Absence of IFNγ expression induces neuronal degeneration in the spinal cord of adult mice

<p>Abstract</p> <p>Background</p> <p>Interferon gamma (IFNγ) is a pro-inflammatory cytokine, which may be up-regulated after trauma to the peripheral or central nervous system. Such changes include reactive gliosis and synaptic plasticity that are considered important responses to the proper regenerative response after injury. Also, IFNγ is involved in the upregulation of the major histocompatibility complex class I (MHC class I), which has recently been shown to play an important role in the synaptic plasticity process following axotomy. There is also evidence that IFNγ may interfere in the differentiation and survival of neuronal cells. However, little is known about the effects of IFNγ absence on spinal cord neurons after injury.</p> <p>Methods</p> <p>We performed a unilateral sciatic nerve transection injury in C57BL/6J (wild type) and IFNγ-KO (mutant) mice and studied motoneuron morphology using light and electron microscopy. One week after the lesion, mice from both strains were sacrificed and had their lumbar spinal cords processed for histochemistry (n = 5 each group) and transmission electron microscopy (TEM, n = 5 each group). Spinal cord sections from non-lesioned animals were also used to investigate neuronal survival and the presence of apoptosis with TUNEL and immunohistochemistry.</p> <p>Results</p> <p>We find that presumed motoneurons in the lower lumbar ventral horn exhibited a smaller soma size in the IFNγ-KO series, regardless of nerve lesion. In plastic embedded sections stained with toluidine blue, the IFNγ-KO mice demonstrated a greater proportion of degenerating neurons in the ventral horn when compared to the control series (p < 0.05). Apoptotic death is suggested based on TUNEL and caspase 3 immunostaining. A sciatic nerve axotomy did not further aggravate the neuronal loss. The cellular changes were supported by electron microscopy, which demonstrated ventral horn neurons exhibiting intracellular vacuoles as well as degenerating nuclei and cytoplasm in the IFNγ-KO mice. Adjacent glial cells showed features suggestive of phagocytosis. Additional ultrastructural studies showed a decreased number of pre-synaptic terminals apposing to motoneurons in mutant mice. Nevertheless, no statistical difference regarding the input covering could be detected among the studied strains.</p> <p>Conclusion</p> <p>Altogether, these results suggest that IFNγ may be neuroprotective and its absence results in neuronal death, which is not further increased by peripheral axotomy.</p

Chemotropic guidance facilitates axonal regeneration and synapse formation after spinal cord injury.

A principal objective of spinal cord injury (SCI) research is the restoration of axonal connectivity to denervated targets. We tested the hypothesis that chemotropic mechanisms would guide regenerating spinal cord axons to appropriate brainstem targets. We subjected rats to cervical level 1 (C1) lesions and combinatorial treatments to elicit axonal bridging into and beyond lesion sites. Lentiviral vectors expressing neurotrophin-3 (NT-3) were then injected into an appropriate brainstem target, the nucleus gracilis, and an inappropriate target, the reticular formation. NT-3 expression in the correct target led to reinnervation of the nucleus gracilis in a dose-related fashion, whereas NT-3 expression in the reticular formation led to mistargeting of regenerating axons. Axons regenerating into the nucleus gracilis formed axodendritic synapses containing rounded vesicles, reflective of pre-injury synaptic architecture. Thus, we report for the first time, to the best of our knowledge, the reinnervation of brainstem targets after SCI and an essential role for chemotropic axon guidance in target selection

Extensive spontaneous plasticity of corticospinal projections after primate spinal cord injury.

Although axonal regeneration after CNS injury is limited, partial injury is frequently accompanied by extensive functional recovery. To investigate mechanisms underlying spontaneous recovery after incomplete spinal cord injury, we administered C7 spinal cord hemisections to adult rhesus monkeys and analyzed behavioral, electrophysiological and anatomical adaptations. We found marked spontaneous plasticity of corticospinal projections, with reconstitution of fully 60% of pre-lesion axon density arising from sprouting of spinal cord midline-crossing axons. This extensive anatomical recovery was associated with improvement in coordinated muscle recruitment, hand function and locomotion. These findings identify what may be the most extensive natural recovery of mammalian axonal projections after nervous system injury observed to date, highlighting an important role for primate models in translational disease research

Serotonergic 5-HT1A receptor agonist (8-OH-DPAT) ameliorates impaired micturition reflexes in a chronic ventral root avulsion model of incomplete cauda equina/conus medullaris injury

Trauma to the thoracolumbar spine commonly results in injuries to the cauda equina and the lumbosacral portion of the spinal cord. Both complete and partial injury syndromes may follow. Here, we tested the hypothesis that serotonergic modulation may improve voiding function after an incomplete cauda equina/conus medullaris injury. For this purpose, we used a unilateral L5-S2 ventral root avulsion (VRA) injury model in the rat to mimic a partial lesion to the cauda equina and conus medullaris. Compared to a sham-operated series, comprehensive urodynamic studies demonstrated a markedly reduced voiding efficiency at 12 weeks after the VRA injury. Detailed cystometrogram studies showed injury-induced decreased peak bladder pressures indicative of reduced contractile properties. Concurrent external urethral sphincter (EUS) electromyography demonstrated shortened burst and prolonged silent periods associated with the elimination phase. Next, a 5-HT1A receptor agonist, 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT), was administered intravenously at 12 weeks after the unilateral L5-S2 VRA injury. Both voiding efficiency and maximum intravesical pressure were significantly improved by 8-OH-DPAT (0.3-1.0 mg/kg). 8-OH-DPAT also enhanced the amplitude of EUS tonic and bursting activity as well as duration of EUS bursting and silent period during EUS bursting. The results indicate that 8-OH-DPAT improves voiding efficiency and enhances EUS bursting in rats with unilateral VRA injury. We conclude that serotonergic modulation of the 5-HT1A receptor may represent a new strategy to improve lower urinary tract function after incomplete cauda equina/conus medullaris injuries in experimental studies

Differential effects of urethane and isoflurane on external urethral sphincter electromyography and cystometry in rats

Urethane is a common and often preferred anesthetic agent for urodynamic recordings in rats, but its use is often restricted to terminal procedures because of a prolonged duration of action and potentially toxic effects. When urodynamic recordings are part of survival procedures in rodent experimental models, inhalation anesthetics, such as isoflurane, are frequently used and generally well tolerated. In this study, we compared the effects of urethane and isoflurane on lower urinary tract function. For this purpose, adult female rats were anesthetized by subcutaneous administration of urethane (n = 6) or by inhalation of isoflurane (n = 5). Micturition reflexes were assessed by concurrent cystometrogram and external urethral sphincter (EUS) electromyography (EMG) recordings to determine bladder contractile properties, EUS activation patterns, and the coordination between bladder contractions and EUS activation. Compared with urethane, isoflurane reduced frequency of bursts, firing frequency, and amplitude of EUS EMG activity during voiding as well as the EUS EMG amplitude during the bladder filling phase. Isoflurane also prolonged the bladder intercontractile intervals. Other several key functional aspects of the bladder contractile properties as well as the coordination between bladder contractions were not different between the two experimental groups. We conclude that micturition reflexes were differentially affected by isoflurane and urethane. Specifically, isoflurane exhibited a significant suppression of the EUS EMG activity and prolonged the bladder intercontractile intervals compared with urethane. We suggest that these anesthetic properties be taken into consideration during the experimental design and interpretation of urodynamic recordings in rodent models

The Suitability of Propofol Compared with Urethane for Anesthesia during Urodynamic Studies in Rats.

Urethane anesthesia preserves many reflex functions and is often the preferred anesthetic for urodynamic studies in rats. Because of the toxicity profile of urethane, its use as an anesthetic typically is limited to acute and terminal investigations. Alternative anesthetic options are needed for longitudinal studies of micturition reflexes in rats. In this study, we evaluated propofol anesthesia administered at constant rate infusion at different planes of anesthesia in rats for combined cystometrography and external urethral sphincter (EUS) EMG in rats. No reflex micturition was noted after rats received 100%, 80%, or 60% of a previously reported anesthetic dose of propofol. At 40% of the standard propofol dose, a subset of rats showed reflex voiding, with bladder contractions and associated EUS EMG activity. In contrast, urethane anesthesia at a surgical plane allowed for reflex voiding with bladder contractions and EUS activation. Latency to leaking or voiding was longer in rats under propofol anesthesia than in those under urethane anesthesia. In a subset of rats with reflex voiding under propofol anesthesia, voiding efficiency was decreased compared with that of rats anesthetized with urethane. We conclude that propofol anesthesia suppresses micturition reflexes in rats more efficiently than did urethane. Propofol is a suitable anesthetic for longitudinal studies in rats, but its use for urodynamic evaluations is limited in these animals due to its marked suppression of both bladder contractions and EUS EMG activation

Ultrastructural synaptic features differ between alpha- and gamma-motoneurons innervating the tibialis anterior muscle in the rat

We investigated the synaptology of retrogradely labeled spinal motoneurons after injection of horseradish peroxidase into the tibialis anterior (TA) muscle of adult rat. In total, 32 TA motoneurons were investigated in the electron microscope and demonstrated a bimodal size distribution with cell diameter peaks at 40 mu m and 20 mu m, likely representing alpha- and gamma-motoneurons, respectively. Both alpha- and gamma-motoneurons were apposed by S- and F-type synaptic boutons, whereas only alpha-motoneurons demonstrated inputs by the large M- and C-type boutons. The proportion of cell body membrane covered by synaptic inputs was surprisingly indistinguishable between alpha-motoneurons (72.2%) and gamma-motoneurons (63.5%). The ratio between the number of F- and S-type boutons in apposition with the motoneuron cell body (F/S ratio) and the ratio between the soma membrane coverage provided by F- and S-type boutons were both significantly higher in alpha- than in gamma-motoneurons. When comparing our data with previous findings in other species, we conclude that rat TA alpha-motoneurons are similar to cat and primate alpha-motoneurons with regard to synaptic terminal morphology, frequency, and distribution. However, rat gamma-motoneurons show a markedly higher total synaptic coverage and frequency than cat gamma-motoneurons, although both species exhibit appositions made by the same synaptic types and similar ratios between inhibitory and excitatory inputs