Clinical Reasoning: Longitudinally Extensive Spinal Cord Lesions in a Middle-aged Man

An immunocompetent 47-year-old man presented with a five-month history of progressive lower limb weakness, back pain, sphincter dysfunction, and intermittent fever, suggesting myelopathy in a chronic deteriorating course. A comprehensive analysis comprising of blood tests, neuroimaging, CSF profiling, molecular analysis, and histopathology was performed. Notably, enhanced spinal cord MRI revealed longitudinally extensive intradural-extramedullary lesions involving the cervical, thoracic, and lumbosacral spinal cord, with homogeneous enhancement and spinal cord compression. Serum TPHA and RPR tests were positive. CSF profiling showed pleocytosis, significant protein elevations, hypoglycorrhachia, and positive TPHA test. 18F-FDG-PET/CT indicated slightly increased intraspinal FDG uptake. Spinal cord biopsy further showed small round blue cells in poorly differentiated tissues. Immunostaining was positive for NKX2.2, CD56, CD99, Synaptophysin, and Ki67 (50%). Molecular analysis detected a novel MALAT-CYSLTR1 fusion protein and variants in oncogenic genes including PTCH1, TERT, CREBBP, SPEN, and STK11. The diagnosis of intraspinal extraosseous Ewing’s sarcoma (ES) was confirmed. Briefly, our case details the diagnosis of a patient with intradural-extramedullary ES and highlights the value of spinal cord biopsy in progressive myelopathy of unknown causes.

Visualizing lower urinary tract afferent projections in the lumbosacral spinal cord in rats v1

This collection includes the protocols required to map the lower urinary tract afferent projections to the lumbosacral spinal cord of male and female Sprague-Dawley rats. Afferents can be visualized using 3D reconstruction of alternating sections in TissueMaker (MBF Bioscience), or through the immunolabelling and clearing method, iDISCO. The following protocols are performed, regardless of endpoint: STAGE 1: Use of cholera toxin subunit B to label neural projections to lower urinary tract organs. STAGE 2: Intracardiac perfusion with fixative for anatomical studies. The next set of protocols pertain to the 3D reconstruction of spinal cord from alternating sections. STAGE 3: Immunohistochemical labelling of lower urinary tract afferents in spinal cord. STAGE 4: Quantitation of lower urinary tract afferents in 3D reconstruction of lumbosacral spinal cord sections For the visualization of lower urinary tract afferents in the intact spinal cord, skip Stages 3 and 4, and instead use Stage 5: STAGE 5: Immunolabelling and clearing of intact spinal cord for visualization of lower urinary tract afferents

Quantitative Assessment of Clinician Assistance During Dynamic Rehabilitation Using Force Sensitive Resistors

Background: Neuromodulation using epidural electrical stimulation (EES) has shown functional restoration in humans with chronic spinal cord injury (SCI). EES during body weight supported treadmill training (BWSTT) enhanced stepping performance in clinical trial participants with paraplegia. Unfortunately, tools are lacking in availability to quantify clinician assistance during BWSTT with and without EES. Force sensitive resistors (FSRs) have previously quantified clinician assistance during static standing; however, dynamic tasks have not been addressed.Objective: To determine the validity of FSRs in measurements of force and duration to quantify clinician assistance and participant progression during BWSTT with EES in participants with SCI.Design: A feasibility study to determine the effectiveness of EES to restore function in individuals with SCI.Methods: Two male participants with chronic SCI were enrolled in a pilot phase clinical trial. Following implantation of an EES system in the lumbosacral spinal cord, both participants underwent 12 months of BWSTT with EES. At monthly intervals, FSRs were positioned on participants' knees to quantity forces applied by clinicians to achieve appropriate mechanics of stepping during BWSTT. The FSRs were validated on the benchtop using a leg model instrumented with a multiaxial load cell as the gold standard. The outcomes included clinician-applied force duration measured by FSR sensors and changes in applied forces indicating progression over the course of rehabilitation.Results: The force sensitive resistors validation revealed a proportional bias in their output. Loading required for maximal assist training exceeded the active range of the FSRs but were capable of capturing changes in clinician assist levels. The FSRs were also temporally responsive which increased utility for accurately assessing training contact time. The FSRs readings were able to capture independent stance for both participants by study end. There was minimal to no applied force bilaterally for participant 1 and unilaterally for participant 2.Conclusions: Clinician assistance applied at the knees as measured through FSRs during dynamic rehabilitation and EES (both on and off) effectively detected point of contact and duration of forces; however, it lacks accuracy of magnitude assessment. The reduced contact time measured through FSRs related to increased stance duration, which objectively identified independence in stepping during EES-enabled BWSTT following SCI.

Kv3 channels contribute to the excitability of sub-populations of spinal cord neurons in lamina VII

Autonomic parasympathetic preganglionic neurons (PGN) drive contraction of the bladder during micturition but remain quiescent during bladder filling. This quiescence is postulated to be due to recurrent inhibition of PGN by fast-firing adjoining interneurons. Here, we defined four distinct neuronal types within lamina VII of the lumbosacral spinal cord, where PGN are situated, by combining whole cell patch clamp recordings with k-means clustering of a range of electrophysiological parameters. Additional morphological analysis separated these neuronal classes into parasympathetic preganglionic populations (PGN) and a fast firing interneuronal population. Kv3 channels are voltage-gated potassium channels (Kv) that allow fast and precise firing of neurons. We found that blockade of Kv3 channels by tetraethylammonium (TEA) reduced neuronal firing frequency and isolated high-voltage-activated Kv currents in the fast-firing population but had no effect in PGN populations. Furthermore, Kv3 blockade potentiated the local and descending inhibitory inputs to PGN indicating that Kv3-expressing inhibitory neurons are synaptically connected to PGN. Taken together, our data reveal that Kv3 channels are crucial for fast and regulated neuronal output of a defined population that may be involved in intrinsic spinal bladder circuits that underpin recurrent inhibition of PGN.

Downstream projection of Barrington's nucleus to the spinal cord in mice

Barrington's nucleus (Bar) which controls micturition behavior through downstream projections to the spinal cord contains two types of projection neurons BarCRH and BarESR1 that have different functions and target different spinal circuitry. Both types of neurons project to the L6-S1 spinal intermediolateral (IML) nucleus while BarESR1 neurons also project to the dorsal commissural nucleus (DCN). To obtain more information about the spinal circuits targeted by Bar, we used patch-clamp recording in spinal slices from adult mice in combination with optogenetic stimulation of Bar terminals. Recording of opto-evoked excitatory post synaptic currents (oEPSCs) in DiI-labeled lumbosacral preganglionic neurons (LS-PGN) revealed that both Bar neuronal populations make strong glutamatergic monosynaptic connections with LS-PGN, while BarESR1 neurons also elicited smaller amplitude glutamatergic polysynaptic oEPSCs or polysynaptic inhibitory post synaptic currents (oIPSCs) in some LS-PGN. Optical stimulation of BarCRH and BarESR1 terminals also elicited monosynaptic oEPSCs and polysynaptic oIPSCs in sacral DCN neurons, some of which must include interneurons projecting either to the IML or ventral horn. Application of capsaicin increased opto-evoked firing during repetitive stimulation of Bar terminals through the modulation of spontaneous post synaptic currents in LS-PGN. In conclusion, our experiments have provided insights into the synaptic mechanisms underlying the integration of inputs from Bar to autonomic circuitry in the lumbosacral spinal cord that may control micturition.

Voluntary Modulation of Evoked Responses Generated by Epidural and Transcutaneous Spinal Stimulation in Humans with Spinal Cord Injury

Transcutaneous (TSS) and epidural spinal stimulation (ESS) are electrophysiological techniques that have been used to investigate the interactions between exogenous electrical stimuli and spinal sensorimotor networks that integrate descending motor signals with afferent inputs from the periphery during motor tasks such as standing and stepping. Recently, pilot-phase clinical trials using ESS and TSS have demonstrated restoration of motor functions that were previously lost due to spinal cord injury (SCI). However, the spinal network interactions that occur in response to TSS or ESS pulses with spared descending connections across the site of SCI have yet to be characterized. Therefore, we examined the effects of delivering TSS or ESS pulses to the lumbosacral spinal cord in nine individuals with chronic SCI. During low-frequency stimulation, participants were instructed to relax or attempt maximum voluntary contraction to perform full leg flexion while supine. We observed similar lower-extremity neuromusculature activation during TSS and ESS when performed in the same participants while instructed to relax. Interestingly, when participants were instructed to attempt lower-extremity muscle contractions, both TSS- and ESS-evoked motor responses were significantly inhibited across all muscles. Participants with clinically complete SCI tested with ESS and participants with clinically incomplete SCI tested with TSS demonstrated greater ability to modulate evoked responses than participants with motor complete SCI tested with TSS, although this was not statistically significant due to a low number of subjects in each subgroup. These results suggest that descending commands combined with spinal stimulation may increase activity of inhibitory interneuronal circuitry within spinal sensorimotor networks in individuals with SCI, which may be relevant in the context of regaining functional motor outcomes.

Quantitation of lower urinary tract afferents in 3D reconstruction of lumbosacral spinal cord sections v1

This protocol details the 3D reconstruction of the lumbosacral spinal cord using alternating cryosections, and then goes through the steps required to quantify lower urinary tract afferents. Using TissueMaker (MBF Bioscience), images of alternating sections can be ordered and aligned prior to the production of a single image stack. In Neurolucida 360 (MBF Bioscience), regions of interest can be defined within the image stack, and the bouton-like immunolabelling of cholera toxin B can be segmented. Once saved, this data can then be extracted using Neurolucida Explorer (MBF Bioscience).

Immunohistochemical labelling of lower urinary tract afferents in spinal cord v1

This protocol is used for immunohistochemical visualisation of cholera toxin subunit B within afferents innervating the lower urinary tract in cryosections of rat lumbosacral spinal cord. Free-floating sections are processed in a double labelling protocol to distinguish regions of innervation by these afferents. Cholera toxin B antibody [lower urinary tract afferents] Choline acetyltransferase antibody [preganglionic autonomic neurons and motoneurons]

Immunolabelling and clearing of intact spinal cord for visualization of lower urinary tract afferents v1

The whole-mount immunolabeling and clearing method (iDISCO) was used to visualize cholera toxin subunit B-labelled lower urinary tract afferents in the lumbosacral spinal cord of the rat. Imaging of spinal cord was performed on a light sheet microscope with a 12x lens. Concurrently, choline acetyltransferase identified preganglionic autonomic neurons and motoneurons within the spinal cord, which were used to confirm the rostrocaudal location of afferents.