Neuronal RARβ signaling modulates PTEN activity directly in neurons and via exosome transfer in astrocytes to prevent glial scar formation and induce spinal cord regeneration

Failure of axonal regeneration in the central nervous system (CNS) is mainly attributed to a lack of intrinsic neuronal growth programs and an inhibitory environment from a glial scar. Phosphatase and tensin homolog (PTEN) is a major negative regulator of neuronal regeneration and, as such, inhibiting its activity has been considered a therapeutic target for spinal cord (SC) injuries (SCIs). Using a novel model of rat cervical avulsion, we show that treatment with a retinoic acid receptor β (RARβ) agonist results in locomotor and sensory recovery. Axonal regeneration from the severed roots into the SC could be seen by biotinylated dextran amine labeling. Light micrographs of the dorsal root entry zone show the peripheral nervous system (PNS)–CNS transition of regrown axons. RARβ agonist treatment also resulted in the absence of scar formation. Mechanism studies revealed that, in RARβ-agonist-treated neurons, PTEN activity is decreased by cytoplasmic phosphorylation and increased secretion in exosomes. These are taken up by astrocytes, resulting in hampered proliferation and causing them to arrange in a normal-appearing scaffold around the regenerating axons. Attribution of the glial modulation to neuronal PTEN in exosomes was demonstrated by the use of an exosome inhibitor in vivo and PTEN siRNA in vitro assays. The dual effect of RARβ signaling, both neuronal and neuronal–glial, results in axonal regeneration into the SC after dorsal root neurotmesis. Targeting this pathway may open new avenues for the treatment of SCIs. SIGNIFICANCE STATEMENT Spinal cord injuries (SCIs) often result in permanent damage in the adult due to the very limited capacity of axonal regeneration. Intrinsic neuronal programs and the formation of a glial scar are the main obstacles. Here, we identify a single target, neuronal retinoic acid receptor β (RARβ), which modulates these two aspects of the postinjury physiological response. Activation of RARβ in the neuron inactivates phosphatase and tensin homolog and induces its transfer into the astrocytes in small vesicles, where it prevents scar formation. This may open new therapeutic avenues for SCIs

COX-2 inhibition by diclofenac is associated with decreased apoptosis and lesion area after experimental focal penetrating traumatic brain injury in rats

Traumatic brain injury (TBI) is followed by a secondary inflammation in the brain. The inflammatory response includes prostanoid synthesis by the inducible enzyme cyclooxygenase-2 (COX-2). Inhibition of COX-2 is associated with improved functional outcome in experimental TBI models, although central nervous system-specific effects are not fully understood. Animal studies report better outcomes in females than males. The exact mechanisms for this gender dichotomy remain unknown. In an initial study we reported increased COX-2 expression in male rats, compared to female, following experimental TBI. It is possible that COX-2 induction is directly associated with increased cell death after TBI. Therefore, we designed a sequential study to investigate the blocking of COX-2 specifically, using the established COX-2 inhibitor diclofenac. Male Sprague-Dawley rats weighing between 250 and 350 g were exposed to focal penetrating TBI and randomly selected for diclofenac treatment (5 ?g intralesionally, immediately following TBI) (n = 8), controls (n = 8), sham operation (n = 8), and normal (no manipulation) (n = 4). After 24 h, brains were removed, fresh frozen, cut into 14?m coronal sections and subjected to COX-2 immunofluorescence, Fluoro Jade, TUNEL, and lesion area analyses. Diclofenac treatment decreased TUNEL staining indicative of apoptosis with a mean change of 54% (p 0.05) and lesion area with a mean change of 55% (p 0.005). Neuronal degeneration measured by Fluoro Jade and COX-2 protein expression levels were not affected. In conclusion, COX-2 inhibition by diclofenac was associated with decreased apoptosis and lesion area after focal penetrating TBI and may be of interest for further studies of clinical applications

Neuropeptide and Small Transmitter Coexistence: Fundamental Studies and Relevance to Mental Illness

Neuropeptides are auxiliary messenger molecules that always co-exist in nerve cells with one or more small molecule (classic) neurotransmitters. Neuropeptides act both as transmitters and trophic factors, and play a role particularly when the nervous system is challenged, as by injury, pain or stress. Here neuropeptides and coexistence in mammals are reviewed, but with special focus on the 29/30 amino acid galanin and its three receptors GalR1, -R2 and -R3. In particular, galanin's role as a co-transmitter in both rodent and human noradrenergic locus coeruleus (LC) neurons is addressed. Extensive experimental animal data strongly suggest a role for the galanin system in depression-like behavior. The translational potential of these results was tested by studying the galanin system in postmortem human brains, first in normal brains, and then in a comparison of five regions of brains obtained from depressed people who committed suicide, and from matched controls. The distribution of galanin and the four galanin system transcripts in the normal human brain was determined, and selective and parallel changes in levels of transcripts and DNA methylation for galanin and its three receptors were assessed in depressed patients who committed suicide: upregulation of transcripts, e.g., for galanin and GalR3 in LC, paralleled by a decrease in DNA methylation, suggesting involvement of epigenetic mechanisms. It is hypothesized that, when exposed to severe stress, the noradrenergic LC neurons fire in bursts and release galanin from their soma/dendrites. Galanin then acts on somato-dendritic, inhibitory galanin autoreceptors, opening potassium channels and inhibiting firing. The purpose of these autoreceptors is to act as a 'brake' to prevent overexcitation, a brake that is also part of resilience to stress that protects against depression. Depression then arises when the inhibition is too strong and long lasting - a maladaption, allostatic load, leading to depletion of NA levels in the forebrain. It is suggested that disinhibition by a galanin antagonist may have antidepressant activity by restoring forebrain NA levels. A role of galanin in depression is also supported by a recent candidate gene study, showing that variants in genes for galanin and its three receptors confer increased risk of depression and anxiety in people who experienced childhood adversity or recent negative life events. In summary, galanin, a neuropeptide coexisting in LC neurons, may participate in the mechanism underlying resilience against a serious and common disorder, MDD. Existing and further results may lead to an increased understanding of how this illness develops, which in turn could provide a basis for its treatment

Assessment of Fluid Cavitation Threshold Using a Polymeric Split Hopkinson Bar-Confinement Chamber Apparatus

The authors would like to acknowledge the Natural Sciences and Engineering Research Council of Canada for financial support, and Compute Canada and Sharcnet for providing the necessary computing resources.Mild Traumatic Brain Injury (mTBI) has been associated with blast exposure resulting from the use of improvised explosive devices (IEDs) in recent and past military conflicts. Experimental and numerical models of head blast exposure have demonstrated the potential for high negative pressures occurring within the head at the contre-coup location relative to the blast exposure, and it has been hypothesized that this negative pressure could result in cavitation of Cerebrospinal Fluid (CSF) surrounding the brain, leading to brain tissue damage. The cavitation threshold of CSF, the effect of temperature, and the effect of impurities or dissolved gases are presently unknown. In this study, a novel Polymeric Split Hopkinson Pressure Bar and confinement chamber apparatus were used to generate loading in distilled water similar to the conditions in the vicinity of the CSF during blast exposure. Cavitation was identified using high-speed imaging of the event, and a validated numerical model of the apparatus was applied to determine the pressure in the fluid during the exposure. Increasing the water temperature resulted in a decrease in the 50% probability of cavitation from 21 °C (−3320 kPa ± 3%) to 37 °C (−3195 kPa ± 5%) in agreement with the theoretical values, but was not statistically significant. Importantly, the effect of water treatment had a significant effect on the cavitation pressure for water with wetting agent (−3320 kPa ± 3%), degassed water (−1369 kPa ± 16%) and untreated distilled water (−528 kPa ± 25%). Thus, reducing dissolved gases through degassing or the use of a wetting agent significantly increases the cavitation pressure and reduces the variability of the cavitation pressure threshold

Identification of Excitatory Interneurons Contributing to Generation of Locomotion in Lamprey: Structure, Pharmacology, and Function

1. In the in vitro preparation of the lamprey spinal cord, paired intracellular recordings of membrane potential were used to identify interneurons producing excitatory postsynaptic potentials (EPSPs) on myotomal motoneurons. 2. Seventy-nine interneurons (8.4% of all neuron-motoneuron pairs tested) elicited unitary EPSPs that followed one-for-one at short, constant latencies and were therefore considered monosynaptic according to conventional criteria. Evidence was obtained for selectivity and divergence of excitatory interneuron (EIN) outputs and for convergence of EIN input to motoneurons. 3. The neurotransmitter released by EINs may be an excitatory amino acid such as glutamate, because the EPSPs were depressed by antagonists of excitatory amino acids. 4. Intracellular dye injection revealed that EINs have small cell bodies (average 11 x 27 microns), transversely oriented dendrites, and thin (less than 3 microns) slowly conducting axons (0.7 m/s) that project caudally and ipsilaterally. One EIN exhibited a system of thin multi-branching axon collaterals with periodic swellings. Ultrastructurally, these swellings contained clear spherical vesicles, and they apposed postsynaptic membrane specializations. 5. During fictive locomotion, the membrane-potential oscillations of EINs were greater in amplitude than, but similar in shape and timing to, those of their postsynaptic motoneurons. EINs fired action potentials during fictive locomotion and contributed to the depolarization of motoneurons. 6. These interneurons are proposed to be a source of excitation to motoneurons and interneurons in the lamprey spinal cord, participating in motor activity including locomotion

The Clemedson Blast Tube

Traumatic brain injuries (TBI) because of detonations have become a significant problem in military medicine. Partly because the use of modern body protection has increased the survival of victims subjected to detonations from landmines or improvised explosive devices. Detonations commonly expose these victims to pressure waves, high speed fragments, and bodily accelerations. The pressure wave itself may result in a mild TBI, commonly referred to as primary blast, while penetration of fragments into the brain and head rotations resulting from body accelerations can lead to more severe forms of TBI. The details of the cellular injury mechanisms of primary blast are still debated and studies are needed to understand the propagation and effects of the pressure waves inside the skull. Laboratory experiments with good control for physical parameters can provide information that is difficult to retrieve from real-life cases of blast injury. This study focused on head kinematics and pressure propagation into the animal brain cavity during simulated blast trauma (part 1) and the behavioral outcome (part 2). The rat blast model presented here produced maximum intracranial pressure increases of 6\ua0bar while minimal pressure drops. Violent head-to-head restraint contact occurred at approximately 1.7\ua0ms after the pressure pulse reached the head; this contact did not produce any high intracranial pressures. Working memory error was not significantly changed between the exposed and controls at 1\ua0week after blast while significantly more reference memory errors at 5\ua0days and 2\ua0weeks following injury compared to sham after blast