The Role of Aquaporin 4 in Lacrimal Gland Ductal Fluid Secretion in Mice.

Purpose: Earlier reports highlighted the predominant presence of aquaporin 4 (AQP4) in the duct cells of rabbit lacrimal glands (LGs). Whereas significant alterations in AQP4 mRNA levels have been observed in experimental dry eye and during pregnancy, the impact of AQP4 in LG ductal fluid production remains unclear. In our recent work, the role of AQP4 in LG ductal fluid secretion was investigated utilizing wild type (WT) and AQP4 knock out (KO) mice. Methods: Tear production was assessed in both WT and KO animals. Immunostaining was used to identify AQP4 protein. Duct segments were harvested from LGs of WT and KO mice. Fluid secretion and filtration permeability (Pf) were quantified using video-microscopy. Ductal tear production, elicited by a cell-permeable cAMP analogue (8-bromo cAMP), carbachol, vasoactive intestinal peptide (VIP), and phenylephrine (PHE), were assessed in both WT and KO ducts. Results: A higher expression of AQP4 protein was noted in the duct cells from WT mice when compared to acinar cells. Pf did not show notable alterations between WT and AQP4 KO ducts. Carbachol elicited comparable secretory responses in ducts from both WT and KO animals. However, 8-bromo cAMP, VIP, and PHE stimulation resulted in decreased secretion in ducts from AQP4 KO LGs. Conclusions: Our findings underscore the functional relevance of AQP4 in the fluid production of mouse LG ducts. AQP4 seems to play different roles in fluid secretions elicited by different secretagogues. Specifically, cAMP-mediated, and adrenergic agonist-related secretions were reduced in AQP4 KO ducts.

Restoration of Motor Function through Delayed Intraspinal Delivery of Human IL-10-Encoding Nucleoside-Modified mRNA after Spinal Cord Injury.

Efficient in vivo delivery of anti-inflammatory proteins to modulate the microenvironment of an injured spinal cord and promote neuroprotection and functional recovery is a great challenge. Nucleoside-modified messenger RNA (mRNA) has become a promising new modality that can be utilized for the safe and efficient delivery of therapeutic proteins. Here, we used lipid nanoparticle (LNP)-encapsulated human interleukin-10 (hIL-10)-encoding nucleoside-modified mRNA to induce neuroprotection and functional recovery following rat spinal cord contusion injury. Intralesional administration of hIL-10 mRNA-LNP to rats led to a remarkable reduction of the microglia/macrophage reaction in the injured spinal segment and induced significant functional recovery compared to controls. Furthermore, hIL-10 mRNA treatment induced increased expression in tissue inhibitor of matrix metalloproteinase 1 and ciliary neurotrophic factor levels in the affected spinal segment indicating a time-delayed secondary effect of IL-10 5 d after injection. Our results suggest that treatment with nucleoside-modified mRNAs encoding neuroprotective factors is an effective strategy for spinal cord injury repair.

The use of a detailed video-based locomotor pattern analysis system to assess the functional reinnervation of denervated hind limb muscles.

BACKGROUND: Spinal cord injuries induce a critical loss of motoneurons followed by irreversible locomotor function impairment. Surgical approaches combined with neuroprotective agents effectively rescue the damaged motoneurons and improve locomotor function. Our aim was to develop a reliable method which is able to provide quantifiable and in-depth data on the locomotor recovery during skeletal muscle reinnervation. NEW METHOD: Sprague-Dawley rats underwent lumbar 4 ventral root avulsion and reimplantation followed by riluzole treatment in order to rescue the injured motoneurons of the damaged pool. Control animals were operated, but received no riluzole treatment. The locomotor pattern of the hind limb was recorded biweekly on a special runway equipped with high resolution and high speed digital cameras producing both lateral and rear views simultaneously. All together 12 parameters of the hind limb movement pattern were evaluated by measuring specific joint angles, footprints and gait parameters in single video frames. Four months after the operation Fast Blue, a fluorescent retrograde tracer was applied to the L4 spinal nerve in order to label the reinnervating motoneurons. RESULTS: Our results confirmed the sensitivity of our arrangement and established strong relationship between the functional improvement and the morphological reinnervation. Moreover, we developed a correction method to make the system tolerant to the differences in the weight, step duration and step length. COMPARISON WITH EXISTING METHODS: There are no commercially available cheap, multi-parametric analysing equipment to characterise the gait in its complexity. CONCLUSIONS: Our system offers a modular, adaptable and expandable analysis on the reinnervation of the limb musculature in rodents.

Grafted human induced pluripotent stem cells improve the outcome of spinal cord injury: modulation of the lesion microenvironment.

Spinal cord injury results in irreversible tissue damage followed by a very limited recovery of function. In this study we investigated whether transplantation of undifferentiated human induced pluripotent stem cells (hiPSCs) into the injured rat spinal cord is able to induce morphological and functional improvement. hiPSCs were grafted intraspinally or intravenously one week after a thoracic (T11) spinal cord contusion injury performed in Fischer 344 rats. Grafted animals showed significantly better functional recovery than the control rats which received only contusion injury. Morphologically, the contusion cavity was significantly smaller, and the amount of spared tissue was significantly greater in grafted animals than in controls. Retrograde tracing studies showed a statistically significant increase in the number of FB-labeled neurons in different segments of the spinal cord, the brainstem and the sensorimotor cortex. The extent of functional improvement was inversely related to the amount of chondroitin-sulphate around the cavity and the astrocytic and microglial reactions in the injured segment. The grafts produced GDNF, IL-10 and MIP1-alpha for at least one week. These data suggest that grafted undifferentiated hiPSCs are able to induce morphological and functional recovery after spinal cord contusion injury.

Novel Insight Into the Role of CFTR in Lacrimal Gland Duct Function in Mice.

Purpose: The role of cystic fibrosis transmembrane conductance regulator (CFTR) in lacrimal gland (LG) function has only recently received some attention, mainly from our group. In the present study, we investigated the potential changes of LG pathology, tear secretion, ocular surface integrity, and fluid secretion in isolated LG ducts from CFTR knockout (KO) mice. Methods: Tear production and ocular surface integrity were investigated in anesthetized wild-type (WT) and KO mice using cotton threads and fluorescein staining, respectively. Immunofluorescence was used to localize CFTR protein in the LGs. Ductal fluid secretions evoked by forskolin (10 µM); cell-permeable cAMP analogue (8-bromo cAMP, 100 µM); or carbachol (100 µM) were measured in isolated LG ducts using video-microscopy. Intracellular Ca2+ homeostasis underlying carbachol stimulation was investigated with microfluorometry. Results: Significant decrease in tear secretion and impaired ocular surface integrity were observed in KO mice. Immunofluorescence demonstrated the predominant presence of CFTR protein in the apical membranes of the duct cells from WT mice. Continuous fluid secretion was evoked by forskolin and 8-bromo cAMP in LG ducts from WT mice, while no secretory response was observed in ducts from KO mice. Carbachol caused similar secretory responses in ducts from WT and KO animals without significant differences in cytosolic Ca2+ signaling. Conclusions: Our results suggest the important role of CFTR in LG ductal secretion and in the maintenance of ocular surface integrity, suggesting that CFTR may be a promising target of novel therapeutic approaches in the treatment of dry eye.

Delayed Spinal Cord-Brachial Plexus Reconnection after C7 Ventral Root Avulsion: The Effect of Reinnervating Motoneurons Rescued by Riluzole Treatment.

Ventral root avulsion induces dramatic loss of the affected spinal cord motoneurons. The neuroprotective effect of riluzole has been previously proven on the injured motoneurons: the vast majority of them can be rescued even when they have no possibility to regenerate their axons. In this study the number of injured motoneurons rescued by riluzole treatment and their capacity to reinnervate the denervated forelimb muscles was investigated. Surgical reconnection with a peripheral nerve graft between the affected spinal cord segment and the C7 spinal nerve was established immediately or with 1- and 3-week delay after avulsion. Avulsion and immediate reconnection of the motoneuron pool to the spinal nerve resulted in moderate reinnervation of the spinal nerve (281 ± 23 standard error of mean [SEM] retrogradely labeled motoneurons), whereas treatment of the injured motoneurons with riluzole yielded considerably higher numbers of reinnervating motoneurons (548 ± 18 SEM). Reconnection of the motor pool with the C7 spinal nerve with 1-week delay allowed fewer motor axons to reinnervate their targets in control and riluzole-treated animals (159 ± 21 vs. 395 ± 16 SEM). A clinically relevant 3-week delay in reconnection further reduced the number of reinnervating motoneurons (76 ± 22 SEM), but riluzole pre-treatment still enabled a significant number of rescued motoneurons (396 ± 17 SEM) to regenerate their axons into the C7 spinal nerve. These results show that those injured adult motoneurons that are rescued by riluzole treatment started immediately after the avulsion injury are able to reinnervate their targets even if they are provided with a conduit several weeks after the primary injury. This finding suggests that partial rescue of injured motoneurons with riluzole in patients who suffered a brachial plexus avulsion injury may provide an available pool of surviving motoneurons for late reconnection/reimplantation surgeries.