Lumbar Intrathecal Injection Research Articles

Lumbar Intrathecal Injection in Adult and Neonatal Mice.

This article describes a step-by-step process of lumbar intrathecal injection of Evans blue dye and AAV9-EGFP in adult (2-month-old) and neonatal (postnatal day 10) mice. Intrathecal injection is a clinically translatable technique that has already been extensively applied in humans. In mice, intrathecal injection is considered a challenging procedure that requires a trained and experienced researcher. For both adult and neonatal mice, lumbar intrathecal injection is directed into the L5-L6 intervertebral space. Intrathecally injected material enters the cerebrospinal fluid (CSF) within the intrathecal space from where it can directly access the central nervous system (CNS) parenchyma. Simultaneously, intrathecally injected material exits the CSF with pressure gradient and enters the endoneurial fluid and ultimately the peripheral nerves. While in the CSF, the injectable material also enters the bloodstream and systemic circulation through the arachnoid villi. A successful lumbar intrathecal injection results in adequate biodistribution of the injectable material in the CNS, PNS, and peripheral organs. When correctly applied, this technique is considered as minimally invasive and non-disruptive and can be used for the lumbar delivery of any solute. © 2024 Wiley Periodicals LLC. Basic Protocol 1: C57BL/6 adult and P10 mice lumbar intrathecal injection Basic Protocol 2: Tissue collection and preparation for evaluating Evans blue dye diffusion Basic Protocol 3: Tissue collection and preparation for immunohistochemistry staining Basic Protocol 4: Tissue collection and vector genome copy number analysis.

Intrathecal Vector Delivery in Juvenile Rats via Lumbar Cistern Injection.

Gene therapy is a powerful technology to deliver new genes to a patient for the treatment of disease, be it to introduce a functional gene, inactivate a toxic gene, or provide a gene whose product can modulate the biology of the disease. The delivery method for the therapeutic vector can take many forms, ranging from intravenous infusion for systemic delivery to direct injection into the target tissue. For neurodegenerative disorders, it is often desirable to skew transduction towards the brain and/or spinal cord. The least invasive approach to target the entire central nervous system involves injection into the cerebrospinal fluid (CSF), allowing the therapeutic to reach a large fraction of the central nervous system. The safest approach to deliver a vector into the CSF is the lumbar intrathecal injection, where a needle is introduced into the lumbar cistern of the spinal cord. This technique, also known as a lumbar puncture, has been widely used in neonatal and adult rodents and in large animal models. While the technique is similar across species and developmental stages, subtle differences in size, structure, and elasticity of tissues surrounding the intrathecal space require accommodations in the approach. This article describes a method for performing lumbar puncture in juvenile rats to deliver an adeno-associated serotype 9 vector. Here, 25-35 µL of vector were injected into the lumbar cistern, and a green fluorescent protein (GFP) reporter was used to evaluate the transduction profile resulting from each injection. The benefits and challenges of this approach are discussed.

In vivo distribution of cerebrospinal fluid tracer in human upper spinal cord and brain stem.

BACKGROUNDIntrathecal injection is an attractive route through which drugs can be administered and directed to the spinal cord, restricted by the blood-spinal cord barrier. However, in vivo data on the distribution of cerebrospinal fluid (CSF) substances in the human spinal cord are lacking. We conducted this study to assess the enrichment of a CSF tracer in the upper cervical spinal cord and the brain stem.METHODSAfter lumbar intrathecal injection of a magnetic resonance imaging (MRI) contrast agent, gadobutrol, repeated blood samples and MRI of the upper cervical spinal cord, brain stem, and adjacent subarachnoid spaces (SAS) were obtained through 48 hours. The MRI scans were then analyzed for tracer distribution in the different regions and correlated to age, disease, and amounts of tracer in the blood to determine CSF-to-blood clearance.RESULTSThe study included 26 reference individuals and 35 patients with the dementia subtype idiopathic normal pressure hydrocephalus (iNPH). The tracer enriched all analyzed regions. Moreover, tracer enrichment in parenchyma was associated with tracer enrichment in the adjacent SAS and with CSF-to-blood clearance. Clearance from the CSF was delayed in patients with iNPH compared with younger reference patients.CONCLUSIONA CSF tracer substance administered to the lumbar thecal sac can access the parenchyma of the upper cervical spinal cord and brain stem. Since CSF-to-blood clearance is highly individual and is associated with tracer level in CSF, clearance assessment may be used to tailor intrathecal treatment regimes.FUNDINGSouth-Eastern Norway Regional Health and Østfold Hospital Trust supported the research and publication of this work.

Clinical significance of Epstein-Barr Virus detection in the cerebrospinal fluid of patients who underwent hematopoietic stem cell transplantation

Objective: To analyze the detection rate, clinical significance, and prognosis of Epstein-Barr virus (EBV) in the cerebrospinal fluid (CSF) of patients following allogeneic hematopoietic stem cell transplantation. Methods: A retrospective analysis was performed on 1100 patients who underwent the CSF virus test after allogeneic hematopoietic stem cell transplantation in Peking University People's Hospital between January 2017 and June 2022. Among them, 19 patients were screened positive for EBV in their CSF, and their clinical characteristics, treatment, and prognosis were analyzed. Results: Among 19 patients with EBV-positive cerebrospinal fluid, 12 were male and 7 were female, with 5 patients aged <18 years and 12 aged ≥18 years, with a median age of 27 (5-58) years old. There were 7 cases of acute myeloid leukemia, 8 of acute lymphocytic leukemia, 2 of aplastic anemia, 1 of Hodgkin's lymphoma, and 1 of hemophagocytic syndrome. All 19 patients underwent haploid hematopoietic stem cell transplantation, including 1 secondary transplant. Nineteen patients had neurological symptoms (headache, dizziness, convulsions, or seizures), of which 13 had fever. Ten cases showed no abnormalities in cranial imaging examination. Among the 19 patients, 6 were diagnosed with EB virus-related central nervous system diseases, with a median diagnosis time of 50 (22-363) days after transplantation. In 9 (47.3%) patients, EBV was detected in their peripheral blood, and they were treated with intravenous infusion of rituximab (including two patients who underwent lumbar puncture and intrathecal injection of rituximab). After treatment, EBV was not detected in seven patients. Among the 19 patients, 2 died from EBV infection and 2 from other causes. Conclusion: In patients who exhibited central nervous system symptoms after allogeneic hematopoietic stem cell transplantation, EBV should be screened as a potential pathogen. EBV detected in the CSF may indicate an infection; however, it does not confirm the diagnosis.

Gene replacement therapy in two Golgi-retained CMT1X mutants before and after the onset of demyelinating neuropathy

X-linked Charcot-Marie-Tooth disease type 1 (CMT1X) is a demyelinating neuropathy resulting from loss-of-function mutations affecting the GJB1/connexin 32 (Cx32) gene. We previously showed functional and morphological improvement in Gjb1-null mice following AAV9-mediated delivery of human Cx32 driven by the myelin protein zero (Mpz) promoter in Schwann cells. However, CMT1X mutants may interfere with virally delivered wild-type (WT) Cx32. To confirm the efficacy of this vector also in the presence of CMT1X mutants, we delivered AAV9-Mpz-GJB1 by lumbar intrathecal injection in R75W/Gjb1-null and N175D/Gjb1-null transgenic lines expressing Golgi-retained mutations, before and after the onset of the neuropathy. Widespread expression of virally delivered Cx32 was demonstrated in both genotypes. Re-establishment of WT Cx32 function resulted in improved muscle strength and increased sciatic nerve motor conduction velocities in all treated groups from both mutant lines when treated before as well as after the onset of the neuropathy. Furthermore, morphological analysis showed improvement of myelination and reduction of inflammation in lumbar motor roots and peripheral nerves. In conclusion, this study provides proof of principle for a clinically translatable gene therapy approach to treat CMT1X before and after the onset of the neuropathy, even in the presence of endogenously expressed Golgi-retained Cx32 mutants.

An ASO therapy for Angelman syndrome that targets an evolutionarily conserved region at the start of the UBE3A-AS transcript.

Angelman syndrome is a devastating neurogenetic disorder for which there is currently no effective treatment. It is caused by mutations or epimutations affecting the expression or function of the maternally inherited allele of the ubiquitin-protein ligase E3A (UBE3A) gene. The paternal UBE3A allele is imprinted in neurons of the central nervous system (CNS) by the UBE3A antisense (UBE3A-AS) transcript, which represents the distal end of the small nucleolar host gene 14 (SNHG14) transcription unit. Reactivating the expression of the paternal UBE3A allele in the CNS has long been pursued as a therapeutic option for Angelman syndrome. Here, we described the development of an antisense oligonucleotide (ASO) therapy for Angelman syndrome that targets an evolutionarily conserved region demarcating the start of the UBE3A-AS transcript. We designed and chemically optimized gapmer ASOs targeting specific sequences at the start of the human UBE3A-AS transcript. We showed that ASOs targeting this region precisely and efficiently repress the transcription of UBE3A-AS, reactivating the expression of the paternal UBE3A allele in neurotypical and Angelman syndrome induced pluripotent stem cell-derived neurons. We further showed that human-targeted ASOs administered to the CNS of cynomolgus macaques by lumbar intrathecal injection repress UBE3A-AS and reactivate the expression of the paternal UBE3A allele throughout the CNS. These findings support the advancement of this investigational molecular therapy for Angelman syndrome into clinical development (ClinicalTrials.gov, NCT04259281).

A translatable RNAi-driven gene therapy silences PMP22/Pmp22 genes and improves neuropathy in CMT1A mice.

Charcot-Marie-Tooth disease type 1A (CMT1A), the most common inherited demyelinating peripheral neuropathy, is caused by PMP22 gene duplication. Overexpression of WT PMP22 in Schwann cells destabilizes the myelin sheath, leading to demyelination and ultimately to secondary axonal loss and disability. No treatments currently exist that modify the disease course. The most direct route to CMT1A therapy will involve reducing PMP22 to normal levels. To accomplish this, we developed a gene therapy strategy to reduce PMP22 using artificial miRNAs targeting human PMP22 and mouse Pmp22 mRNAs. Our lead therapeutic miRNA, miR871, was packaged into an adeno-associated virus 9 (AAV9) vector and delivered by lumbar intrathecal injection into C61-het mice, a model of CMT1A. AAV9-miR871 efficiently transduced Schwann cells in C61-het peripheral nerves and reduced human and mouse PMP22 mRNA and protein levels. Treatment at early and late stages of the disease significantly improved multiple functional outcome measures and nerve conduction velocities. Furthermore, myelin pathology in lumbar roots and femoral motor nerves was ameliorated. The treated mice also showed reductions in circulating biomarkers of CMT1A. Taken together, our data demonstrate that AAV9-miR871–driven silencing of PMP22 rescues a CMT1A model and provides proof of principle for treating CMT1A using a translatable gene therapy approach.

Incidence and management of surgical site infection in the cervical spine following a transoral approach.

Transoral approach can accomplish ventral decompression directly. However, surgical site infection (SSI) cannot be ignored. This paper aims to review the prevalence of infection and conduct advice for the treatment of SSI in the cervical spine following the transoral approach. A retrospective analysis of patients with SSI after transoral atlantoaxial reduction plate (TARP) surgery was performed. SSI was classified into three kinds according to the modified American CDC criteria. 2.9% (17/581) patients who underwent TARP surgery, experienced SSI, of which five had superficial SSI (SI), eight had deep SSI (DI), and four had organ/space SSI (O/SI). The patients with SI underwent intravenous antibiotic treatment and were ultimately cured. Among the remaining 12 patients with DI and O/SI, 11 underwent reoperation for TARP system removal and subsequently one-stage posterior occipitocervical fixation, and one patient experienced infection twomonths post-operatively and died without receiving treatment. Among patients who underwent revision surgery, three experienced intracranial infection due to intra-operative dural tears, and continuous lumbar cerebrospinal fluid drainage and intrathecal injection of antibiotics were used as effective and appropriate treatments, with outcomes of one recovery and two deaths. All patients with SSI were begun on intravenous antibiotics with conversion to oral antibiotics. The incidence of SSI was 2.9% (17/581). Adequate peri-operative preparation, early diagnosis, and appropriate treatment of SSI require further research.

Emerging treatments for hereditary demyelinating neuropathies

Inherited neuropathies known as Charcot-Marie-Tooth (CMT) disease are genetically heterogeneous, progressive disorders without effective therapy. Several demyelinating CMT types result from cell-autonomous loss of function mechanisms specifically in myelinating Schwann cells. Therefore, we have developed cell-targeted gene replacement or gene silencing approaches to treat demyelinating CMT. Using clinically relevant adeno-associated viral (AAV9) vectors and lumbar intrathecal injection we achieved widespread vector biodistribution throughout the PNS. Delivery of the GJB1 gene encoding the gap junction protein connexin32 (Cx32), which is associated with X-linked CMT (CMT1X), resulted in restoration of Cx32 expression in Schwann cells. GJB1 gene replacement in a model of CMT1X provided phenotypic improvement in motor performance, nerve conduction velocities, and peripheral nerve morphology both at pre- as well as post-onset stages of the neuropathy. Likewise, replacement of the SH3TC2 gene associated with recessively inherited CMT4C neuropathy in a model of the disease resulted in improvement of functional and morphological abnormalities. Finally, a gene silencing approach was used to treat an overexpressing model of CMT1A, the commonest demyelinating CMT type caused by PMP22 gene duplication. AAV9-mediated delivery of PMP22-targeting microRNA resulted in efficient silencing of PMP22 expression in PNS tissues, along with improvement of functional and morphological abnormalities. Treatment-responsive blood biomarkers of CMT neuropathies were identified and validated across different disease models. Our studies provide proof of principle for the therapeutic potential of gene replacement or gene silencing to treat demyelinating inherited neuropathies.

Biodistribution of Adeno-Associated Virus Serotype 5 Viral Vectors Following Intrathecal Injection.

The pharmacokinetic profile of AAV particles following intrathecal delivery has not yet been clearly defined. The present study evaluated the distribution profile of adeno-associated virus serotype 5 (AAV5) viral vectors following lumbar intrathecal injection in mice. After a single bolus intrathecal injection, viral DNA concentrations in mouse whole blood, spinal cord, and peripheral tissues were determined using quantitative polymerase chain reaction (qPCR). The kinetics of AAV5 vector in whole blood and the concentration over time in spinal and peripheral tissues were analyzed. Distribution of the AAV5 vector to all levels of the spinal cord, dorsal root ganglia, and into systemic circulation occurred rapidly within 30 min following injection. Vector concentration in whole blood reached a maximum 6 h postinjection with a half-life of approximately 12 h. Area under the curve data revealed the highest concentration of vector distributed to dorsal root ganglia tissue. Immunohistochemical analysis revealed AAV5 particle colocalization with the pia mater at the spinal cord and macrophages in the dorsal root ganglia (DRG) 30 min after injection. These results demonstrate the widespread distribution of AAV5 particles through cerebrospinal fluid and preferential targeting of DRG tissue with possible clearance mechanisms via DRG macrophages.

The AAV9 Variant Capsid AAV-F Mediates Widespread Transgene Expression in Nonhuman Primate Spinal Cord After Intrathecal Administration.

Intrathecal delivery of AAV9 into the subarachnoid space has been shown to transduce spinal cord and brain and be less affected by preexisting antibodies, which are lower in cerebral spinal fluid. Still, efficiency of transduction needs to be improved. Recently, we identified a new capsid from a library selection in mice, called AAV-F, that allowed robust transduction of the spinal cord gray matter after lumbar injection. In this study, we test transduction of spinal cord by AAV-F (n = 3) compared to AAV9 (n = 2), using a reporter gene, in cynomolgus monkeys after lumbar intrathecal injection. Using an automated image analysis (IA) approach to sensitively quantitate reporter gene expression in spinal cord, we found that AAV-F capsid mediated slightly higher transgene expression (both in percentages of cells and intensity of immunostaining) in motor neurons and interneurons, in the lumbar and thoracic regions, compared to AAV9. Interestingly, although AAV-F mediated higher transgene expression in spinal cord, the number of genomes in spinal cord and periphery were on average lower for AAV-F than AAV9, which suggest that lower numbers of genomes were able to mediate higher transgene expression in spinal cord with this capsid. In contrast, dorsal root ganglion transduction efficiency was lower for AAV-F compared to AAV9 on average. Interestingly, we also observed transduction of Schwann cells in sciatic nerve in two nonhuman primates injected with AAV-F, but none with AAV9. Overall, our data demonstrate the utility of automated IA for quantitation of AAV transduction in the spinal cord and the favorable on-target:off-target transduction profile suggests that the AAV-F capsid be considered for gene therapy applications focused on treating the spinal cord after intrathecal delivery.

AAV9-mediated Schwann cell-targeted gene therapy rescues a model of demyelinating neuropathy

Mutations in the GJB1 gene, encoding the gap junction (GJ) protein connexin32 (Cx32), cause X-linked Charcot-Marie-Tooth disease (CMT1X), an inherited demyelinating neuropathy. We developed a gene therapy approach for CMT1X using an AAV9 vector to deliver the GJB1/Cx32 gene under the myelin protein zero (Mpz) promoter for targeted expression in Schwann cells. Lumbar intrathecal injection of the AAV9-Mpz.GJB1 resulted in widespread biodistribution in the peripheral nervous system including lumbar roots, sciatic and femoral nerves, as well as in Cx32 expression in the paranodal non-compact myelin areas of myelinated fibers. A pre-, as well as post-onset treatment trial in Gjb1-null mice, demonstrated improved motor performance and sciatic nerve conduction velocities along with improved myelination and reduced inflammation in peripheral nerve tissues. Blood biomarker levels were also significantly ameliorated in treated mice. This study provides evidence that a clinically translatable AAV9-mediated gene therapy approach targeting Schwann cells could potentially treat CMT1X.

Analysis on endoscopic endonasal approach combined with the frontotemporal orbitozygomatic approach under the microscope for skull base tumor with intra and extra-cranial involvement

Objective: To evaluate the application of combination use of endoscopic endonasal approach combined with the frontotemporal orbitozygomatic approach microscopically in skull base tumor with intra and extra-cranial involvement. Methods: A total of 7 patients (4 males and 3 females, aging from 27 to 65 years old, with a medium age of 48) undergone complicated skull base surgeries via endoscopic endonasal approach combined with the frontotemporal orbitozygomatic approach microscopically from May 2016 to January 2018 were reviewed respectively. The patients included 2 cases of recurrent invasive pituitary adenoma, 3 cases of basal skull meningiomas, 1 case of clivus chondrosarcoma, and 1 case of recurrent nasopharyngeal carcinoma. The lesion extensively infiltrated nasal cavity, extending to the paranasal sinus, bilateral cavernous sinus, sellar region, suprasellar, superior clivus, temporal lobe, pterygopalatine fossa, infratemporal fossa and important intracranial vessels. All the 7 patients were treated under general anesthesia by endoscopic endonasal approach combined with frontotemporal orbitozygomatic approach under the microscope. Total excision rate, intraoperative and postoperative complications and postoperative curative effect were observed. All of them were followed up for 6 to 12 months. The Glasgow Outcome Scale (GOS) was used to evaluate the prognosis. Result: Total tumor removal was performed in 5 cases, subtotal removel in 2 cases. There was no complication during the operation. Postoperative severe complications occurred in 2 cases, including 1 case of cerebrospinal fluid rhinorrhea and intracranial infection, which was cured by lumbar cistern drainage and intrathecal injection; 1 case occurred oculomotor nerve paralysis, which recovered during follow-up. Postoperative complications occurred in 1 case of trochlear nerve dysfunction, 2 cases of facial numbness, and 1 case of tinnitus. During follow-up, all patients recovered to varying degrees. There was no bleeding and death after the operation. No tumor recurred during the follow-up period. All patients were recovered well with GOS grade Ⅳ-Ⅴ. Conclusions: Endoscopic transnasal approach combined with microscopic frontotemporal orbitozygomatic approach can remove tumors in one stage, reduce surgical complications and improve surgical effect. It has good application prospects and is suitable for excising complex intracranial and extracranial communicating tumors of widely involving sellar, clivus and petrous apex area.

Save Your Maximum Tolerated Dose: How to Diagnose Procedure-Related Spinal Cord Lesions After Lumbar Intrathecal Bolus Administration of Oligonucleotides in Cynomolgus Monkeys.

Many potential drugs for treatment of neurodegenerative diseases, particularly antisense oligonucleotides (ASOs), are administered via lumbar intrathecal injection, because these drugs do not cross the blood-brain barrier. Intrathecal injection is a well-established method in cynomolgus monkeys, a species that is used in preclinical safety assessment when other nonrodent species cannot be used. The authors completed intrathecal ASO administration in over 30 preclinical safety studies (>1000 animals and >4500 dose administrations) during which we observed 3 cases of procedure-related spinal cord necrosis (incidence <0.1%). We describe clinical symptoms, diagnostic approaches, morphological features, and prognosis of this rare injury, and compare these findings with typical drug-related findings of ASOs dosed by intrathecal injection. The low incidence of procedure-related and dose-limiting lesions confines this analysis to a small sample set. The pattern of effects is similar across all monkeys despite differences in age, body weight, and intrathecal injection site. All 3 cases presented a combination of the following findings: blood in cerebrospinal fluid at time of injection, clinical signs that increase in severity within a day of dosing, lameness of both hind limbs, reduced muscle tone, and loss of patellar, foot grip, and/or anal reflexes. In all cases, magnetic resonance imaging (MRI) showed a linear hyperintense lesion in the lumbar spinal cord. In 2 cases, this hyperintensity was associated with evidence of spinal cord edema. We conclude that a pattern of in-life and pathology findings, including noninvasive MRI assessment, is indicative of procedure-related effects.

MRI characterization of early CNS transport kinetics post intrathecal gadolinium injection: Trends of subarachnoid and parenchymal distribution in healthy volunteers

PurposeTo quantify CSF transport kinetics and brain glymphatic distribution using MRI following intrathecal injection of gadolinium contrast in healthy adults. Subjects and methodsEight completely healthy volunteer subjects underwent intrathecal injection of gadolinium via image guided lumbar puncture and serial MRI's at six subsequent time points up to 11 h post-injection. Rate of enhancement and deposition were calculated for various regions and lobes of the brain. ResultsNormalized cranial data revealed that gadolinium in the intracranial CSF spaces peaked within 1–3 h and started to decrease at 7 h. In some regions of the brain parenchyma, such as the cerebral cortex and white matter, enhancement was increasing after 11 h. Differential rates of uptake between the parietal and frontal (p = 0.0003), cingulate (p = 0.002) and temporal (p = 0.018) lobes were shown as well as a several fold change between various cortical regions. Lastly, a linear regression comparing laterality between 35 cortical regions yielded (R2 = 0.90, p < 0.001) with a slope of 1.01 showing symmetry in uptake. ConclusionsGadolinium enhancement after lumbar intrathecal injection demonstrated differential CSF flow and brain parenchymal penetration, which illustrated the distributory function of the glymphatic system.

Effect of Neuraxial Analgesic Procedures on Intraoperative Hemodynamics During Routine Clinical Care of Gynecological and General Surgeries: A Case-Control Query of Electronic Data.

BackgroundThe purpose of this study was to determine whether neuraxial analgesic procedures affect intraoperative hemodynamics and/or postoperative outcomes. Previous studies have examined effects in small samples of patients in highly controlled research environments. This study examined “real-world” data from a large sample of subjects receiving routine clinical cares.MethodsA matched case–control analysis of electronic medical records from a large, academic hospital was performed. Patients who underwent neuraxial procedures preoperatively for postoperative analgesia for abdominal surgery (n=1570) were compared with control patients matched according to age, sex, ASA class and type of surgical procedure. Intraoperative hemodynamic measures, fluids and pressor utilization were quantified. Postoperative outcomes were determined based on the changes in laboratory values, the ordering of imaging studies and admission to an intensive care unit during the seven days following surgery as well as 30-day mortality.ResultsMedical records of 1082 patients who received an epidural catheter placement and 488 patients who received a lumbar intrathecal morphine injection were compared with an equal number of matched control patients. Preoperative placement of an epidural catheter for the management of postoperative pain was demonstrated to be associated with significant reductions in mean arterial pressure intraoperatively and poorer postoperative outcomes (more intensive care unit [ICU] admissions, more myocardial injuries) when compared with controls. A similar analysis of preoperatively administered intrathecal morphine injections was not associated with intraoperative alterations in blood pressure and had improved outcomes (less ICU admissions) in comparison with controls.ConclusionIn a “real-world” sample, intrathecal morphine administration proved to be highly beneficial as a neuraxial analgesic procedure as it was not associated with intraoperative hypotension and was associated with improved clinical outcomes, in contrast to opposite findings associated with epidural catheter placement. There should be a careful consideration of elective neuraxial method utilized for postoperative pain control, with the present study raising significant concerns related to the use of epidural analgesia and its potential effect on clinical outcomes.

Gene replacement therapy after neuropathy onset provides therapeutic benefit in a model of CMT1X.

X-linked Charcot-Marie-Tooth disease (CMT1X), one of the commonest forms of inherited demyelinating neuropathy, results from GJB1 gene mutations causing loss of function of the gap junction protein connexin32 (Cx32). The aim of this study was to examine whether delayed gene replacement therapy after the onset of peripheral neuropathy can provide a therapeutic benefit in the Gjb1-null/Cx32 knockout model of CMT1X. After delivery of the LV-Mpz.GJB1 lentiviral vector by a single lumbar intrathecal injection into 6-month-old Gjb1-null mice, we confirmed expression of Cx32 in lumbar roots and sciatic nerves correctly localized at the paranodal myelin areas. Gjb1-null mice treated with LV-Mpz.GJB1 compared with LV-Mpz.Egfp (mock) vector at the age of 6months showed improved motor performance at 8 and 10months. Furthermore, treated mice showed increased sciatic nerve conduction velocities, improvement of myelination and reduced inflammation in lumbar roots and peripheral nerves at 10months of age, along with enhanced quadriceps muscle innervation. Plasma neurofilament light (NEFL) levels, a clinically relevant biomarker, were also ameliorated in fully treated mice. Intrathecal gene delivery after the onset of peripheral neuropathy offers a significant therapeutic benefit in this disease model, providing a proof of principle for treating patients with CMT1X at different ages.

Gene replacement therapy in a model of Charcot-Marie-Tooth 4C neuropathy.

Charcot-Marie-Tooth disease type 4C is the most common recessively inherited demyelinating neuropathy that results from loss of function mutations in the SH3TC2 gene. Sh3tc2-/- mice represent a well characterized disease model developing early onset progressive peripheral neuropathy with hypo- and demyelination, slowing of nerve conduction velocities and disturbed nodal architecture. The aim of this project was to develop a gene replacement therapy for treating Charcot-Marie-Tooth disease type 4C to rescue the phenotype of the Sh3tc2-/- mouse model. We generated a lentiviral vector LV-Mpz.SH3TC2.myc to drive expression of the human SH3TC2 cDNA under the control of the Mpz promoter specifically in myelinating Schwann cells. The vector was delivered into 3-week-old Sh3tc2-/- mice by lumbar intrathecal injection and gene expression was assessed 4-8 weeks after injection. Immunofluorescence analysis showed presence of myc-tagged human SH3TC2 in sciatic nerves and lumbar roots in the perinuclear cytoplasm of a subset of Schwann cells, in a dotted pattern co-localizing with physiologically interacting protein Rab11. Quantitative PCR analysis confirmed SH3TC2 mRNA expression in different peripheral nervous system tissues. A treatment trial was initiated in 3 weeks old randomized Sh3tc2-/- littermate mice which received either the full or mock (LV-Mpz.Egfp) vector. Behavioural analysis 8 weeks after injection showed improved motor performance in rotarod and foot grip tests in treated Sh3tc2-/- mice compared to mock vector-treated animals. Moreover, motor nerve conduction velocities were increased in treated Sh3tc2-/- mice. On a structural level, morphological analysis revealed significant improvement in g-ratios, myelin thickness, and ratios of demyelinated fibres in lumbar roots and sciatic nerves of treated Sh3tc2-/- mice. Finally, treated mice also showed improved nodal molecular architecture and reduction of blood neurofilament light levels, a clinically relevant biomarker for axonal injury/degeneration. This study provides a proof of principle for viral gene replacement therapy targeted to Schwann cells to treat Charcot-Marie-Tooth disease type 4C and potentially other similar demyelinating inherited neuropathies.

Identifying the role of group III/IV muscle afferents in the carotid baroreflex control of mean arterial pressure and heart rate during exercise.

We investigated the contribution of group III/IV muscle afferents to carotid baroreflex resetting during electrically evoked (no central command) and voluntary (requiring central command) isometric knee extension exercise. Lumbar intrathecal fentanyl was used to attenuate the central projection of μ-opioid receptor-sensitive group III/IV leg muscle afferent feedback. Spontaneous carotid baroreflex control was assessed by loading and unloading the carotid baroreceptors with a variable pressure neck chamber. Group III/IV muscle afferents did not influence spontaneous carotid baroreflex responsiveness at rest or during exercise. Afferent feedback accounted for at least 50% of the exercise-induced increase in the carotid baroreflex blood pressure and heart rate operating points, adjustments that are critical for an appropriate cardiovascular response to exercise. These findings suggest that group III/IV muscle afferent feedback is, independent of central command, critical for the resetting of the carotid baroreflex blood pressure and heart rate operating points, but not for spontaneous baroreflex responsiveness. This study sought to comprehensively investigate the role of metabolically and mechanically sensitive group III/IV muscle afferents in carotid baroreflex responsiveness and resetting during both electrically evoked (EVO, no central command) and voluntary (VOL, requiring central command) isometric single-leg knee-extension (15% of maximal voluntary contraction; MVC) exercise. Participants (n=8) were studied under control conditions (CTRL) and following lumbar intrathecal fentanyl injection (FENT) to inhibit μ-opioid receptor-sensitive lower limb muscle afferents. Spontaneous carotid baroreflex control of mean arterial pressure (MAP) and heart rate (HR) were assessed following rapid 5s pulses of neck pressure (NP, +40mmHg) or suction (NS, -60mmHg). Resting MAP (87±10mmHg) and HR (70±8bpm) were similar between CTRL and FENT conditions (P>0.4). In terms of spontaneous carotid baroreflex responsiveness, FENT did not alter the change in MAP or HR responses to NP (+13±5mmHg, P=0.85; +9±3bpm; P=0.99) or NS (-13±5mmHg, P=0.99; -24±11bpm; P=0.49) at rest or during either exercise protocol, which were of a remarkably similar magnitude to rest. In contrast, FENT administrationreduced the exercise-induced resetting of the operating point for MAP and HR during both EVO (116±10mmHg to 100±15mmHg and 93±14bpm to 82±10bpm) and VOL (107±13mmHg to 100±17mmHg and 89±10bpm to 72±10bpm) exercise bouts. Together, these findings document that group III/IV muscle afferent feedback is critical for the resetting of the carotid baroreflex MAP and HR operating points, independent of exercise-induced changes in central command, but not for spontaneous carotid baroreflex responsiveness.

Intrathecal gene therapy rescues a model of demyelinating peripheral neuropathy

Inherited demyelinating peripheral neuropathies are progressive incurable diseases without effective treatment. To develop a gene therapy approach targeting myelinating Schwann cells that can be translatable, we delivered a lentiviral vector using a single lumbar intrathecal injection and a myelin-specific promoter. The human gene of interest, GJB1, which is mutated in X-linked Charcot-Marie-Tooth Disease (CMT1X), was delivered intrathecally into adult Gjb1-null mice, a genetically authentic model of CMT1X that develops a demyelinating peripheral neuropathy. We obtained widespread, stable, and cell-specific expression of connexin32 in up to 50% of Schwann cells in multiple lumbar spinal roots and peripheral nerves. Behavioral and electrophysiological analysis revealed significantly improved motor performance, quadriceps muscle contractility, and sciatic nerve conduction velocities. Furthermore, treated mice exhibited reduced numbers of demyelinated and remyelinated fibers and fewer inflammatory cells in lumbar motor roots, as well as in the femoral motor and sciatic nerves. This study demonstrates that a single intrathecal lentiviral gene delivery can lead to Schwann cell-specific expression in spinal roots extending to multiple peripheral nerves. This clinically relevant approach improves the phenotype of an inherited neuropathy mouse model and provides proof of principle for treating inherited demyelinating neuropathies.