Continuous intrathecal orexin delivery inhibits cataplexy in a murine model of narcolepsy

Abstract

Narcolepsy-cataplexy is a chronic neurological disorder caused by loss of orexin (hypocretin)-producing neurons, associated with excessive daytime sleepiness, sleep attacks, cataplexy, sleep paralysis, hypnagogic hallucinations, and fragmentation of nighttime sleep. Currently, human narcolepsy is treated by providing symptomatic therapies, which can be associated with an array of side effects. Although peripherally administered orexin does not efficiently penetrate the blood-brain barrier, centrally delivered orexin can effectively alleviate narcoleptic symptoms in animal models. Chronic intrathecal drug infusion through an implantable pump is a clinically available strategy to treat a number of neurological diseases. Here we demonstrate that the narcoleptic symptoms of orexin knockout mice can be reversed by lumbar-level intrathecal orexin delivery. Orexin was delivered via a chronically implanted intrathecal catheter at the upper lumbar level. The computed tomographic scan confirmed that intrathecally administered contrast agent rapidly moved from the spinal cord to the brain. Intrathecally delivered orexin was detected in the brain by radioimmunoassay at levels comparable to endogenous orexin levels. Cataplexy and sleep-onset REM sleep were significantly decreased in orexin knockout mice during and long after slow infusion of orexin (1 nmol/1 µL/h). Sleep/wake states remained unchanged both quantitatively as well as qualitatively. Intrathecal orexin failed to induce any changes in double orexin receptor-1 and -2 knockout mice. This study supports the concept of intrathecal orexin delivery as a potential therapy for narcolepsy-cataplexy to improve the well-being of patients.

Keywords: lumbar spinal cord; non-REM sleep; orexin knockout mice; slow infusion.

Fig. 1.

Contrast medium and orexin delivered at lumbar spinal cord level were detected in the brain. (A–D) Representative CT scan of a mouse whereby contrast medium was infused via intrathecal route. Two-dimensional axial cross-sectional sagittal (A) and coronal (B) image showing contrast medium in brain ventricles and spinal cord (arrows). (C and D) The 3D reconstructed image showing contrast medium at brain ventricles and spinal cord (arrows). (E–G) CT scan of a control mouse with a misplaced catheter (E). Two-dimensional coronal (F) and 3D sagittal (G) brain images where no contrast medium was detected. (H) Orexin peptide content in brains of orexin knockout mice with and without orexin-A infusion. Circles represent values of the individual mouse and gray bars show median values. n = 5; **P ≤ 0.01 by using one-way ANOVA, followed by Fisher’s probable least-squares difference (LSD) test. (I) Schematic representation of the construction of the intrathecal catheter. (Upper) Catheter dimensions; (Lower Left) an overview sketch of catheter implantation; and (Lower Right) picture of intact animal after surgery connected to recording leads and infusion system. ID, inside diameter; n.s., not significant; OD, outside diameter.

Fig. 2.

Intrathecally delivered orexin-A suppressed cataplexy and SOREM in orexin knockout mice. (A and B) Typical examples of EEG delta power (0.5–4 Hz), EEG theta power (4–10 Hz), EMG integral, and hypnograms of an orexin knockout mouse during aCSF (vehicle) (A) and orexin-A (B) infusion with chocolate provided in the cage. Hypnograms represent concatenated 10-s epochs of EEG/EMG activity, scored as wake, REM, NREM (gray), and SOREM and cataplexy (black). Twelve hours per mouse, starting at the beginning of the dark phase, are shown. The shaded area in B shows the duration of orexin-A infusion. CP, cataplexy; NR, NREM sleep; R, REM sleep; SR, SOREM; W, wake.

Fig. 3.

Intrathecal orexin-A infusion suppressed cataplexy and SOREM in orexin knockout mice. (A and B) Hourly plots of SOREM and cataplexy during vehicle and orexin-A infusion within the 12-h dark phase. The shaded area on graphs represents the duration of orexin-A infusion. (C) Total amounts of time spent in SOREM and cataplexy within the 12-h dark phase, during vehicle and orexin-A infusion. (D and E) The number of bouts (D) and stage duration (E) of SOREM and cataplexy during a vehicle and orexin-A infusion. (F and G) EEG power densities of SOREM (F) and cataplexy (G) episodes during vehicle and orexin-A infusion. Values are presented as mean ± SEM; n = 4; *P ≤ 0.05, **P ≤ 0.01 by using two-way ANOVA (A, B, F, and G) and paired t test (C–E). (H) Dose-dependent changes in SOREM and cataplexy in orexin knockout mice during/after vehicle, orexin-A (100 pmol/1 µL/h and 1 nmol/1 µL/h, blue bars and red bars, respectively). Circles represent individual animal values. Values are mean ± SEM; n = 3–5; *P ≤ 0.05, **P ≤ 0.01 by using one-way ANOVA, followed by Scheffé’s post hoc test. CP, cataplexy; orexin-A, colored lines and bars; SR, SOREM; vehicle, black lines and gray bars.

Fig. 4.

Intrathecally delivered orexin-A did not alter sleep/wake stages in orexin knockout mice. (A) Hourly plots of wake (W), REM (R), and NREM (NR) sleep during a vehicle and orexin-A infusion are shown. (B) Total amounts of time spent in the wake, REM, and NREM sleep in dark and light phases. (C) Stage transitions from NR to W, W to NR, NR to R, R to W, and W to cataplexy (CP) during a vehicle and orexin-A infusion. (D and E) Changes in the number of bouts (D) and stage duration (E) of the wake, REM, and NREM sleep, during vehicle and orexin-A infusion. (F and G) EEG power densities of REM and NREM episodes during a vehicle and orexin-A infusion. The shaded area on graphs represents the duration of orexin-A infusion, while nonshaded area represents aCSF infusion. Values are mean ± SEM; n = 4; *P ≤ 0.05, **P ≤ 0.01 by using two-way ANOVA (A, B, F, and G) and paired t test (C–E). Orexin-A, colored lines and bars; vehicle, black lines and gray bars.

Fig. 5.

Intrathecal orexin-A infusion in OXR-DKO mice did not inhibit SOREM and cataplexy. Hourly plots of SOREM (A), cataplexy (B), and the total amount of time spent in SOREM and cataplexy (C) over a 12-h dark phase during a vehicle and orexin-A infusion are shown. The number of bouts (D) and stage duration (E) of SOREM and cataplexy during/after a vehicle and orexin-A infusion are shown. (F) EEG power densities of SOREM and cataplexy episodes during/after a vehicle and orexin-A infusion. Values presented are mean ± SEM; n = 5. Orexin-A, colored lines and bars; vehicle, black lines and gray bars.