SUMMARY Spinal cord injury (SCI) above cervical level 4 disrupts descending axons from the medulla that innervate phrenic motor neurons, causing permanent paralysis of the diaphragm. Using an ex vivo preparation in neonatal mice, we have identified an excitatory spinal network which can direct phrenic motor bursting in the absence of medullary input. After complete cervical SCI, blockade of fast inhibitory synaptic transmission caused spontaneous, bilaterally-coordinated phrenic bursting. Here, spinal cord glutamatergic neurons were both sufficient and necessary for induction of phrenic bursts. Direct stimulation of phrenic motor neurons was insufficient to evoke burst activity. Transection and pharmacological manipulations showed that this spinal network acts independently of medullary circuits which normally generate inspiration, suggesting a distinct non-respiratory function. We further show that this “latent” network can be harnessed to restore diaphragm function after high cervical spinal cord injury in adult mice and adult rats.

Neuronal KCNQ channels mediate the M-current, a key regulator of membrane excitability in the central and peripheral nervous systems. Mutations in KCNQ2 channels cause severe neurodevelopmental disorders, including epileptic encephalopathies. However, the impact that different mutations have on channel function remains poorly defined, largely because of our limited understanding of the voltage sensing mechanisms that trigger channel gating. Here, we define parameters of voltage sensor movements in wt-KCNQ2 and channels bearing epilepsy-associated mutations using cysteine accessibility and voltage clamp fluorometry (VCF). Cysteine modification reveals that a stretch of 8-9 amino acids in the S4 become exposed upon VSD activation of KCNQ2 channels. VCF shows that the voltage dependence and the time course of S4 movement and channel opening/closing closely correlate. VCF reveals different mechanisms by which different epilepsy-associated mutations affect KCNQ2 channel voltage-dependent gating. This study provides insight into KCNQ2 channel function, which will aid in uncovering the mechanisms underlying channelopathies.

PurposeTo determine the feasibility and accuracy of spectral-domain optical coherence tomography (SD-OCT) based grading of anterior chamber cell, using aqueous sampling as a standard, in a rabbit model of anterior uveitis.MethodsAdult Dutch-belted rabbits were preimmunized with M. tuberculosis (Tb) H37RA antigen, 1 week prior to induction of anterior uveitis with an intracameral injection of Tb antigen. The anterior chamber was imaged with SD-OCT, followed by a slit lamp examination. Two independent, trained graders recorded their estimate of anterior chamber cell count using the Standardization of Uveitis Nomenclature (SUN) scores for each eye prior to performing an anterior chamber tap to determine the aqueous cell density using a hemocytometer. Using the aqueous cell density as a standard, correlation with SD-OCT counts were compared to those with SUN scores.ResultsOverall, SD-OCT correlated well with the hemocytometer counts (Spearman coefficient = 0.53, P < 0.001) compared with SUN grading and hemocytometer counts (Spearman coefficient = 0.02, P = 0.88). The correlation improved to 0.65 (P < 0.001) when we excluded eyes with corneal thickness ≥ 470 μm. Eyes with corneal thickness ≥ 470 μm exhibited the greatest degree of ocular inflammation and corneal opacity.ConclusionsIn our rabbit model, SD-OCT grading of anterior chamber cell correlated significantly better with aqueous cell counts, compared to traditional slit lamp grading. Spectral-domain optical coherence tomography grading of anterior chamber cell may be a good alternative to SUN grading. Although SUN grading remains the clinical gold standard, alternative quantitative methods to assess ocular inflammation could provide insight into disease mechanism and aid in measuring treatment response.

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