Is Innervation of the Neuromuscular Junction at the Diaphragm Modulated by sGC/cGMP Signaling?

We previously reported NO/sGC signaling in the upper respiratory pathway, receiving input from the respiratory neurons of the brainstem to phrenic motoneurons in the C3-C6 spinal cord. In order to assess whether innervation of the neuromuscular junction (NMJ) at the diaphragm is modulated by sGC/cGMP signaling, we performed unilateral 8-day continuous ligation of the phrenic nerve in rats. We examined sGCß1 within the lower bulbospinal pathway (phrenic motoneurons, phrenic nerves and NMJs at the diaphragm) and the cGMP level in the contra- and ipsilateral hemidiaphragm. Additionally, we characterized the extent of phrenic nerve axonal degeneration and denervation at diaphragm NMJs. The results of our study show that continuous 8-day phrenic nerve ligation caused a marked increase in sGCß1 (immunoreactivity and the protein level) in the ipsilateral phrenic motor pool. However, the protein sGCß1 level in the phrenic nerve below its ligation and the cGMP level in the ipsilateral hemidiaphragm were evidently decreased. Using confocal analysis we discovered a reduction in sGCß1-IR boutons/synaptic vesicles at the ipsilateral MNJs. These findings are consistent with the marked axonal loss (∼47%) and significant NMJs degeneration in the ipsilateral diaphragm muscle. The remarkable unilateral decrease in cGMP level in the diaphragm and the failure of EMG recordings in the ipsilateral hemidiaphragm muscle can be attributed to the fact that sGC is involved in transmitter release at the diaphragm NMJs via the sGC-cGMP pathway.

The Characterization of AT1 Expression in the Dorsal Root Ganglia After Chronic Constriction Injury.

To clarify the role of Angiotensin II in the regulation of sensory signaling, we characterized the AT1 expression in neuronal subpopulation of lower lumbar dorsal root ganglia under normal conditions and its alteration in neuropathic pain model. The characterization of AT1 expression was done under control and after the chronic constriction injury induced by four loose ligatures of the sciatic nerve representing the model of posttraumatic painful peripheral neuropathy. Major Angiotensin II receptor type was expressed in approximately 43 % of small-sized and 62 % of large-sized neurons in control. The AT1 overexpression after sciatic nerve ligation lasting 7 days was detected predominantly in small-sized AT1 immunoreactive neurons (about 38 % increase). Chronic constriction injury caused a statistically marked increase in number of the small-sized peptidergic (CGRP immunoreactive) neuronal subpopulation expressing AT1 (about 64 %). The subpopulations of AT1-immunoreactive and nonpeptide-containing primary sensory neurons revealed by IB4 binding, tyrosine hydroxylase- and parvalbumin-immunoreactive neurons were not markedly changed. Our results indicate that: (1) the AT1 overexpression after the chronic constriction injury is an important factor in Angiotensin II-potentiated pain perception; (2) Angiotensin II is involved in pathological mechanisms of neuropathic pain and this effect can be mediated perhaps in combination with other neuropeptides synthesized in the primary sensory neurons.

Baclofen or nNOS inhibitor affect molecular and behavioral alterations evoked by traumatic spinal cord injury in rat spinal cord.

BACKGROUND CONTEXT: The loss of descending control after spinal cord injury (SCI) and incessant stimulation of Ia monosynaptic pathway, carrying proprioceptive impulses from the muscles and tendons into the spinal cord, evoke exaggerated α-motoneuron activity leading to increased reflex response. Previous results from our laboratory have shown that Ia monosynaptic pathway is nitrergic. PURPOSE: The aim of this study was to find out whether nitric oxide produced by neuronal nitric oxide synthase (nNOS) plays a role in setting the excitability of α-motoneurons after thoracic spinal cord transection. STUDY DESIGN: We tested the hypothesis that the inhibition of nNOS in α-motoneurons after SCI could have a neuroprotective effect on reflex response. METHODS: Rats underwent spinal cord transection at Th10 level followed by 7, 10, and 14 days of survival. The animals were treated with Baclofen (a gamma aminobutyric acid B receptor agonist, 3 µg/two times per day/intrathecally) applied for 3 days from the seventh day after transection; N-nitro-l-arginine (NNLA) (nNOS blocator) applied for the first 3 days after injury (20 mg/kg per day, intramuscularly); NNLA and Baclofen; or NNLA (60 mg/kg/day, single dose) applied on the 10th day after transection. We detected the changes in the level of nNOS protein, nNOS messenger RNA, and nNOS immunoreactivity. To investigate the reflex response to heat-induced stimulus, tail-flick test was monitored in treated animals up to 16 days after SCI. RESULTS: Our data indicate that Baclofen therapy is more effective than the combined treatment with NNLA and Baclofen therapy. The single dose of NNLA (60 mg/kg) applied on the 10th day after SCI or Baclofen therapy reduced nNOS expression in α-motoneurons and suppressed symptoms of increased reflex activity. CONCLUSIONS: The results clearly show that increased nNOS expression in α-motoneurons after SCI may be pharmacologically modifiable with Baclofen or bolus dose of nNOS blocker.

Repeated Baclofen treatment ameliorates motor dysfunction, suppresses reflex activity and decreases the expression of signaling proteins in reticular nuclei and lumbar motoneurons after spinal trauma in rats.

The interruption of supraspinal input to the spinal cord leads to motor dysfunction and the development of spasticity. Clinical studies have shown that Baclofen (a GABAB agonist), while effective in modulating spasticity is associated with side-effects and the development of tolerance. The aim of the present study was to assess if discontinued Baclofen treatment and its repeated application leads antispasticity effects, and whether such changes affect neuronal nitric oxide synthase (nNOS) in the brainstem, nNOS and parvalbumin (PV) in lumbar α-motoneurons and glial fibrillary acidic protein in the ventral horn of the spinal cord. Adult male Wistar rats were exposed to Th9 spinal cord transection. Baclofen (30mg/b.w.) diluted in drinking water, was administered for 6 days, starting at week 1 after injury and then repeated till week 4 after injury. The behavior of the animals was tested (tail-flick test, BBB locomotor score) from 1 to 8 weeks. Our results clearly indicate the role of nitric oxide, produced by nNOS in the initiation and the maintenance of spasticity states 1, 6 and 8 weeks after spinal trauma. A considerable decrease of nNOS staining after Baclofen treatment correlates with improvement of motor dysfunction. The findings also show that parvalbumin and astrocytes participate in the regulation of ion concentrations in the sub-acute phase after the injury.

Effect of subpressor dose of angiotensin II on pain-related behavior in relation with neuronal injury and activation of satellite glial cells in the rat dorsal root ganglia.

To clarify the role of angiotensin II (Ang II) in the regulation of sensory signaling, we studied the effect of subpressor dose (150 ng/kg/min) of Ang II on pain-related behavior in relation with neuronal injury and activation of satellite glial cells (SGCs) in the dorsal root ganglia (DRGs) after chronic constriction injury (CCI). Systemic continuous delivery of Ang II induced the tactile, heat and cold hyperlagesia, when measured at 7 days ofpost-injury. Blockade of the AT1 receptor with losartan (2.5 mg/kg/day) prevented tactile hyperalgesia and attenuated cold hyperalgesia, but did not affect the response to noxious heat stimulus. A marked increase of large-sized injured primary afferent neurons, detected by ATF3 immunolabeling, was seen in lower lumbar DRGs on ipsilateral side after Ang II treatment. Subpressor dose of Ang II induced an increase of activated SGCs (detected by GFAP immunolabeling) enveloping large-diameter neurons. Our results suggested that Ang II through the AT1 receptor activation is an important regulatory factor in neuropathic pain perception and plays an important role in the injury of large-sized primary afferent neurons and activation of SGCs elicited by the CCI.

The vulnerability of nitrergic neurons to transient spinal cord ischemia: a quantitative immunohistochemical and histochemical study.

Spinal cord ischemia belongs to serious and relatively frequent diseases of CNS. The aim of the present study was to find out the vulnerability of nitrergic neurons to 15 min transient spinal cord ischemia followed by 1 and 2 weeks of reperfusion. We studied neuronal nitric oxide synthase (nNOS) and nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) in structural elements of lumbosacral spinal cord along its rostrocaudal axis. In addition, a neurological deficit of experimental animals was evaluated. Spinal cord ischemia, performed on the rabbit, was induced by abdominal aorta occlusion using Fogarty catheter introduced into the right femoral artery for a period of 15 min. After surgical intervention the animals survived for 7 and 14 days. nNOS-immunoreactivity (nNOS-IR) was measured by immunohistochemical and NADPHd-positivity by histochemical method, and both immunohistochemical and histochemical stainings were quantified by densitometric analyses. Neurological deficit was evaluated according Zivin's criteria. The number of nNOS-IR and/or NADPH-d positive neurons and the density of neuropil were markedly increased in superficial dorsal horn (laminae I-III) after 15 min ischemia and 7 days of reperfusion. However, ischemia followed by longer time of survival (14 days) returned the number of nNOS-IR and NADPH-d positive neurons to control. In the pericentral region (lamina X) containing interneurons and crossing fibers of spinal tracts, than in lamina VII and in dorsomedial part of the ventral horn (lamina VIII) we recorded a decreased number of nNOS-IR and NADPH-d positive neurons after both ischemia/reperfusion periods. In the medial portion of lamina VII and dorsomedial part of the ventral horn (lamina VIII) we observed many necrotic loci. This area was the most sensitive to ischemia/reperfusion injury. Fifteen minute ischemia caused a marked deterioration of neurological function of hind limbs, often developing into paraplegia. A quantitative immunohistochemical and histochemical study have shown a strong vulnerability of nitrergic neurons in intermediate zone to transient spinal cord ischemia.

Spinal cord transection modifies neuronal nitric oxide synthase expression in medullar reticular nuclei and in the spinal cord and increases parvalbumin immunopositivity in motoneurons below the site of injury in experimental rabbits.

Using immunohistochemistry, we detected the expression of neuronal nitric oxide synthase (nNOS) in ventral medullary gigantocellular reticular nuclei and in the lumbosacral spinal cord 10 days after thoracic transection in experimental rabbits. We tried to determine whether neurons located below the site of injury are protected by the calcium binding protein parvalbumin (PV). Changes of nNOS immunoreactivity (IR) in spinal cord were correlated with the level of nNOS protein in dorsal and ventral horns. Ten days after transection, nNOS was upregulated predominantly in lateral gigantocellular nuclei. In the spinal cord, we revealed a significant increase of nNOS protein in the dorsal horn. This is consistent with a higher density of punctate and fiber-like immunostaining for nNOS in laminae III-IV and the up-regulation of nNOS-IR in neurons of the deep dorsal horn. After surgery, the perikarya of motoneurons remained nNOS immunonegative. Contrary to nNOS, the PV-IR was upregulated in α-motoneurons and small-sized neurons of the ventral horn. However, its expression was considerably reduced in neurons of the deep dorsal horn. The findings indicate that spinal transection affects nNOS and PV in different neuronal circuits.

The impact of short-lasting repeated vibrations on retrograde axonal transport, the expression of CGRP and parvalbumin in lower lumbar dorsal root ganglia.

A prolonged exposure to vibration stimuli triggers pathological changes with many later manifested symptoms. Early vibration-induced changes are still not very well explored. Therefore, short 30 min vibration period per day with frequency 60 Hz repeated for 10 days was used, and the retrograde axonal transport from the sciatic nerve, the expression of calcitonin gene-related peptide (CGRP) and parvalbumin (PV) were studied in the dorsal root ganglia (DRGs) corresponding to lower lumbar spinal levels. Repeated vibration markedly decreased (25 and 34%) the accumulation of retrogradely transported Fluorogold to spinal motor neurons, whereas a significant increase (35 and 25%) was seen in the DRG primary sensory neurons corresponding to the L4 and L5 spinal level. Immunohistochemical studies showed a significant reduction of CGRP-positive small-sized neuronal cells in both DRGs. Fluoro-Jade labeling revealed that marked loss of CGRP-imunoreactive DRG sensory neurons is not due to neuronal degeneration. CGRP protein expression determined by Western blot analysis and optical density measurement, and NGF level measured by ELISA have been decreased, markedly only at the L4 DRG. PV protein expression was not affected by short repeated vibrations. Our results indicate that (a) short-lasting repeated vibrations affect the retrograde axonal transport in the DRG sensory neurons differently than in spinal motor neurons; (b) a decreased NGF-dependent CGRP production in the DRG primary sensory neurons plays an important role in early vibration-induced pathological mechanisms.