Intrathecal Delivery Research Articles

Prepontine baclofen catheter placement: A case report and technical illustration

BackgroundIntrathecal baclofen (ITB) is used for medically refractory spasticity and dystonia. There are times when intraventricular baclofen (IVB) is used as a last resort for patients who cannot tolerate or cannot receive intrathecal baclofen through a traditional route. We present a patient with a history of cerebral palsy and multiple ITB and IVB revisions who presented with medically refractory spasticity and severe autonomic storming. Other options are needed for patients with persistent autonomic storming, dystonia and spasticity following IVB and spinal intrathecal baclofen treatment. MethodsWe propose a technical variation and case illustration of a prepontine catheter placement through an endoscopic third ventriculostomy. Through this approach, a ventricular catheter was used to deliver baclofen into the intrathecal space. ResultsFollowing the procedure, the patient showed improvement in Ashworth score and autonomic storming. ConclusionThis technique could provide an alternative and superior therapeutic option to traditional catheter placement for intrathecal baclofen delivery.

Intrathecal AAV9-Neprilysin Protects Against Perivascular Amyloid-β Accumulation in a Mouse Model of Cerebral Amyloid Angiopathy

Targeting amyloid-β (Aβ) accumulation seems a promising strategy for the treatment of cerebral amyloid angiopathy (CAA) and neurodegenerative diseases. However, it is still challenging to develop a safe and efficient technology for brain-wide perivascular Aβ clearance. Using intrathecally injected adeno-associated virus 9 (AAV9) encoding soluble form of neprilysin (sNEP), we showed that AAV-sNEP reduced Aβ accumulation and improved cognitive deficits in Tg-SwDI transgenic mice. Meningeal cells, adventitial cells and smooth muscle cells in leptomeningeal arteries and penetrating arterioles were transfected by intrathecal injection of AAV9-sNEP, hence generating sufficient sNEP to exert beneficial effects by perivascular Aβ clearance, protecting against smooth muscle cell damage and pericyte loss, maintaining blood-brain barrier (BBB) integrity, and eventually improving cognitive decline in Tg-SwDI mice. This study provides pre-clinical evidence that intrathecal delivery of AAV-mediated sNEP may be a promising therapeutic strategy for the treatment of CAA. Funding Statement: This study was supported by the Science and Technology Development Fund, Macau SAR (020/2017/A1 and 039/2017/AFJ); multi-year research grant, University of Macau, (MYRG2016-00184-ICMS-QRCM and MYRG2018-00242-ICMS); Science and Technology Program of Guangzhou (No. 2014J4500031) and Guangzhou Clinical Research and Translational Center for Major Neurological Disease (No. 201604020010). Declaration of Interests: The authors declared no conflicts of interest. Ethics Approval Statement: All animal experiments were approved by the ethics committee at the University of Macau (Macau, China) and Sun Yat-Sen University, and performed in compliance with the institutional guidelines.

Lot-to-Lot Variation in Adeno-Associated Virus Serotype 9 (AAV9) Preparations.

Viral vectors are complex drugs that pose a particular challenge for manufacturing. Previous studies have shown that, unlike small-molecule drugs, vector preparations do not yield a collection of identical particles. Instead, a mixture of particles that vary in capsid stoichiometry and impurities is created, which may differ from lot to lot. The consequences of this are unclear, but conflicting reports regarding the biological properties of vectors, including transduction patterns, suggest that this variability may have an effect. However, other variables, including differences in animal strains and techniques, make it difficult to identify a cause. Here, we report lot-to-lot variation in spinal cord gray matter transduction following intrathecal delivery of self-complementary adeno-associated virus serotype 9 vectors. Eleven lots of vector were evaluated from six vector cores, including one preclinical/Good Laboratory Practice lot. Eight of the lots, including the preclinical lot, failed to transduce the gray matter, whereas the other three provided robust transduction. The cause for this variation is unknown, but it did not correlate with vector titer, buffer, or purification method. These results highlight the need to identify the cause of this variation and to develop improved production and quality control methods to ensure lot-to-lot consistency of vector potency.

IFNβ Treatment Inhibits Nerve Injury-induced Mechanical Allodynia and MAPK Signaling By Activating ISG15 in Mouse Spinal Cord

Neuropathic pain is difficult to treat and remains a major clinical challenge worldwide. While the mechanisms which underlie the development of neuropathic pain are incompletely understood, interferon signaling by the immune system is known to play a role. Here, we demonstrate a role for interferon β (IFNβ) in attenuating mechanical allodynia induced by the spared nerve injury in mice. The results show that intrathecal administration of IFNβ (dosages up to 5,000 U) produces significant, transient, and dose-dependent attenuation of mechanical allodynia without observable effects on motor activity or feeding behavior, as is common with IFN administration. This analgesic effect is mediated by the ubiquitin-like protein interferon-stimulated gene 15 (ISG15), which is potently induced within the spinal cord following intrathecal delivery of IFNβ. Both free and conjugated ISG15 are elevated following IFNβ treatment, and this effect is increased in UBP43−/− mice lacking a key deconjugating enzyme. The IFNβ-mediated analgesia reduces MAPK signaling activation following nerve injury, and this effect requires induction of ISG15. These findings highlight a new role for IFNβ, ISG15, and MAPK signaling in immunomodulation of neuropathic pain and may lead to new therapeutic possibilities. PerspectiveNeuropathic pain is frequently intractable in a clinical setting, and new treatment options are needed. Characterizing the antinociceptive potential of IFNβ and the associated downstream signaling pathways in preclinical models may lead to the development of new therapeutic options for debilitating neuropathies.

Nerve Injury-Induced Neuronal PAP-I Maintains Neuropathic Pain by Activating Spinal Microglia.

Pancreatitis-associated proteins (PAPs) display multiple functions in visceral diseases. Previous studies showed that the expression level of PAP-I was low in the DRG of naive rats but was de novo expressed after peripheral nerve injury. However, its role in neuropathic pain remains unknown. We found that PAP-I expression was continuously upregulated in the DRG neurons from rat spared nerve injury models, and transported toward the spinal dorsal horn to act as a proinflammatory factor. Intrathecal delivery of PAP-I enhanced sensory hyperalgesia, whereas PAP-I deficiency by either gene knockout or antibody application alleviated tactile allodynia at the maintenance phase after spared nerve injury. Furthermore, PAP-I functioned by activating the spinal microglia via C-C chemokine receptor Type 2 that participated in neuropathic pain. Inhibition of either microglial activation or C-C chemokine receptor Type 2 abolished the PAP-I-induced hyperalgesia. Thus, PAP-I mediates the neuron-microglial crosstalk after peripheral nerve injury and contributes to the maintenance of neuropathic pain.SIGNIFICANCE STATEMENT Neuropathic pain is maladaptive pain condition, and the maintaining mechanism is largely unclear. Here we reveal that, after peripheral nerve injury, PAP-I can be transported to the spinal dorsal horn and is crucial in the progression of neuropathic pain. Importantly, we prove that PAP-I mainly functions through activating the spinal microglia via the CCR2-p38 MAPK pathway. Furthermore, we confirm that the proinflammatory effect of PAP-I is more prominent after the establishment of neuropathic pain, thus indicating that microglia also participate in the maintenance phase of neuropathic pain.

Application of Hepatocyte Growth Factor for Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is an adult onset neurodegenerative disorder characterized by the death of upper and lower motor neurons. About 10% of all ALS cases are familial, and we have identified SOD1 and FUS mutations as the most common causes in a consecutive series of 111 familial ALS pedigrees in Japan (Nishiyama A, 2017). From studies of the TDP43, FUS, and C9orf72 genes, perturbations of RNA processing can be highly adverse in motor neurons. Hepatocyte growth factor (HGF) is one of the most potent survival-promoting factors for motor neurons. We administered human recombinant HGF (hrHGF) by continuous intrathecal delivery to transgenic rats at onset of paralysis for 4 weeks. Intrathecal administration of hrHGF attenuated motor neuron degeneration and prolonged the duration of the disease by 63% (Ishigaki A, 2007). To translate this strategy to human treatment, we induced a contusive cervical spinal cord injury in the common marmoset, a primate, and then administered hrHGF intrathecally. We conducted a first-in-human phase I trial of intrathecal hrHGF in 15 Japanese patients with ALS (Warita H, 2019). Based on the results, we are conducting a phase II trial of intrathecal hrHGF for patients with ALS.

Intrathecal Delivery of Antisense Oligonucleotides in the Rat Central Nervous System

Intrathecal Delivery of Antisense Oligonucleotides in the Rat Central Nervous System

Intrathecal Delivery of Antisense Oligonucleotides in the Rat Central Nervous System.

The blood brain barrier (BBB) is an important defense against the entrance of potentially toxic or pathogenic agents from the blood into the central nervous system (CNS). However, its existence also dramatically lowers the accessibility of systemically administered therapeutic agents to the CNS. One method to overcome this, is to inject those agents directly into the cerebrospinal fluid (CSF), thus bypassing the BBB. This can be done via implantation of a catheter for either continuous infusion using an osmotic pump, or for single bolus delivery. In this article, we describe a surgical protocol for delivery of CNS-targeting antisense oligonucleotides (ASOs) via a catheter implanted directly into the cauda equina space of the adult rat spine. As representative results, we show the efficacy of a single bolus ASO intrathecal (IT) injection via this catheterization system in knocking down the target RNA in different regions of the rat CNS. The procedure is safe, effective and does not require expensive equipment or surgical tools. The technique described here can be adapted to deliver drugs in other modalities as well.

Development of an AAV-Based MicroRNA Gene Therapy to Treat Machado-Joseph Disease

Spinocerebellar ataxia type 3 (SCA3), or Machado-Joseph disease (MJD), is a progressive neurodegenerative disorder caused by a CAG expansion in the ATXN3 gene. The expanded CAG repeat is translated into a prolonged polyglutamine repeat in the ataxin-3 protein and accumulates within inclusions, acquiring toxic properties, which results in degeneration of the cerebellum and brain stem. In the current study, a non-allele-specific ATXN3 silencing approach was investigated using artificial microRNAs engineered to target various regions of the ATXN3 gene (miATXN3). The miATXN3 candidates were screened in vitro based on their silencing efficacy on a luciferase (Luc) reporter co-expressing ATXN3. The three best miATXN3 candidates were further tested for target engagement and potential off-target activity in induced pluripotent stem cells (iPSCs) differentiated into frontal brain-like neurons and in a SCA3 knockin mouse model. Besides a strong reduction of ATXN3 mRNA and protein, small RNA sequencing revealed efficient guide strand processing without passenger strands being produced. We used different methods to predict alteration of off-target genes upon AAV5-miATXN3 treatment and found no evidence for unwanted effects. Furthermore, we demonstrated in a large animal model, the minipig, that intrathecal delivery of AAV5 can transduce the main areas affected in SCA3 patients. These results proved a strong basis to move forward to investigate distribution, efficacy, and safety of AAV5-miATXN3 in large animals.

Brain pharmacology of intrathecal antisense oligonucleotides revealed through multimodal imaging.

Intrathecal (IT) delivery and pharmacology of antisense oligonucleotides (ASOs) for the CNS have been successfully developed to treat spinal muscular atrophy. However, ASO pharmacokinetic (PK) and pharmacodynamic (PD) properties remain poorly understood in the IT compartment. We applied multimodal imaging techniques to elucidate the IT PK and PD of unlabeled, radioactively labeled, or fluorescently labeled ASOs targeting ubiquitously expressed or neuron-specific RNAs. Following lumbar IT bolus injection in rats, all ASOs spread rostrally along the neuraxis, adhered to meninges, and were partially cleared to peripheral lymph nodes and kidneys. Rapid association with the pia and arterial walls preceded passage of ASOs across the glia limitans, along arterial intramural basement membranes, and along white-matter axonal bundles. Several neuronal and glial cell types accumulated ASOs over time, with evidence of probable glial accumulation preceding neuronal uptake. IT doses of anti-GluR1 and anti-Gabra1 ASOs markedly reduced the mRNA and protein levels of their respective neurotransmitter receptor protein targets by 2 weeks and anti-Gabra1 ASOs also reduced binding of the GABAA receptor PET ligand 18F-flumazenil in the brain over 4 weeks. Our multimodal imaging approaches elucidate multiple transport routes underlying the CNS distribution, clearance, and efficacy of IT-dosed ASOs.

Effects of ghrelin on pGSK-3\u03b2 and \u03b2-catenin expression when protects against neuropathic pain behavior in rats challenged with chronic constriction injury

Ghrelin has been shown to alleviate neuropathic pain by inhibiting the release of proinflammatory cytokines. The purpose of this study was to investigate the role of GSK-3β/β-catenin signaling in mediating the effect of ghrelin on neuropathic pain and to understand the associated mechanisms. Chronic constriction injury (CCI) of the sciatic nerve was used to establish a rat model of neuropathic pain. Hyperalgesia and allodynia were evaluated by observing the mechanical withdrawal threshold and the thermal withdrawal latency. Wnt3a and β-catenin protein expression and GSK-3β phosphorylation were detected by western blotting analysis. The levels of tumor necrosis factor-α and IL-1β were determined using an enzyme-linked immunosorbent assay. In addition, we used immunohistochemical analysis to determine the levels of GSK-3β phosphorylation in the dorsal horn of the spinal cord. Intrathecal delivery of ghrelin effectively ameliorated CCI-induced mechanical allodynia and thermal hyperalgesia at 7 and 14 days and reduced the levels of tumor necrosis factor-α. Ghrelin inhibited CCI-induced GSK-3β activation and β-catenin overexpression in the spinal dorsal horn. Moreover, intrathecal injection of ghrelin suppressed the activation of GSK-3β in the spinal dorsal horn of CCI rats, as assessed by immunohistochemical analysis. Our data indicated that ghrelin could markedly alleviate neuropathic pain by inhibiting the expression of β-catenin, via the suppression of GSK-3β activation, in the spinal cord of CCI rats.

Neurotensin Analogues Containing Cyclic Surrogates of Tyrosine at Position 11 Improve NTS2 Selectivity Leading to Analgesia without Hypotension and Hypothermia.

Neurotensin (NT) exerts its analgesic effects through activation of the G protein-coupled receptors NTS1 and NTS2. This opioid-independent antinociception represents a potential alternative for pain management. While activation of NTS1 also induces a drop in blood pressure and body temperature, NTS2 appears to be an analgesic target free of these adverse effects. Here, we report modifications of NT at Tyr11 to increase selectivity toward NTS2, complemented by modifications at the N-terminus to impair proteolytic degradation of the biologically active NT(8-13) sequence. Replacement of Tyr11 by either 6-OH-Tic or 7-OH-Tic resulted in a significant loss of binding affinity to NTS1 and subsequent NTS2 selectivity. Incorporation of the unnatural amino acid β3hLys at position 8 increased the half-life to over 24 h in plasma. Simultaneous integration of both β3hLys8 and 6-OH-Tic11 into NT(8-13) produced a potent and NTS2-selective analogue with strong analgesic action after intrathecal delivery in the rat formalin-induced pain model with an ED50 of 1.4 nmol. Additionally, intravenous administration of this NT analogue did not produce persistent hypotension or hypothermia. These results demonstrate that NT analogues harboring unnatural amino acids at positions 8 and 11 can enhance crucial pharmacokinetic and pharmacodynamic features for NT(8-13) analogues, i.e., proteolytic stability, NTS2 selectivity, and improved analgesic/adverse effect ratio.

Effect of Long-Term Intrathecal Bupivacaine Infusion on Blood Pressure

The local anesthetic bupivacaine is a common analgesic adjuvant medication used in combination with opioids in intrathecal drug delivery systems (IDDSs). While the acute effects of spinal bupivacaine injection on blood pressure (BP) have been studied, there is a dearth of data regarding long-term effects of continuous intrathecal bupivacaine infusion. A retrospective review of all noncancer pain patients receiving bupivacaine through IDDSs from January 2013 to November 2017 was performed. Blood pressure values before implantation, and after implant at the 1-week, 3-month, 6-month and 1-year time points are recorded as well as corresponding bupivacaine and opioid doses in the IDDS. Eighty-two patients were included in the study; median patient age was 64 years and intrathecal catheter tips were located predominantly in the lower thoracic spine. Significant decreases in systolic blood pressure (SBP) and mean arterial pressure (MAP) relative to baseline occurred in patients receiving chronic lower thoracic intrathecal bupivacaine delivery at 3, 6, and 12 months post-implant (-12.96 ± 18.21; p < 0.001 and - 6.38 ± 12.04; p < 0.001, respectively). The only significant decrease in DBP was observed at 1 year post-implant (-3.09 ± 11.76; p < 0.05). Similar decreases in BP values occurred in patients with or without hypertension. Long-term thoracic intrathecal infusion of bupivacaine likely exerts a lowering effect on BP, primarily because of a significant decrease in SBP. This effect is likely due to efferent sympathetic blockade by bupivacaine.

Intrathecal administration of AVXS-101 gene-replacement therapy (GRT) for spinal muscular atrophy type 2 (SMA2): Phase 1/2A study (strong)

The one-time survival motor neuron (SMN) GRT AVXS-101 treats the genetic root cause of SMA. In a phase 1/2a study (CL-101; NCT02122952), intravenous AVXS-101 administration improved outcomes in SMA type 1 patients. STRONG is a multicenter, open-label phase 1/2a study (NCT03381729) assessing intrathecal AVXS-101 in SMA2 patients. SMA2 patients (biallelic SMN1 loss, 3xSMN2) aged ≥6–<60 months who could sit but not stand/walk received an intrathecal AVXS-101 dose (dose A: 6.0x10e13; B: 1.2x10e14; C: 2.4x10e14 vg). Dose B enrollment was completed following demonstration of acceptable safety in dose A and B. Endpoints included safety/tolerability, optimal dose, unsupported standing ≥3 s, and change in baseline Hammersmith Functional Motor Scale-Expanded (HFMSE) score. By 24 March 2019, 31 patients were enrolled (11 sites). Enrollment is complete for dose A (n = 3, ≥6–<24 months) and B (n = 25; n = 13, ≥6–<24 months; n = 12, ≥24–<60 months), and ongoing for dose C (3 patients dosed [≥6–<24 months]; 12 planned each ≥6–<24 and ≥24–<60 months). As of 8 March 2019, no fatal treatment-emergent adverse events (TEAEs) have occurred; 7 serious TEAEs occurred in 4 patients, all of which resolved. Patients aged ≥24–<60 months showed rapid HFMSE responses (half had ≥3-point increase from baseline by 1 month post-treatment). Patients aged ≥6–<24 and ≥24–<60 months averaged a 4.2-point increase from baseline HFMSE score at last follow-up. Ten patients achieved 22 additional motor milestones. Interim STRONG data show intrathecal AVXS-101 delivery is feasible, well tolerated, and appears to improve motor function in SMA2 patients.

SFKs/p38 Pathway is Involved in Radicular Pain by Promoting Spinal Expression of Pro-Inflammatory Cytokines in a Rat Model of Lumbar Disc Herniation.

A controlled, randomized, animal study. The aim of this study was to investigate the role of src-family kinases/p38 pathway in a rat model of lumbar disc herniation (LDH). LDH always generates radicular pain, and the mechanism remains unclear. We have reported that spinal src-family kinases (SFKs) may be involved in the process, but the downstream mechanism needs further investigation. LDH was induced by implantation of autologous nucleus pulposus (NP), harvest from the tail, in lumbar 4/5 spinal nerve roots of rat. Von Frey filaments and radiant heat tests were performed to determine mechanical and thermal pain threshold respectively. Basso, Beattie, and Bresnahan (BBB) scale was assessed to test the locomotor function. The protein level of p-SFKs, t-SFKs, p-p38, t-p38 in spinal cord was examined by western blotting analysis. Cellular location of p-p38 was determined by immunochemistry staining. Spinal tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β, and IL-6 levels were detected by enzyme-linked immunosorbent assay (ELISA). Rats with NP implantation showed persistent ipsilateral mechanical allodynia and thermal hyperalgesia, which manifested as obvious decrease of paw withdrawal threshold (PWT) and paw withdrawal latency (PWL). BBB scale indicated the locomotor function of hindpaws in rats with NP implantation kept intact. Western blotting and immunohistochemistry staining revealed that phosphorylated SFKs (p-SFKs) and phosphorylated p38 MAPK (p-p38) were sequentially upregulated in ipsilateral spinal dorsal horn, but not in contralateral side of rats with NP. Intrathecal delivery of SFKs inhibitor reduced spinal p-p38 expression. Both SFKs and p38 inhibitors alleviated pain behaviors in a dose-responsive manner without disturbing locomotor function and reduced spinal expression of TNF-α, IL-1β, and IL-6 in rats with NP. Spinal SFKs contribute to radicular pain by activation of p38 MAPK and increasing pro-inflammatory cytokines expression in rats with NP implantation. Targeting SFKs/p38 pathway may be helpful for alleviating radicular pain. N/A.

Lovastatin inhibits Toll-like receptor 4 signaling in microglia by targeting its co-receptor myeloid differentiation protein 2 and attenuates neuropathic pain

There is growing interest in drug repositioning to find new therapeutic indications for drugs already approved for use in people. Lovastatin is an FDA approved drug that has been used clinically for over a decade as a lipid-lowering medication. While lovastatin is classically considered to act as a hydroxymethylglutaryl (HMG)-CoA reductase inhibitor, the present series of studies reveal a novel lovastatin effect, that being as a Toll-like receptor 4 (TLR4) antagonist. Lovastatin selectively inhibits lipopolysaccharide (LPS)-induced TLR4-NF-κB activation without affecting signaling by other homologous TLRs. In vitro biophysical binding and cellular thermal shift assay (CETSA) show that lovastatin is recognized by TLR4′s coreceptor myeloid differentiation protein 2 (MD-2). This finding is supported by molecular dynamics simulations that lovastatin targets the LPS binding pocket of MD-2 and lovastatin binding stabilizes the MD-2 conformation. In vitro studies of BV-2 microglial cells revealed that lovastatin inhibits multiple effects of LPS, including activation of NFkB; mRNA expression of tumor necrosis factor-a, interleukin-6 and cyclo-oxygenase 2; production of nitric oxide and reactive oxygen species; as well as phagocytic activity. Furthermore, intrathecal delivery of lovastatin over lumbosacral spinal cord of rats attenuated both neuropathic pain from sciatic nerve injury and expression of the microglial activation marker CD11 in lumbar spinal cord dorsal horn. Given the well-established role of microglia and proinflammatory signaling in neuropathic pain, these data are supportive that lovastatin, as a TLR4 antagonist, may be productively repurposed for treating chronic pain.

SETD7 mediates spinal microgliosis and neuropathic pain in a rat model of peripheral nerve injury

Gene transcription regulation is critical for the development of spinal microgliosis and neuropathic pain after peripheral nerve injury. Using a model of chronic constriction injury (CCI) of the sciatic nerve, this study characterized the role of SET domain containing lysine methyltransferase 7 (SETD7) which monomethylates histone H3 lysine 4 (H3K4me1), a marker for active gene transcription. SETD7 protein expression in the spinal dorsal horn ipsilateral to nerve lesion was increased from one day to 14 days after CCI, concomitantly with the expression of inflammatory genes, Ccl2, Il-6 and Il-1β. The CCI-induced SETD7 expression was predominantly localized to microglia, as demonstrated by immunohistochemistry and western blot from magnetic activated cell sorted spinal microglia. SETD7 knockdown by intrathecal lentivirus shRNA delivery prior to CCI prevented spinal microgliosis and neuropathic pain, whereas lentiviral SETD7 transduction exacerbated these symptoms. In addition, SETD7 regulated H3K4me1 level and expression of inflammatory mediators both in CCI rats and in the HAPI rat microglia cell line. Accordingly, PFI-2, a specific inhibitor of SETD7 monomethylation activity, suppressed the lipopolysaccharides-induced amoeboid morphology of primary microglia and the expression of inflammatory genes, Ccl2, Il-6 and Il-1β. Moreover, intrathecal administration of PFI-2 alleviated CCI-induced neuropathic pain. However, this effect was observed in male but not in female rats. These results demonstrate a critical role of SETD7 in the development of spinal microgliosis and neuropathic pain subsequently to peripheral nerve injury. The pharmacological approach further suggests that SETD7 is a new target for the treatment of neuropathic pain. The underlying mechanisms may involve H3K4me1-dependent regulation of inflammatory gene expression in microglia.

A multidisciplinary approach to dosing nusinersen for spinal muscular atrophy.

In December 2016, nusinersen gained FDA approval as the first pharmacologic treatment for spinal muscular atrophy (SMA), a disorder of motor neurons and the leading genetic cause of infant mortality. Nusinersen's intrathecal delivery requirement, strict dosage protocol, and accelerated FDA approval presented a challenge to health care centers hoping to implement treatment of patients with SMA. Scheduling logistics, combined with the specific ventilatory, anesthetic, and spinal access needs of this patient population, requires extensive coordination of care. This complexity, in addition to the high cost of treatment, may lead to overburdening of an institution's dosing resources, causing delays in treatment initiation and limiting patients' access to therapy and may result in barriers to coverage. We initiated a comprehensive stepwise protocol to maximize patient inclusion, as well as safety and efficiency outcome measures. This retrospective cohort study reviews the dosing process. As a result of immense collaborative efforts involving care coordination of patients and families, in addition to health providers in the divisions of neurology, anesthesiology, pulmonology, orthopedics, interventional radiology, physical therapy, and neurosurgery, we have successfully dosed 62 SMA patients. Throughout this process, we have improved anesthetic techniques, as well as minimized procedural complications and missed scheduled doses. We present here recommendations for safe and effective implementation of nusinersen utilizing a multidisciplinary approach, based on our 1 and a half year experience at a tertiary care children's hospital.

Increased ER–mitochondria tethering promotes axon regeneration

Translocation of the endoplasmic reticulum (ER) and mitochondria to the site of axon injury has been shown to facilitate axonal regeneration; however, the existence and physiological importance of ER–mitochondria tethering in the injured axons are unknown. Here, we show that a protein linking ER to mitochondria, the glucose regulated protein 75 (Grp75), is locally translated at axon injury site following axotomy, and that overexpression of Grp75 in primary neurons increases ER–mitochondria tethering to promote regrowth of injured axons. We find that increased ER–mitochondria tethering elevates mitochondrial Ca2+ and enhances ATP generation, thereby promoting regrowth of injured axons. Furthermore, intrathecal delivery of lentiviral vector encoding Grp75 to an animal with sciatic nerve crush injury enhances axonal regeneration and functional recovery. Together, our findings suggest that increased ER–mitochondria tethering at axonal injury sites may provide a therapeutic strategy for axon regeneration.

Epigenetic reduction of miR-214-3p upregulates astrocytic colony-stimulating factor-1 and contributes to neuropathic pain induced by nerve injury

Emerging evidence has indicated that colony-stimulating factor-1 (CSF1) modulates neuroinflammation in the central nervous system and the development of neuropathic pain, while the underlying mechanism remains unknown. Here, we identified the increased expression of CSF1 derived from activated astrocytes in the ipsilateral dorsal horn in rats with spinal nerve ligation (SNL). Suppression of CSF1 expression alleviated neuroinflammation, neuronal hyperexcitability, and glutamatergic receptor subunit upregulation in the dorsal horn and improved SNL-induced pain behavior. We also found reduced miR-214-3p expression in the ipsilateral dorsal horn following an SNL procedure; miR-214-3p directly bound to the 3'-UTR of CSF1 mRNA and negatively regulated CSF1 expression. Intrathecal delivery of miR-214-3p mimic reversed the enhanced expression of CSF1 and astrocyte overactivity and alleviated the IL-6 upregulation and pain behavior induced by SNL. Moreover, suppression of spinal miR-214-3p increased astrocyte reactivity, promoted CSF1 and IL-6 production, and induced pain hypersensitivity in naive animals. Furthermore, SNL induced the expression of DNA methyltransferase 3a (DNMT3a) that was associated with the hypermethylation of the miR-214-3p promoter, leading to reduced miR-214-3p expression in the model rodents. Treatment with the DNMT inhibitor zebularine significantly reduced cytosine methylation in the miR-214-3p promoter; this reduced methylation consequently increased the expression of miR-214-3p and decreased the content of CSF1 in the ipsilateral dorsal horn and, further, attenuated IL-6 production and pain behavior in rats with SNL. Together, our data indicate that the DNMT3a-mediated epigenetic suppression of miR-214-3p enhanced CSF1 production in astrocytes, which subsequently induced neuroinflammation and pain behavior in SNL model rats.