there is limited understanding of how the cerebellar circuitry affects motor recovery after PNS injury. We, therefore, examine the role of DCN in motor recovery after PNI by electrical DCN lesions or to lesion a non-motor area such as hippocampus as control. Successful DCN lesion was confirmed by motor coordination deficit assessed by accelerated rotarod test and histological analysis, and only those DCN-lesioned mice with ataxia symptom were used for SNC study , which has been shown to produce a selective removal of the hippocampus and dentate gyrus, resulting in severe spatial cognitive deficits44. Microinjection of NMDA was performed 3 weeks before the SNC, and learning and memory deficits were validated in hippocampus-lesioned mice by Y-maze and Morris water maze43, and histological analysis  followed by CMAP amplitude recording in the hindlimb gastrocnemius muscle  (i.e. interposed nucleus of DCN) and pharmacologic intervention (i.e. baclofen) have been shown to improve ataxia symptoms by restoring aberrant hyperexcitability in the cerebellum52 using multielectrode arrays for 21 consecutive days following a single SNC  in the cerebellum using non-invasive MRS during the course of baclofen treatment and recovery from a single SNC Fig. 53,54. Glice Fig. . To ensuice Fig.  and EMG ice Fig. , compareice Fig. . The numice Fig.  and inteice Fig.  returnedd-aspartate (NMDA)  receptors in DCN. Compared to the uninjured mice, we found that the mRNA expression levels of Gria3, Gria4, Grin2b, and Grin2c were markedly increased in DCN at day 7 post-SNC in non-ataxia control and ataxia mice  and N-methyl-Gria1 in PNIs, Gria1 was blocked pharmacologically in wild-type C57BL/6 mice using DNQX55, a selective antagonist of the ionotropic glutamate receptors, AMPA receptors are the most abundant ionotropic glutamate receptors in the brain. DNQX was administered through a cannula implanted into the DCN immediately after the sciatic nerve crush for 3 weeks  and functional recovery tests were performed on alternate days  overexpressing mouse Gria1 (AAV-Gria1)  in the cerebellum of patients with ataxia-telangiectasia (A-T), compared with the age-matched healthy individuals  patient iPSC-derived Purkinje cells also showed significant downregulation of GRIA1 , when compared with Purkinje cell derived from healthy individuals , when compared with healthy human iPSC-derived neurons 59 and Friedreich’s ataxia 60 patient-derived fibroblasts compare to healthy controls  63, A-T patient-derived fibroblast 64 as well as fibroblast derived from patient with oxidative phosphorylation disease, who exhibited a wide variety of clinical manifestations including ataxia   Fig. 61. We nost) Fig. 65. TogetWe next sought to investigate the spontaneous motor function recovery in ataxia patients after PNIs by conducting a large-scale analysis of 9,655,320 patients from the Mayo Clinic Epic electronic database (non-public). The initial search identified 108,677 patients with PNIs and 587 of them have been diagnosed with cerebellar ataxia. Patients with median or tibial neuropathy were selected since they both are the most commonly encountered PNIs in humans. Patients with median neuropathy at the wrist  are curable by surgical intervention , and usually no treatment is needed for most tibial neuropathy cases. Only the patients with precise date of peripheral nerve lesion diagnosis and those with at least one follow-up visit with a proper nerve conduction study done after the surgical intervention (for CTS patients) or being diagnosed with tibial neuropathy, were selected for analysis Fig. . A total67); however, ataxia patients recovered to a lesser extent  after carpal tunnel decompression surgery  were conducted on non-ataxia patients and 6 NCSs were done on ataxia patients after surgery. The median nerve CMAP amplitude of non-ataxia patients returned back to normal values of 8.1 ± 1.89 mV  in 2–20 months after CTS surgery , which was much lower than the non-ataxia patients  Fig. . In one B) receptor agonist baclofen, we were able to reverse motor deficits in ataxia mice after PNIs. mRNA expression of key glutamine receptors were upregulated in both control and ataxia mice after PNI except Gria1. AAV-mediated cerebellar gene delivery of Gria1 was able to rescue the motor deficits of ataxia mice after PNI. We systematically identified downregulated human GRIA1 expression in patients with ataxic manifestations across several human GEO datasets. These findings, in line with our clinical data, reflect what happens in ataxia patients after PNIs. We demonstrate a concept that ataxia patients, which has a 93% chance of having at least one fall in a year68, could be more prone to have irreversible motor deficits whereas the majority of non-ataxia patients are able to recover spontaneously from PNIs such as CTS and tibial neuropathy.Our results establish a causal relationship between cerebellar circuitry integrity and motor recovery after PNIs. The study of distinct mouse models and ataxia patients with PNIs demonstrates the increased risk of motor deficits associated with cerebellar dysfunction after PNIs. The MRS findings of elevated Glx levels in the cerebellum of ataxia mice suggests the possibility of their connection to the disruption of glutamatergic system. We further demonstrated that by restoring cerebellar circuitry using cerebellar DBS or gamma-aminobutyric acid 50. In Parkinson’s disease patients, it has been demonstrated that switching off a DBS would not stop the beneficial effect abruptly72. Functional MRI studies show that there is a remarkable long-term effect of DBS on structural and functional connectivity (brain rewiring or neuroplasticity)73. Our electrophysiology data showed that DBS of DCN normalized aberrant neural firing leading to complete motor recovery in ataxia mice after a single and multiple SNC, whereas we performed DCN-DBS for 21 consecutive days which is the time period for active axon regeneration and function recovery74. Therefore, we show that an abnormal pattern of activity that is transmitted from Purkinje cells downstream to the DCN in ataxia mice can be restored for better motor recovery from PNI.We first set out to explore the possibility that aberrant hyperexcitability of cerebellar DCN is the primary cause of poor motor recovery after PNIs in ataxia mice and found that disruption of cerebellar circuitry severely affects motor recovery. DCN function as a relay center for inhibitory inputs from Purkinje cells in the cerebellar cortex and excitatory inputs from mossy and climbing fibers, and project to various premotor areas to fine-tune motor activity. Abnormal high frequency burst neural firing pattern in DCN has been linked to ataxia, dystonic Parkinsonism, and tremor78. Purkinje cells are GABAergic neurons and the GABAergic axons of the Purkinje cells that form synapses on DCN are greatly reduced in our ataxia mice as a result of dendritic thinning23. The removal of inhibition of excitatory synapses due to the reduction of GABAergic inhibition has been suggested to be the most important cause of acute neuroplasticity changes in the cerebellum79. In addition, peripheral deafferentation often results in a reduction in the number of neurons containing GABA as well as the glutamic acid decarboxylase (GABA synthesizing enzyme) in the cortical area81. In humans and primates, finger deafferentation by anesthetic block induces not only rapid functional changes but also the partial expansion of the cortical representations of intact fingers into the cortical map of denervated finger within minutes83. Consistent with these studies, we found that by restoration of cerebellar circuitry through administration of baclofen, a clinically available GABAB receptor agonist84, restored normal neural firing pattern of DCN and recovered motor function in ataxia mice after SNC. Baclofen treatment also restored Glx level in the DCN of ataxia mice as determined by using noninvasive MRS. In fact, there is an increasing interest in the roles of brain metabolites such as Glx in spinal cord injury and neuropathic pain since glutamate and glutamine are relatively abundant amino acids in the brain that are critical for neuronal function85. It has been shown that an elevated level of Glx in the anterior cingulate cortex is associated with SCI-related neuropathic pain in patients86. In rats, Glx decreases dramatically in the cortex and gradually returns to normal levels after a low thoracic spinal cord injury87.GABA is the primary inhibitory neurotransmitter in the CNS and abnormalities in GABAergic system have been widely reported in ataxia88. Purkinje cells express all four AMPA receptor subunits GluA1-490, and intense GluR1 immunoreactivity and mRNA expression are detected in the Purkinje cells92. We therefore speculate that synaptic dysfunction associated with downregulation of cerebellar Gria1 in ataxia mice and patients with ataxic manifestations, leading to motor deficits after PNI. PNI induces not only substantial changes in neural plasticity in the CNS93 but also transcriptomic changes in a number of key genes involved in the regulation of synaptic plasticity13. There is growing evidence that Gria1 expression and the balance of glutamatergic throughput via AMPA receptors are crucial for neural plasticity95. In rats, PNI up-regulated gene expression of ion channels, AMPA receptors , GABA receptors, and signaling transduction molecules in the dorsal spinal cord98. Co-deletion of two AMPA receptor subunits Gria1 and Gria4 in cerebellar Bergmann glial cells, leads to impairments in fine motor coordination99. To this end, we conclude that an intact cerebellar circuitry and a proper AMPA receptor-mediated glutamatergic neurotransmission are crucial pre-requisite to the success of motor function recovery after PNIs. In line with our hypothesis, the interruption of the hippocampal circuit (non-motor performance brain area) does not affect spontaneous motor recovery indicating that synaptic integrity of the glutaminergic system in the cerebellum is necessary for complete motor recovery after PNI. Future research examining the temporal correlations between glutamatergic neurotransmission and motor recovery would be useful to better understand the links between brain circuitry and PNS regeneration.In this study, we report for the first time that there is a potential mechanistic link between Gria1 and spontaneous motor recovery in patients with ataxia due to the glutamatergic dysfunction. GRIA1 encodes the GluA1 subunit of the AMPA subtype of glutamine receptor, which is an essential component of synaptic plasticity, especially for learning and memory functions in AD. Disruption of AMPA receptor trafficking by Abeta oligomers in the hippocampus is believed to be a major contributor to synaptic dysfunction in AD, leading to cognitive deficits12. Remodeling of spinal and brain circuits after PNIs usually do not allow for a meaningful functional recovery, especially after sever PNIs13. The conceptualization of improving CNS plasticity for better functional recovery in the PNS has been emerged for decades14. Promoting spinal plasticity by chondroitinase improve recovery of forelimb function after PNI15. Our work on the single and multiple SNC, that adult mice usually take 4–8 weeks to regain full motor function74, suggests that disruption of cerebellar circuitry leads to permanent motor deficits. This raise the intriguing possibility that the major cause of motor deficits in patients with cerebellar disorders might not be the diseases themselves but perhaps the unrecognized PNI sustained by the patients. Our clinical data support these notions are correlational, the patients with ataxia did not reach full motor recovery after carpal tunnel decompression surgery or recover spontaneously after tibial nerve neuropathy as observed in the non-ataxia patients  occurs depending on the severity of the injury and the distance of axon regeneration to accurately reinnervate their original targets. Our multiple SNC model, a form of severe PNI, extends the traveling distance as well as the time for regenerating axons to reach the original targets50% Fig. . In pati50% Fig. . These d100, suggesting the use of AMPA receptor potentiator for the treatment of cognitive impairments associated with schizophrenia. Additionally, PF-04958242 has been shown to improve auditory function significantly in age-related sensorineural hearing loss101. Another AMPA receptor potentiator, LY451395, which has been in Phase II development with possible use in Alzheimer’s disease whereas abnormalities of glutamatergic homeostasis also occur. A significant improvement in the neuropsychiatric symptoms was evident in patients with Alzheimer’s disease102. In mice, enhancement of AMPA receptor signaling by AMPA receptor agonists (CX 1837 and CX 1739) induced brain-derived neurotrophic factor secretion in periinfarct cortex and promoted motor function recovery after ischemic stroke. In contrast, pharmacological blocking of AMPA receptor signaling by AMPA receptor antagonist (CFM2) worsened motor task performance that normally recovers after stroke103. A novel AMPA receptor potentiator, TAK-137, not only enhanced cognitive function in naïve rats but also improved psychostimulant-induced hyperlocomotion104. These studies collectively suggest that the restoration of AMPA receptor function is critical to the maintenance of glutamatergic homeostasis that might show similarly favorable therapeutic benefits to improve motor function in patients with cerebellar dysfunctions and damages, and therefore warrant further investigation in a clinical setting.In summary, we believe that it is imperative to raise awareness among healthcare providers which concerns not only the link between ataxia and irreversible motor deficits but also has additional implications in the context of movement disorders associated with cerebellar dysfunctions or physical damage to the cerebellum that might cause disruption to the glutamatergic system and descending motor circuits results in poor clinical outcomes following PNIs. One potential therapeutic strategy is to stimulate AMPA receptors, given that the clinical application of AMPA receptor potentiators has been reported for treating psychiatric diseases. A recent clinical trial demonstrated that AMPA receptor potentiator (PF-04958242) attenuated the schizophrenia-relevant ketamine-induced cognitive impairment in verbal learning and working memoryPcp2-Cre mice (Pcp2-Cre+/−) with Lhx1 fl and Lhx5 fl (Lhx1fl/fl/Lhx5fl/fl) mice to obtain Pcp2-Cre+/−/Lhx1fl/fl/Lhx5fl/fl (ataxia mice) and Pcp2-Cre−/−/Lhx1fl/fl/Lhx5fl/fl (littermate mice) served as control mice23. Adult (8–10 weeks old) male double knockout mice and age-matched male littermates, and C57BL/6 wild-type adult mice were used for all experiments. Mouse lines were maintained on the C57BL6 background.All animal experimental protocols were approved by the Animal Research Ethics Sub-Committee at the City University of Hong Kong (ref. A-0017) and in compliance with the American Veterinary Medical Association (AVMA) Guidelines. Mice had free access to food and water and were maintained on a 12:12-h light–dark cycle. We made our best effort to reduce the number of animals used in current study. To generate Lhx1/5 double knockout mice, we crossed 37. To delay muscle reinnervation for a specific period of time , we crushed the sciatic nerves four times at 2-, 4-, and 5-day intervals for 16, 22, and 25 days of muscle denervation, respectively7. Animals were allowed to recover on heated pads before returning to their home cages. The surgeon who performed the surgery was blinded to the mouse genotypes and treatments.Adult male mice were anesthetized with 2.5% isoflurane, and the left sciatic nerve was crushed once with 5/45 smooth forceps (Fine Science Tools) for 15 s at the level of the external rotator muscles distal to the sciatic notch37. The observers were blinded to the mouse genotypes, surgery, and treatment groups.On day 3 after the last crush in single or multiple crush mice, we first performed a sensory pinprick assay followed by toe spreading reflex, grip strength measurement, and accelerated rotarod test every other day at least 30 min apart from each testA sensory pinprick assay was performed to measure the successful regeneration of sensory axons into hindlimb skin, mice were placed on wire mesh cages and habituated for at least 30 min. An insect pin (FST) was gently applied from the most distal toe (score 5) to the heel (score 0) (divided into 5 areas), briskly withdrawal of ipsilateral hindlimb indicated a positive response. A score of 5 indicated a positive response from every tested area and complete recovery of sensory function. The area innervated by the saphenous nerve of the ipsilateral hindlimb was tested as a positive control if there was no positive response from these five areas.To examine motor function recovery, we assessed spontaneous toe spreading reflex by covering the mice gently with a piece of cloth and picked up by the tail for clear observation. The toe spreading reflex was scored as: 0—no spreading; 1—intermediate spreading with all toes; and 2—full spreading was defined as a complete wide and sustained spreading of all toes for at least 2 seconds. Mice were scored when a full response was observed on the contralateral side to the injury. Mice were evaluated twice in each experimental session with at least a 45-min interval.To determine the recovery of hindlimb grip strength, forelimbs were rested on a plastic bar and the hindlimbs were positioned to grip the metal T-bar, and gently pulled off. Values (in grams) at which the mice were released from the T-bar were recorded by a grip strength meter . Animals were trained for three alternative days to get accustomed to the grip strength machine before taking the baseline values. Five readings were taken from each mouse and average values were presented.Motor coordination was assessed using the accelerated rotarod  test. Mice were trained for 2 days at a constant (4 rpm) or accelerating speed (4–40 rpm in 5 min) for 2 min. Mice were placed on the rotating wheel and latency (retention time) for the mice to fall off from the rotating wheel was recorded in accelerated mode. A 15-min rest interval was provided between each set of 5 trials.37.Mice were deeply anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg) and placed on a heating pad to maintain body temperature. A monopolar needle (27-gauge) electrode for EMG recording was custom-made with stainless steel wire  and the impedance was tested before use (Gamry Instruments). Sciatic nerve stimulation  was performed using a stimulator . The stimulating electrode was placed at the sciatic notch and the reference electrode was placed at the tail base. The EMG of the gastrocnemius muscle was recorded by inserting a recording electrode into the belly of gastrocnemius muscle and an Achilles tendon electrode as a reference. For EMG recording of interosseous muscle, the active and reference two-pin electrodes were inserted into the first and fourth interosseous muscles of the same hindlimb, respectively. EMG signals were sampled at 30 kHz , amplified, and bandpass-filtered with low and high frequency cutoffs of 10 and 250 Hz, respectively. The amplified EMG signal was then low-pass filtered  to remove aliasing in the 2000- to 3000-Hz range; any DC offset was also removed. CMAP amplitude was measured from the peak of the negative deflection to that of the positive deflection using Spike2. CMAP amplitude data are presented as mean ± SEM of 20 peaks/mice2. Under light anesthesia (1% isoflurane), a series of pinches were delivered to the most distal part of the sciatic nerve and gradually moving proximally to the crushed site at 3 days post-injury. The distance (mm) from the injury site to the most distal point on the nerve that produces a reflex withdrawal when pinched was recorded. The distance represents the length of in vivo axon regeneration within 72 h from the crush site to the response site. The person who performed the nerve pinch test and the second observer who verified the reflex withdrawal, were both blinded to the mouse genotypes.A nerve pinch test was performed to quantify the rate of in vivo axon regeneration. Left sciatic nerves were crushed for 15 s6. Preconditioned DRGs were digested with collagenase/dispase II (Roche Diagnostics), trypsinized, and dissociated mechanically using Pasteur pipettes. A total of 1500 DRG neurons were plated onto 8-well chamber slides (Millipore) precoated with poly-d-lysine and laminin (Sigma-Aldrich). Neurobasal (NB) medium supplemented with B27, 200 mM l-glutamine, penicillin/streptomycin, 50 ng/mL nerve growth factor (Gibco), 2 ng/mL glial cell line-derived neurotrophic factor, and 10 μM cytosine arabinoside (Sigma-Aldrich) were used. The cultures were allowed to grow for 17 h and fixed with 4% paraformaldehyde (PFA), and immunostained with anti-β tubulin III antibody. Non-overlapping images were taken using a motorized fluorescence microscope (Nikon Eclipse 90i). Longest neurite length of individual neurons from each mouse genotype was measured by automated WIS-NeuroMath software and averaged from at least 180 neurons per genotype. Data were obtained from three separate experiments repeated in duplicates.Sciatic nerve transection was performed in adult control and ataxia mice by ligating the left sciatic nerve with 6-0 suture followed by complete transection distal to the ligature. Ipsilateral and contralateral L4 and L5 DRGs were harvested after 5 days (preconditioning) of nerve transection and primary dissociated neuronal culture was prepared47. Bilateral electrical lesions were produced by passing a current of 0.75 mA for 10 s. For the sham-operated mouse group, electrodes were placed in the DCN without passing any current. All animals were observed daily to monitor health and allowed 2 weeks to recover from the surgery. An accelerated rotarod test was performed to validate motor coordination deficits in the DCN lesion mice. The sensory and motor function were tested in the DCN-lesioned and sham-operated mice before performing the sciatic nerve injury, to confirm that both mouse groups have similar baseline values before the SNC.Bipolar electrodes were prepared by perfluoroalkoxy-insulated stainless steel wires  and were positioned stereotaxically in the DCN of ketamine/xylazine anesthetized adult mice. Training and behavioral baselines were taken the week before the lesion. The electrodes were bilaterally inserted into the DCN at the following coordinates relative to bregma: anteroposterior (AP) = −6.4 mm, mediolateral (ML) = −1.3 mm, and dorsoventral (DV) = +2.5–3 mm from the skull surface44. There were five infusions sites in each hemisphere centering on the hippocampus, which targeted CA1–CA3 area . Lesion was produced by infusions of 0.1 μL of NMDA (10 mg/mL) over 1 min and the syringe was left in place for an additional 1 min before being slowly withdrawn. The mice were then allowed to recover for 3 weeks and were examined for spatial memory using the Morris water maze task and Y-Maze test. Baseline measurements of sensory and motor functions, EMG activities, and motor coordination (accelerated rotarod test), were taken from the hippocampus-lesioned mice with memory and learning deficits, and sham-operated mice for comparison.Adult mice were anesthetized with ketamine/xylazine and their heads were fixed using the stereotaxic apparatus. Animals were given multiple intracerebral infusions of NMDA) (Sigma-Aldrich)Three weeks after the hippocampal lesion, mice were subjected to Morris water maze task and Y-maze test. The same individual who handled the animals during the training period also performed all behavioral tests. The observer was blinded to the surgery and treatment groups.43.Spatial memory and learning were assessed by using the Morris water maze. A round tank with a diameter of 150 and 60 cm deep was filled approximately half-way with water, and the water temperature was maintained at 25 °C. The maze was divided into four equal quadrants by two principal axes perpendicular to one another, and four visual cues were placed on the walls of the water maze room. Mice were trained for 5 days with 4 trials per day and a minimum of 15 min inter-trial interval for each mouse. During training, the starting position was alternated between trials but hidden platform was placed in the center of one of the quadrants. Mice were allowed to swim freely to a small hidden platform (10 cm in diameter) submersed 1.5 cm under the water surface. The platform was camouflaged by adding a non-toxic paint in the water and making it nearly invisible. This opaque color also provides an efficient background for the ANY-maze tracking system. If the mice did not find the hidden platform during a period of 90 s, it was gently guided to the platform, and allowed to remain on the platform for 15 s to get familiarize with their surroundings. A probe test was performed 24 h after the last training day to assess the learning memory by removing the platform and allowing the mice to swim freely for 90 s. The latency to reach the platform area was recorded and analyzed by the ANY-maze tracking system (Stoelting)43.Spatial working memory was assessed by Y-maze test using a symmetrical Y-shaped maze with arms of equal length and angle. Each arm was 30 cm long, 10 cm wide, 20 cm height and interconnected with each other at a 120° angle. Y-maze arms were randomly divided into three arms—starting arm (SA), other arm (OA), and novel arm (NA). The test consisted of two trials of 5 min each with an inter-trial interval of 30 min. During the first trial, NA was blocked by a central partition and mice were placed at the end of SA, away from the center. The mice were allowed free access to the two arms (SA and OA) in the first trial and after that, it was returned to the home cage for the inter-trial interval. During the second trial, the central partition of NA was removed and the mice were allowed to explore all three arms. After each trial, the Y-maze was wiped with 75% ethanol to avoid the odor cues. During the experiment, the Y-maze was video recorded and the time spent in NA (%) were analyzed using the ANY-maze tracking software (Stoelting)47. Screw and the MEA were affixed to the skull with dental cement  to enhance the signal-to-noise ratio. The reference electrode was placed over the frontal bone. The exposed area of the skull was kept moist using artificial cerebrospinal fluid. The animals were observed daily and allowed to recover for 1 week after the surgery. This recovery period was based on the observation that no differences in motor behavior parameters were detected before and after electrode implantation at 5–7 days post-implantation.Customized multielectrode arrays were fabricated with nichrome wire  and tested for impedance before use . A 10-mm midline skin incision was made and bregma and lambda were located for precise stereotactic electrode placement. MEA electrodes were bilaterally inserted into the interposed nucleus of DCN at the following coordinates relative to bregma: AP = −6.4 mm, ML = −1.3 mm, and DV = +2.5–3 mm from the skull surfacePost-implantation electrophysiology and animal behavioral baselines were taken before the DCN-DBS. SNC was performed and electrode-implanted mice were divided into four groups: (1) Control mice with no DCN-DBS; (2) Control mice with DCN-DBS; (3) Ataxia mice with no DCN-DBS and (4) Ataxia mice with DCN-DBS. DCN stimulation  was performed for 1 h per day for 21 consecutive days after a single SNC, and for 33 consecutive days after the first SNC in the 22-day muscle denervation model. DCN stimulation  at a frequency of 130 Hz was triggered using a stimulator .Weekly MEA recordings of DCN were performed in both single and multiple SNC models after the sciatic nerve injury. Neural spike in DCN was recorded in freely-moving mice using the Apollo II acquisition system . Data were analyzed using Spike2 software, Blackrock offline spike sorting (BOSS) software (Blackrock Microsystem) for neurospike sorting. The sorted spike was further analyzed based on the principal component analysis (PCA) method to subtract error arising from electrode drifting and background signals. Our results showed good spike clustering and minimal data point drifting, which indicated low background noise levels. A customized MATLAB program was used to determine the frequency of neurospike, bursts, and interspike interval. We blinded the investigator who performed DCN neurospike data analysis by replacing the names of data files with a code name; this was done by a technician who was unaware of the mouse genotypes and treatments (with or without DCN-DBS).105. The Glx level in the cerebellum of the same mice was measured by MRS before baclofen treatment, 7 days after baclofen treatment, and 7 days after the SNC. Mice received a total of 21 consecutive days of baclofen treatment  was intraperitoneally injected into control and ataxia mice for 7 consecutive days before the SNCent Fig. . Ataxia T2 weighted images were firstly acquired using rapid acquisition with refocused echoes (RARE) sequence, to localize a voxel of interest in cerebrum of each mouse for MRS data acquisition  = 10 ms, TR = 2300 ms, scan time = 9 min 49 s. Water suppression was achieved using the variable pulse power and optimized relaxation (VAPOR) method54. MRS data processing and analysis were performed by MATLAB (MathWorks). MRS spectra were aligned by setting the N-acetylaspartate (NAA) peak at 2 parts per million (ppm). The peak areas for NAA at 2.02 ppm and Glx at 2.34 ppm were determined by Gaussian line shape fitting. The Glx to NAA ratio of each mouse brain was calculated for comparison.Magnetic resonance imaging was performed on a horizontal bore 3 T Bruker BioSpec system (Bruker). An 82 mm quadrature volume resonator was used as a transmitter, and a single surface coil was used for receiving signals. During magnetic resonance scanning, mouse anesthesia was induced and maintained using isoflurane at 2% and 1.5%, respectively. The sagittal and axial 47. A set of five stimuli was delivered for each stimulation current at 1 Hz and increased by increments of 25 µA ranging from 25 to 200 µA triggered by a stimulator   for the implantation of the MEA electrode. After repeating the input/output protocol, motor cortex was stimulated  with simultaneous recording of EMG for 30 min.Lastly, the skull was closed by suturing the skin, and a laminectomy between T12 and L5 was carried out to exposed the lumbar spinal cord. MEA electrode was placed in the ventral horn of the spinal cord  stereotaxically and covered with small cotton balls wetted with saline. A reference electrode was placed subcutaneously in the contralateral side of the recording electrode. After the input/output protocol for electrical stimulation of the spinal cord was validated, ventral horn of the spinal cord (near to L2–L5) was stimulated  with simultaneous recording of EMG for 30 min.To calculate nerve conduction latency, the stimulation marker channel was superimposed with EMG response, and the time difference between electrical stimulation and EMG response was recorded. An average of 25–30 EMG responses for each mouse were used for CMAP amplitudes and latency calculation. The raw data of EMG recordings were examined by a second person who was blinded to the treatment groups.Mice were anesthetized with ketamine/xylazine and placed on a stereotaxic surgery apparatus. A midline incision (10 mm) was made to locate the bregma as a reference. DCN  position was located on the right cerebellum and a 0.5 mm craniotomy was performed. Dual cannula system for mice  was secured in DCN using dental acrylic. One week after implantation, mice were randomly assigned into control and treatment groups.107. Aliquots were stored at −80 °C and diluted (10 times) to final concentrations in ACSF on the day of use. DNQX (1 µL) and ACSF (1 µL) were infused using a 26-gauge needle attached to a 10-µL Hamilton syringe at a slow rate of 0.2 µL/min  and the syringe was left in place for an additional 5 min before being slowly withdrawn. Mice received three injections per week for 3 consecutive weeks after SNC.Stock solution of DNQX  was prepared in artificial cerebrospinal fluid −ΔΔCt formula, and Gapdh was used as the internal standard for the normalization108. The sequences of forward and reverse primers used in the study are listed in Supplementary Table Total RNA was extracted from the cerebellum of injured mice 7 days after SNC using Trizol reagent (Takara), and the concentration of RNA was determined using Nanodrop 2000. Reverse transcription was performed using PrimeScript RT Reagent Kit with gDNA Eraser (Takara). qPCR analysis was performed in triplicate using TB Green Premix Ex Taq qPCR Kit (Takara) on a QuantStudio 12K Flex Real-Time PCR system. The Ct-value of each gene was recorded from each replicate and used to calculate the relative fold change using 212 vg/mL. AAV-ChR2-mCherry, AAV2-Gria1-P2A-eGFP, or AAV2-eGFP (control) (1 µL) was infused using a 26-gauge needle attached to a 10-µL Hamilton syringe at a slow rate of 0.2 µL/min , and the syringe was left in place for an additional 5 min before being slowly withdrawn. Mice were then allowed to recover for 2 weeks after AAV-ChR2-mCherry or AAV2-Gria1-P2A-eGFP injection.To overexpress ChR2 and mouse Gria1 in DCN, a midline incision (10 mm) was made to locate the bregma and lambda sutures for precise microinjection, and a 0.5 mm craniotomy was performed on the right hemisphere as described above. Mice received injection of adeno-associated virus serotype 2 (AAV2)-ChR2-mCherry, AAV2-Gria1-P2A-eGFP or AAV2-eGFP (control) into the DCN  at a viral titer of 5 × 10109. The optical fiber was coupled to the electrophysiology data-acquisition system (Blackrock microsystem) driven by software-generated transistor-to-transistor logic pulses, to align laser stimulation and EMG recording. DCN was stimulated with simultaneous EMG recording of gastrocnemius muscle for 30 min. An average of 25–30 EMG responses for each mouse were used for CMAP amplitudes and latency calculation48.For optical stimulation of DCN, optical fiber with blue laser at 473 nm (Inper Inc.) was illuminated at 50 μm above the injection site with 10 ms pulse and constant laser light power at 1 mW. Intensity output from the optical fiber was verified before and after each experiment, to be about 1 mW by a PM20A fiber power meter (Thorlabs)The research protocol for this retrospective study was reviewed by the Mayo Clinic Institutional Review Board Committee and was exempt from the requirement for IRB approval (IRB decision 20-006692).Mayo Clinic electronic medical record database EPIC was searched with Advanced Cohort Explorer (ACE) software. This program interrogates the electronic medical record in a similar fashion to an internet search. The search was conducted using the following inclusive search terms: peripheral nerve damage, peripheral neuropathy, facial nerve neuropathy, median nerve neuropathy, radial neuropathy, ulnar neuropathy, sciatic nerve neuropathy, tibial neuropathy, and peroneal neuropathy. All these search terms were then cross-referenced with a second search term which included cerebellar stroke, spinocerebellar ataxia, cerebellar ataxia, cerebellar damage, cerebellar injury, cerebellar atrophy, cerebellar degeneration, olivo-ponto-cerebellar atrophy, and multiple system atrophy. We focused further analysis on those ataxia patients and non-ataxia patients (controls) who underwent a corrective surgical procedure for CTS release or after being diagnosed with tibial neuropathy. In the final manual selection stage, only the patients with a precise date of peripheral nerve lesion diagnosis and those with at least one follow-up visit with a proper nerve conduction study (NCS) done after the surgical intervention (for CTS patients) or being diagnosed with tibial neuropathy, were selected for analysis.110 for CMAP amplitude and motor nerve conduction velocity measurements.For the control group, each patient from the ataxia study group was matched with at least one patient with a similar peripheral nerve injury without any cerebellar damage (non-ataxia patient). The control patients were selected manually from the group of patients obtained in the primary search through ACE. All ataxia and control patients had brain imaging (MRI or computerized tomography) performed, and they underwent identical Mayo Clinic standardized NCS protocolhttps://www.ncbi.nlm.nih.gov/geo/), using ‘ataxia’ as a keyword. Initial screening was performed in November 2021 and identified a total of 121 datasets that were derived from human samples. Samples that were derived from the patient's peripheral blood, blood plasma, and immune cells were excluded from the gene expression analysis. We considered samples derived from ataxia patients such as the cerebellum (GSE61019), patient iPSC-derived Purkinje cell and neuron (GSE85349 and GSE96826), patient-derived iPSCs , and patient-derived fibroblasts  and their pre-processed Series Matrix Files (expression quantification matrices) were extracted from NCBI GEO databases for subsequent analysis. The gene expression level of human GRIA1 from each sample was normalized to the average GRIA1 expression level in healthy individuals for relative log2 fold change calculation.To determine the expression profiles of human GRIA1 (NM_000827) in ataxia patients, gene expression datasets from ataxia patient samples were first identified from GEO . For multiple comparisons in behavioral and electrophysiology study, two-way ANOVA was used followed by appropriate post hoc analysis stated in the figure legends. Unpaired Student’s Further information on research design is available in the Supplementary InformationReporting Summary


MT1G , which has been shown to be highly expressed in ECs from parts of aorta  where atherosclerosis is more likely to occur. In conclusion, epinephrine has a greater effect than IH on EC gene expression in terms of number of genes and their expression level. IH inhibited the epinephrine-induced transcriptional response. Further investigation of the interaction between IH and epinephrine is needed to better understand how OSA causes cardiovascular disease.Obstructive sleep apnea (OSA) is an independent risk factor for cardiovascular disease. While intermittent hypoxia (IH) and catecholamine release play an important role in this increased risk, the mechanisms are incompletely understood. We have recently reported that IH causes endothelial cell (EC) activation, an early phenomenon in the development of cardiovascular disease, via IH-induced catecholamine release. Here, we investigated the effects of IH and epinephrine on gene expression in human aortic ECs using RNA-sequencing. We found a significant overlap between IH and epinephrine-induced differentially expressed genes (DEGs) including enrichment in leukocyte migration, cytokine-cytokine receptor interaction, cell adhesion and angiogenesis. Epinephrine caused higher number of DEGs compared to IH. Interestingly, IH when combined with epinephrine had an inhibitory effect on epinephrine-induced gene expression. Combination of IH and epinephrine induced  A recent analysis of global data estimated that close of 1 billion adults between the ages of 30 and 69 have OSA of which approximately half have moderate to severe OSA3. OSA is an independent risk factor for cardiovascular disease including hypertension, stroke, heart failure, coronary artery disease and atrial fibrillation6. Patients with cardiovascular disease have high prevalence of OSA (40–60%)7. In addition, severe OSA  is associated with increased cardiovascular and all-cause mortality8. However, the mechanisms by which OSA causes cardiovascular disease are not completely understood.Obstructive sleep apnea (OSA) is a highly prevalent sleep disorder in the adult population. The prevalence of OSA has been estimated to be up to 34% of adult men and 17% in adult women12. IH is a hallmark manifestation of OSA13. The severity of hypoxia or the hypoxia burden has recently been shown to be a strong predictor of cardiovascular and all-cause mortality in patients with OSA14. IH episodes lead to increased sympathetic tone and elevation of circulating and urinary catecholamine levels19. IH may thus mediate its effects either directly through the effects of hypoxia, or indirectly via catecholamines.The pathophysiology of OSA includes intermittent hypoxia (IH), intermittent hypercapnia, intrathoracic pressure swings, arousals, and sleep fragmentation24. OSA may affect EC function as they are directly exposed to the changes in oxygen concentration as well as to circulating catecholamines. In fact, OSA patients exhibit markers of EC activation, which are reversed by CPAP treatment27.Endothelial cells (ECs) play an important role in the pathogenesis of cardiovascular disease. Activation of ECs characterized by the expression of pro-inflammatory cytokines and adhesion molecules, is an early process in the development of cardiovascular disease33. Using gene array, Polotsky et al. found that short-term (8 h) exposure to human aortic endothelial cells (HAECs) led to increased expression of pro-inflammatory cytokines particularly IL-8 and NRF2 target genes; however, IH changed neither hypoxia-inducible factor-1α (HIF1A) nor HIF target gene expression28. Similar to HAECs, murine lung ECs also increased the expression of cytokines in response to IH29. Interestingly, in this case, IH reduced the expression of genes involved in the management of oxidative stress. While short-term (4–24 h) exposure to IH was well-tolerated, longer exposures to IH led to cell death. Exposure of human umbilical vein ECs (HUVECs) to IH induced the expression of adhesion molecules and endothelial-cell-specific molecule-130. In contrast to Polotsky et al., IH-induced changes were found to be HIF-dependent. Other studies using HUVECs31 and HAECs32 showed increased permeability evidenced by decreased transendothelial electrical resistance and increased expression of vascular endothelial cadherin (VE-cadherin). IH-induced increased permeability in HAECs was both HIF and oxidant dependent32.Since EC activation or dysfunction may precede the development of cardiovascular disease, there has been significant interest into determining mechanisms by which OSA affects EC function. Studies evaluating the effects of IH on ECs have shown increased production of pro-inflammatory cytokines, and adhesion molecules consistent with EC activation34. We found that EC activation in mice subjected to IH mimicking changes in blood O2 saturation in OSA was abolished when catecholamine release was inhibited by carotid body ablation, adrenalectomy or by chemical sympathectomy and thus, was not due to direct effect of IH, but rather indirectly through IH-induced sympathetic activation-derived epinephrine34. While our recent findings provide a novel mechanistic role for IH-induced sympathetic activation in EC activation, our previous study analyzed a limited number of genes involved in inflammation and immune cell adhesion that were implicated in EC activation. Building on our recent findings, in this study we sought to determine the gene expression changes in ECs induced by IH and epinephrine alone or together using an unbiased approach with RNA-seq, and to test the hypothesis that epinephrine modulates the effect of IH-induced gene expression changes in ECs.In contrast to these studies showing that IH may directly cause EC activation, we have recently shown that EC activation occurs indirectly via catecholamines in a murine model of OSA2 desaturations encountered during OSA activates ECs as evidenced by increased expression of pro-inflammatory cytokines and adhesion molecules34. Moreover, our studies using clinically relevant hypoxia levels showed that IH does not cause EC activation directly but indirectly via sympathetic nervous system activation and catecholamine release. We sought to expand this initial study by using RNA-sequencing to determine the effects of IH and epinephrine on EC gene expression in an unbiased manner. We first exposed human aortic endothelial cells (HAECs) to normoxia (20% O2) or IH (5% O2) for 60 cycles and collected RNA for sequencing. Control cells were exposed to normoxia (20% O2) contemporaneously. We did not observe any morphological changes in EC exposed to IH compared to control cells -saturated). In the hypoxia media, the dissolved O2 content ranged from 1.4 to 1.7 mg/l (20.81–25.27% hypoxia (5% O2-saturated).We have recently shown that exposure of mice to IH to model blood OHIF1A was not included among the DEGs under IH. We confirmed that IH did not induce HIF-1α protein or gene expression , Cytosolic phospholipase A2 gamma (PLA2G4C), Ephrin A1 (EFNA1), and Hematopoietic progenitor cell antigen CD34 (CD34). FABP4 plays an important role in angiogenesis. Increased expression causes endothelial dysfunction and is associated with cardiovascular disease37. EFNA1 was initially discovered as a TNF-induced protein in human umbilical vein endothelial cells and increases adhesion and migration of ECs38. CD34 is an important adhesion molecule that regulates immune cell migration. Using qPCR, we confirmed the IH-induced increase in the expression of these genes except for EFNA1  plot showed linearly separable groups of gene expressions between normoxia- and IH-exposed ECs Fig. a. AnalysTo better interpret the gene expression data, we performed gene ontology biological process (GO BP) enrichment analysis Fig. d and KEGTo determine the effect of epinephrine on EC gene expression, we treated HAECs cultured under normoxia with epinephrine (10 µM) or control for 7 h, which corresponds to the time of 60 cycles of IH and collected RNA for sequencing. MDS plot clearly showed separate gene expressions clusters of control and epinephrine treatment Fig. a. AnalysEFNA1 was the top upregulated gene by epinephrine  is required for endothelial morphogenesis39. NR4A1 (Nuclear receptor subfamily 4 group A member 1) is expressed highly in aortic ECs in response to oxidized low-density lipoprotein in vitro or isolated from high-fat treated mice in vivo40. It plays an important role in atherosclerosis formation. IRF1 (Interferon regulatory factor 1) is induced in ECs by inflammatory stimuli such as LPS and regulates VCAM-1 expression independent of NF-κB, but not E-selectin expression41. Genes that were upregulated by epinephrine were validated by qPCR . Furthermore, comparison of the expression levels of top significantly upregulated genes by IH showed that the extent of upregulation of the majority of genes were higher in epinephrine treated cells compared to IH , which is a member of low- molecular-weight cysteine-rich metallothionein protein family, and is involved in zinc and redox metabolism, and displays immunomodulatory functions in inflammatory diseases42  is highly expressed in endothelial cells. Its expression has been linked to blood pressure regulation and aging related changes in vasculature43. KITLG (KIT Ligand), also known as stem cell factor, is regulated by VEGF receptor blockade44. Collectively, IH reduced the effect of epinephrine on EC gene expression.The most significantly upregulated gene by the addition IH to epinephrine treatment was metallothionein-1G  of significant DEGs we identified under IH were also seen in response to epinephrine. Analysis of Gene Ontology Biologic Processes (GO_BPs) showed that the genes that were common between IH and epinephrine were enriched in leukocyte migration, extracellular matrix organization, cell adhesion and blood vessel development. Using KEGG pathway enrichment analysis we identified several pathways specific for epinephrine, including Ras signaling, MAPK signaling, PI3K-Akt signaling, and Phospholipase D signaling pathways in addition to cytokine-cytokine receptor interaction. Collectively, these results suggested that (1) epinephrine has a greater impact than IH on gene expression levels and profiles, (2) IH- and epinephrine-induced gene expression profiles are similar in terms of enrichment of biological processes (GO_BP), and (3) epinephrine has a greater effect than IH on gene expression associated with signaling pathways as assessed by KEGG pathway enrichment analysis.Endothelial cells are important in cardiovascular homeostasis, including regulation of blood pressure. It is not surprising that EC dysfunction and activation play a critical role in the development of cardiovascular disease and precedes clinically overt disease32. Importantly, these pathways and molecular events are responsible for EC dysfunction, hypertension, and atherosclerosis in OSA. Both patients with OSA and animals exposed to IH have elevated oxidative stress with higher production of inflammatory cytokines and adhesion molecules and antioxidant agents have been shown to improve oxidative stress-associated endothelial dysfunction32.We found that IH or epinephrine alone regulates leukocyte migration, angiogenesis, and cell matrix organization (GO_BP and KEGG) pathways in HAECs. These pathways are known to be regulated by various pathophysiologic changes such as oxidative stress, chronic inflammation, and sympathetic activation that occur in patients with OSA, and animals exposed to IH to model OSA33. Our studies did not show activation of HIF-1α or upregulation of HIF target genes. While the lack of upregulation in our studies is consistent with the results from Polotsky et al.28, other studies have shown that IH activates HIF in ECs. The disagreement among studies raises questions about the differences in IH models employed by studies including differences in the severity of hypoxia (0–10% O2), duration of exposure to hypoxia, duration of IH  and the type of ECs used . Some of the studies used intermittent anoxia, which induced the expression of proinflammatory cytokines at the levels similar to what was observed with continuous hypoxia29. The effect of IH was reported to depend on the duration of exposure to IH29. Studies in HUVECs showing IH-induced increased permeability and expression of adhesion molecules used longer duration of exposure to hypoxia (30–40 min) to model OSA31 limiting the relevance of findings to humans with OSA. Lastly, Kaczmarek et al. exposed human coronary artery EC and dermal microvascular ECs to IH (1% O2) and found that IH induced the gene expression of adhesion molecules and inflammatory cytokines as well as HIF-1α in coronary ECs, nut not in dermal microvascular ECs suggesting that the response to IH may be vascular bed dependent33. These findings do not agree with the other studies that showed that IH may directly induce EC activation33 likely due to the differences in IH models used. Collectively, further studies are needed to standardize IH models to best mimic the changes in O2 levels that occur in patients with OSA. Such an approach is necessary to improve our understanding about the mechanisms and will also allow comparison between different studies, which is not possible due to substantial differences in the models used at this time.Pathways we identified following IH are similar to the data from previous studies which evaluated the effect of IH on EC function showing upregulation of cytokines, adhesion molecules and antioxidant pathways47, which then may provide protection by inhibiting catecholamine-induced oxidative stress50. Contrary to the notion that OSA, which is characterized by IH as its hallmark, is an independent risk factor for cardiovascular disease, several studies have shown that IH conditioning may be cardioprotective both in experimental models as well as humans51. IH conditioning reduces infarct size and cardiac arrhythmias, and improves postischemia myocardial function. While the mechanisms are not completely understood, the duration and intensity of IH is thought to play a role in its cardioprotective effects as opposed to the unwanted effects attributed in OSA51. Consistent with our findings on the effects of IH on the epinephrine-induced transcriptional response, a recent study showed that IH exposure reduced markers of endothelial dysfunction in humans52. Furthermore, our findings corroborate data from clinical trials that compared supplemental oxygen, which treats IH, with CPAP therapy, which treats both IH and catecholamine release55. These trials failed to demonstrate cardiovascular benefit of supplemental oxygen (treatment of IH only) compared to CPAP, which remains the gold standard therapy emphasizing the importance of other pathophysiologic changes including catecholamines playing a bigger role than IH in the pathogenesis of cardiovascular disease. Further studies are needed to better understand the specific roles and complex interaction between IH and catecholamines not only on ECs, but also other cells within the cardiovascular system and other organs. Although the oxygen tension in our model was lower than those used for IH conditioning, it remains to be determined what the optimal oxygen concentration is to achieve IH conditioning. Future studies using a range of oxygen concentrations in IH models will be required to determine whether the inhibitory effect of IH on epinephrine-induced gene expression.Interestingly, although epinephrine and IH alone regulated many genes similarly, when exposed simultaneously, the combinatory effect of IH on epinephrine-induced gene expression was inhibitory. In fact, IH had an opposite effect on the epinephrine-induced gene expression and attenuated the effects of epinephrine. As expected from these data, when we compared the gene expression under IH with IH and epinephrine together, there were only few genes that were differentially expressed consistent with loss of epinephrine-effect on gene expression. While further studies will be needed to understand the exact mechanisms behind these unexpected findings, we speculate that one possibility is that IH may exert beneficial effects on the cardiovascular system by activating antioxidant pathwaysMT1G was identified as one of the few genes that is induced by combination of IH and epinephrine. MT1G encodes metallothionein, which is a metal binding protein involved in regulation of free levels of zinc42. Metallothionein has been shown to play an important role in oxidative stress, inflammation, and stress-related cell damage56. EC metallothionein expression has been shown to be regulated by blood flow pattern. ECs in parts of aorta where atherosclerosis is more likely to occur  are exposed to disturbed flow with low sheer stress. ECs from these atheroprone areas have higher expression of metallothionein compared to ECs in other parts of aorta where blood flow pattern is normal57. Exposure of ECs to disturbed flow in vitro also causes increased expression of metallothionein, which leads to increased binding of zinc and consequently decreases levels of free zinc promoting adhesion of monocytes and supporting a role for EC activation and dysfunction57. Interestingly, atherosclerotic areas have been shown to contain higher levels of zinc58. Our results suggest that metallothionein induced by IH and epinephrine may play a role in the pathogenesis of cardiovascular disease in OSA. Future experiments will be required to determine whether MT1G or metallothionein plays a causal role in the effects of IH on endothelial activation in vivo.In our analysis, 34 is within the dosing range used in in vitro studies61. It is possible that that different concentrations of epinephrine may have different effects and the observed effects on gene expression may be dose dependent.Our manuscript has several limitations. Our OSA model focused primarily on IH as the severity of hypoxia is associated with cardiovascular disease and did not include other pathophysiologic changes that occur in OSA including intermittent hypercapnia. Therefore, we cannot exclude the possibility of intermittent hypercapnia or other pathophysiologic changes that occur during an apnea having an effect on EC gene expression. As mentioned above, we only employed a single IH model in our experiments and did not study the impact of duration or the severity of hypoxia on EC function and gene expression. Furthermore, while the epinephrine dose used in our studiesIn conclusion, we found that both IH and epinephrine induce changes in gene expression in ECs. The effect of epinephrine on gene expression is greater than IH alone in terms of number of genes and the changes in the level of expression. Addition of IH attenuated and reversed the epinephrine-induced changes in gene expression in ECs. Our studies also identified new pathways affected by IH and epinephrine. Further investigation of these pathways will increase understanding of the mechanisms by which OSA causes cardiovascular disease.We used only primary human aortic endothelial cells (HAECs) in our studies. HAECs were purchased from Lonza  . Cells were grown in EGM-2 supplemented with SingleQuots from Lonza (CC-3156 and CC-4176) and Penicillin/Streptomycin/Amphotericin B Solution (Sigma). Epinephrine hydrochloride (Sigma) was solubilized in water and kept in − 20 °C in aliquots of 10 mM of concentration. All primary cultures were used from passage 5–7.2 for 2 min followed by 20% O2 for 5 min on a cyclic perfusion system with a flow rate of 5 ml/min. HAECs were exposed to either normoxia (control) or IH contemporaneously. Normoxia (control) cells were also exposed to flow like IH-exposed cells under the same perfusion system. There is no sheer stress due to perfusion system. Hypoxic and normoxic media were prepared by bubbling of appropriate gases prior to experiments. Both hypoxic and normoxic circuit have independent pumps ensuring intermittent, but continuous flow of the conditioned media on a 4-chamber interconnected culture dish. The whole system was operated at 37 °C. The CO2 level in the control normoxia and IH exposure was at 5% and balance was N2. O2 levels in the individual hypoxic and normoxic medium were monitored by a galvanic dissolved oxygen probe .For in vitro studies, HAECs were exposed to alternating cycles of 5% O62. The Kallisto index was made with default parameters and the GENCODE (Release 39 (GRCh38.p13) and was run in quant mode with default parameters. Following pseudoalignment, we computed gene abundances using R package tximport v.1.18.063. Differential expression was calculated between normoxia and hypoxia groups using R package edgeR64. edgeR performs read count filtering, normalization, estimating dispersion, and identification of differentially expressed genes. Differential gene expression was considered significant for genes with an FDR-adjusted p value ≤ 0.05 and fold change (FC) > 2. An absolute fold change ≥ 2 and false discovery rate (FDR) adjusted p value ≤ 0.05 were used to select and classify the significant DEGs. The raw RNA-seq data are accessible via GEO (GSE205050). https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE205050.Total RNA was isolated from HAECs using, GenElute™ Mammalian Total RNA Miniprep Kit and submitted to the University of Chicago Genomics Core Facility for sequencing with the Illumina NovaSEQ6000 sequencer (100 bp paired-end). Sequencing read (FASTQ) files were generated and assessed for per base sequence quality using FastQC. RNA-seq reads were pseudoaligned using Kallisto v.0.44.0 the at University of Chicago, CRI Gardner high performance computing clusterhttps://cran.r-project.org/web/packages/pheatmap/pheatmap.pdf Pathway enrichment analyses were performed, and plots were created using R clusterProfiler package65 or using Enrichr search engine web interface on Enrichr database66. All packages were run on RStudio (2021.09.0 Build 351) with R version 4.0.3. We also performed gene set enrichment analysis using KEGG data67.All volcano plots were drawn using ggplot2 R package. All heatmaps were generated with Pretty heatmaps R package pheatmap package from Z-score normalized expression values. RPL19 was used as a housekeeping gene, and gene expression was quantified using the ΔΔct method to determine relative fold-change. Primer sequences were used in this study are:CD34 EFNA1 FABP4 IRF1 JUNB MT1G NR4A1 PLA2G4C RPL19 SELE RNA was isolated GenElute™ Mammalian Total RNA Miniprep Kit and reverse transcribed using iScript Reverse Transcription Supermix . Quantitative mRNA expression was determined by real-time quantitative PCR (qPCR) using iTaq Universal SYBR Green Supermix . p < 0.05.The qPCR data were analyzed in Prism 9 . All data are shown as mean ± standard deviation (SD). To determine significance, we used non-parametric tests including Mann–Whitney to compare two groups and Kruskal–Wallis test to analyze data with more than two groups requiring multiple comparisons. Statistical significance was defined as *Supplementary Figures.


Background and purposeCraniocervical dissociation injuries encompass a spectrum of osteoligamentous injuries between the skull base and C1-C2 that may be treated via prolonged external immobilization versus occipital cervical fusion depending on the risk of persistent craniocervical instability. However, the presence of atlantoaxial instability (AAI) at C1-C2, as determined by transverse atlantal ligament (TAL) integrity with or without a C1 fracture, may guide the neurosurgical management of craniocervical dissociation spectrum injuries (CDSI) since it implies an overall greater degree of instability at the craniocervical junction (CCJ).Materials and methodsAdult trauma patients who suffered a transverse atlantal ligament injury on cervical magnetic resonance imaging (MRI) were identified retrospectively. The cervical computed tomography (CT) and magnetic resonance imaging examinations for these patients were reviewed for additional traumatic findings. Demographic information, treatment, and outcome information were recorded.ResultsTwenty-nine trauma patients presented to the emergency department (ED) with an acute, midsubstance transverse atlantal ligament tear on cervical magnetic resonance imaging. Thirty-one percent of patients demonstrated a tear in at least one major craniocervical ligament  with 14% demonstrating a tear in two major craniocervical ligaments and no patients demonstrating a tear in all three major craniocervical ligaments. Minor craniocervical ligament injuries  were common and observed in 76% of patients.ConclusionsOur study suggests that multiple major craniocervical junction ligamentous injuries on cervical magnetic resonance imaging are relatively uncommon in the setting of transverse atlantal ligament injury. Craniocervical dissociation spectrum injuries (CDSI) represent a spectrum of increasing hyperflexion-hyperextension forces applied to the osteoligamentous structures of the craniocervical junction (CCJ) that may range in severity from isolated craniocervical ligament tears to overt atlanto-occipital dislocation (AOD) ,2. StricThe stabilizing structures of the CCJ proper are anatomically and physiologically distinct from the major stabilizing structure at C1-C2. In contrast to the other osteoligamentous CCJ structures, the transverse atlantal ligament (TAL) extends horizontally between the medial tubercles of the C1 lateral masses, extending posterior to the C2 dens, and locks the median atlantoaxial joint in place . There iThe complex interplay between the craniocervical junction, including the atlanto-occipital joint and its surrounding supportive ligaments and muscles, and the atlantoaxial joint with its strong, stabilizing transverse atlantal ligament is not well understood. Unstable traumatic injuries of the atlanto-occipital and atlantoaxial joint often occur in isolation . RegardlTherefore, the purpose of our study is to identify adult trauma patients with a confirmed acute, post-traumatic tear of the transverse atlantal ligament on cervical MRI and evaluate for concomitant craniocervical junction ligamentous injuries. We hypothesize that atlantoaxial instability more commonly occurs in isolation from atlanto-occipital instability on cervical MRI. A thorough understanding and discussion of the complex interplay between instability at the CCJ proper and the atlantoaxial joint may allow for improved consensus regarding operative management of these injuries.A waiver of informed consent was granted by the institutional review board (IRB) chairman to retrospectively evaluate the imaging and clinical findings of adult trauma patients (>18 years old) with TAL injuries. Thirty patients who presented to the emergency department (ED) with an intrasubstance tear of the TAL on cervical MRI were identified retrospectively by using the keywords “transverse ligament,” “transverse band,” “cruciform ligament,” or “transverse atlantal ligament” included in cervical MRI reports between January 2015 and January 2021 using Nuance mPower  software. As a level I trauma center and tertiary care trauma spine center that captures all high-speed motor vehicle accidents (MVA) for a large geographic region, we were able to perform an analytic search of more than 15,000 cervical MRI examinations that presented to the ED for trauma during this time frame. The evaluation of the major and minor CCJ ligaments was performed, including the evaluation of the atlanto-occipital capsular ligaments, alar ligaments, tectorial membrane, and anterior and posterior atlanto-occipital membranes. Bony fractures of the craniocervical junction on preceding cervical spine computed tomography (CT) in addition to evaluation for intracranial trauma and cervical cord injury were recorded. Neurosurgical management and clinical outcomes for these injuries were analyzed.Patient selectionInclusion criteria for our patient cohort included all adult trauma patients who presented to our level I trauma center with a high-velocity injury with a confirmed TAL tear on cervical MRI. Exclusion criteria included pediatric patients less than 16 years old, non-trauma patients, and patients without a preceding cervical spine CT. Medial tubercle avulsion fracture of C1 without a midsubstance TAL tear was also excluded from the study. Patients with excessive inflammatory pannus or osteoarthritis of the atlantoaxial joint on cervical MRI that obscured the TAL and CCJ ligaments were also excluded from the study. The confirmation of a TAL tear was agreed upon in consensus by two certificate of additional qualification (CAQ)-certified neuroradiologists with long-term experience and prior research in craniocervical trauma.Imaging protocolsCT and MRI examinations were performed using the standard departmental protocols. CT images were generated with 0.625 mm slice thickness and reconstructed using multiplanar bone and soft tissue algorithms . Evaluation for CCJ bony trauma utilized multiplanar, orthogonal reconstructions using a thin-section bone kernel window. MRI studies were performed on a 1.5 Tesla magnet with a head and neck coil . Slice thickness was 3 mm, and sagittal T1, T2, and short tau inversion recovery (STIR), as well as axial T2 and T2 multi-echo data image combination (MEDIC) sequences, were obtained. All MRI sequences were comprehensively reviewed, and TAL injury location and type were agreed upon in consensus.MRI criteria for injuryAll cervical MRI examinations were graded as non-diagnostic, limited, or diagnostic quality. TAL injuries were confirmed primarily on T2 and STIR axial and sagittal MRI sequences when there was clear disruption of the normal dark T2 hypointense ligament posterior to the C2 dens and increased T2 signal within the ligament. The TAL was also considered injured if the ligament was not visible and replaced with hematoma (ruptured ligament). The location of the TAL injury was recorded as right, left, midline, or bilateral in relation to the C2 dens. Injury of the capsular ligaments was inferred on cervical CT and MRI as widening of the atlanto-occipital joint greater than 3 mm. Alar ligament injury was directly evaluated on axial and sagittal T2-weighted sequences as increased T2 signal and focal disruption of the normal T2 hypointense band. Tectorial membrane (TM) injuries were confirmed primarily on T2 and STIR axial and sagittal imaging according to the classification system developed by Fiester et al. when there was disruption of the normal dark T2 hypointense ligament on cervical MRI and increased T2 signal within the ligament (type 2 tear) or the TM demonstrated increased T2 signal with greater than 50% decrease in TM thickness on sagittal T2-weighted imaging (type 3 tear). Anterior and posterior atlanto-occipital membrane tears were considered injured on axial and sagittal T2 and STIR sequences when there was clear disruption of the ligament and/or increased STIR signal within the ligament.Supplemental dataElectronic patient records were reviewed for the following: 1) age and sex of patient, 2) mechanism of trauma, 3) management , and 4) clinical outcome . A good clinical outcome/full recovery was defined as the absence of persistent neurologic deficits on follow-up evaluation by the neurosurgery department at least four months following the initial injury.A total of 29  patients were identified with TAL tears on cervical MRI between January 2015 and January 2021. Thirteen TAL tears were left-sided, 12 injuries were right-sided, two injuries were bilateral, and two injuries were midline , 14% of patients demonstrated a tear in two or more major craniocervical ligaments, and no patients demonstrated a tear in all three major ligaments. Increased STIR signal and/or frank disruption involving at least one minor craniocervical ligament was more common and present in 76% of patients Figure -1D.A total of 14 patients demonstrated an anterior atlanto-occipital membrane injury, 20 patients demonstrated a posterior atlanto-occipital membrane complex (PAOMc) injury, and 13 patients demonstrated an injury in both minor ligaments Figure -2D.Fifty-nine percent of patients demonstrated a C1 and/or C2 fracture (10 patients with C1 burst fractures and eight patients with type II/III dens fracture). Seventy-six percent of patients demonstrated intracranial hemorrhage on concurrent head CT. There was zero case of cervical cord contusion. All TAL injuries were the result of an MVA, either a primary MVA (25 patients) or a pedestrian/bicyclist struck by a motor vehicle (four patients). Surgical management varied, depending on the extent of the CCJ injury, patient comorbidities, and other post-traumatic injuries at the time of the trauma. Two patients underwent occipital cervical fusion (both occiput-C3), four patients underwent occipital-sparing posterior cervical fusion (three patients with C1-C2 fusion and one patient with C1-C6 fusion), two patients underwent prolonged external immobilization with halo bracing, and the remaining patients were treated with Miami J  collar placement. Seven patients experienced persistent neurologic deficits (weakness and/or spasticity) at least four months following the initial trauma.Despite their distinct anatomic and biomechanical properties and differences in the types of post-traumatic injuries sustained, the interrelation between atlantoaxial instability and the instability of the CCJ proper is poorly understood. This negatively impacts clinical management and has hindered the ability to reach a consensus regarding appropriate neurosurgical care. Our study demonstrates that a minority of patients with atlantoaxial instability (defined by a midsubstance TAL injury) had concomitant major CCJ ligamentous injury with only 14% of patients demonstrating an additional injury of at least two major CCJ ligaments and zero patient demonstrating a tear of all three major CCJ proper ligaments. Moreover, no patients exhibited imaging findings of overt atlanto-occipital dislocation, typically defined as atlanto-occipital joint space widening greater than 2 mm. Minor CCJ ligament injuries involving the anterior atlanto-occipital membrane complex (AAOMc) or posterior atlanto-occipital membrane complex (PAOMc) were more common and present in 76% of patients Figure -3D.However, Chang et al. recently investigated the utility of isolated increased STIR signal in the PAOMc on MRI as a secondary finding of C1-C2 fractures with a high sensitivity (89.7%) . Given tAs injuries to the atlantoaxial joint and CCJ proper more commonly occur in isolation, redefining the radiologic and neurosurgical definition of the CCJ by separating the CCJ proper  from the atlantoaxial joint  would have several positive effects. With the increasing availability and utilization of cervical MRI in the ED trauma setting, the radiologist now has the opportunity to accurately define CDSI by highlighting injuries to the specific major and minor CCJ ligaments and TAL . UnderstCDSI encompasses a spectrum of osteoligamentous injuries at the CCJ and atlantoaxial joint that range from isolated CCJ proper major or minor ligamentous injuries to overt AOD with atlanto-occipital dislocation. In our patient cohort, injuries to the atlantoaxial joint were much more commonly isolated injuries without injury of the major and minor CCJ proper ligaments. Many trauma centers employ a high-resolution isotropic sequence as part of their trauma cervical MRI protocols that allow multiplanar, three-dimensional reconstructions and improved anatomic resolution of the CCJ ligaments and TAL. Along with specialized isotropic imaging, increasing magnet field strength allows improved anatomic resolution of the complex osteoligamentous anatomy of the CCJ . These dIn addition to recognizing the spectrum of post-traumatic imaging appearances of CDSI, understanding the unique biomechanical role of each ligament in maintaining CCJ and atlantoaxial integrity is necessary to optimize clinical management. Regarding the CCJ proper, three major ligaments maintain CCJ integrity: the atlanto-occipital joint and enveloping capsular ligament, the bilateral alar ligaments, and the tectorial membrane ,5. InjurFiester et al. identified nine patients who underwent emergent occipital cervical fusion for acute CDSI and found that all nine patients demonstrated a tear in the tectorial membrane and five patients sustained an alar ligament tear . The impIn contrast to the numerous contributory ligaments for CCJ stability, biomechanical research studies indicate that the TAL acts as the primary stabilizer of the atlantoaxial joint and the sole preventer of anterior subluxation of C1 on C2 ,7. RadioConsensus regarding the appropriateness and necessity of operative fusion and length versus conservative management for CDSI is debated in the neurosurgical literature especially in the absence of overt AOD, which, if survivable, is almost always treated with prompt surgical stabilization. However, the patient’s age and comorbidities play a major role in the overall morbidity and mortality of craniocervical and atlantoaxial trauma . One recThe limitations of our study were subject to those of a retrospective review and included the possibility of reinforcement bias and interpreter error. However, image quality on cervical MRI was graded as diagnostic in all cases to identify a TAL injury. Then, by nature of a retrospective review, we may not have captured all TAL injuries and did not specifically identify C1 medial tubercle avulsion fractures; however, this was deliberate since we wanted to concentrate solely on patients with midsubstance TAL tears. Finally, our results are limited from the small sample size, and although we collected one of the largest cohorts of midsubstance TAL injuries on cervical MRI in the literature, a multiregional research study with a larger sample size investigating the relationship of CCJ and atlantoaxial trauma in CDSI would improve the accuracy of our findings and assist in targeted research studies comparing operative to non-operative management in the patients with different CDSI injury types on cervical CT and MRI.CDSI is a spectrum of increasing, post-traumatic hyperflexion-hyperextension forces applied to the osteoligamentous CCJ and encompasses both the anatomy and biomechanics of the CCJ proper and the atlantoaxial joint. Given the variability and range of imaging findings on CT and MRI, significant controversy exists in the literature regarding the proper clinical management of patients with traumatic CDSI. Our study suggests that major CCJ proper ligamentous injury on cervical MRI is a relatively uncommon finding in the setting of transverse atlantal ligament injury. A thorough understanding and separation of the distinct anatomic and biomechanical properties of the CCJ proper from the atlantoaxial joint would be beneficial to optimize clinical management and guide future investigative research of craniocervical and upper cervical spine trauma.