Bulbospinal noradrenergic fibers contain and release NA, ATP, and neuropeptide Y, factors which regulate neuronal and glial activation state and responses. The key finding of the current study is that presence of these fibers is not necessary for NA plasticity in analgesia after nerve injury and a suggestion that they may be important in braking glial activation. Spinal NA regulation of nociception has been studied for 3 decades, but many investigators consider it of minor importance in the pathophysiology of neuropathic pain. This conclusion is derived from the moderate effect of destruction of these noradrenergic pathways on hypersensitivity after nerve injury, 22
(as reproduced in the current study) and from the lack of effect of intrathecally injected α2-adrenoceptor antagonists on this hypersensitivity. 14, 24
These observations are, however, weakened by the inability to see exacerbated hypersensitivity in an already hypersensitive state after NA destruction and by the assumption that NA release in the spinal cord produces an effect which can be immediately reversed with an antagonist. If NA release, as we postulate, alters glial and neuronal plasticity which require hours or days to achieve, one would not necessarily anticipate an acute inhibition of this effect within minutes of administration of a NA antagonist.
In the current study, intrathecal injection of DβH-saporin completely depleted noradrenergic axons in the spinal cord. We also observed partial reduction of noradrenergic neurons and axons in the noradrenergic nuclei (LC and A5) and paraventricular nucleus by this DβH-saporin treatment, consistent with previous observations 22, 25
. These results indicate that intrathecal injection of DβH-saporin not only depletes spinal noradrenaline but also reduces supra-spinal noradrenergic innervation to some extent. Clearly, extensive supraspinal noradrenergic denervation has profound effects on various physiologic states, and such extensive denervation or silencing, by intracerebroventricular injection of DβH-saporin or intra-LC injection of lidocaine, reduces hypersensitivity after nerve injury 2
. In contrast, more selective ablation of noradrenergic innervation to the spinal cord by intrathecal injection of DβH-saporin as in the current and previous studies 22
, results in a small increase rather than decrease in hypersensitivity in rats after spinal nerve ligation. This protective effect at a time of extreme hypersensitivity after nerve injury is consistent with laboratory and clinical observations that activation of LC or spinal noradrenergic pathway reduces hypersensitivity in rats and humans with chronic pain 8, 9, 12–14, 29, 30
. Nonetheless, a clear limitation of this study is the partial reduction in supraspinal NA innervation induced by intrathecal DβH-saporin injected, which could have affected behavior and descending influences on glial and neuronal plasticity.
In rodents, activation of microglia and astrocytes in the spinal cord contributes to the development and maintenance of hypersensitivity after peripheral nerve injury 38
. Consistent with behavioral observations, depletion of spinal NA in the current study did not affect immunohistochemical markers of activation of microglia and astrocytes in the normal spinal cord 22
, but increased expression of these markers after nerve injury. We recognize that static examination of IBA-1 and GFAP do not adequately characterize activation state of glia or their anti- or pro-nociceptive or –inflammatory effects. Nonetheless, the approach used in the current study is widely applied to provide a rough gauge of glial activation. Previous studies demonstrated that α2-adrenoceptor agonists inhibit activation of microglia and astrocytes in the spinal cord in rats after peripheral nerve injury or inflammation 10, 41
. We have recently demonstrated that peripheral nerve injury enhances spinal noradrenergic inhibition by increasing content and basal release of noradrenaline in the spinal dorsal horn 14, 16
. These results suggest that peripheral nerve injury enhances endogenous spinal noradrenergic tone which, over time, directly or indirectly modulates the degree of glial activation and hypersensitivity, although this degree of endogenous noradrenergic tone itself is not sufficient to relieve neuropathic pain. The current study cannot determine whether increased glial activation in noradrenaline-depleted animals reflects reduced inhibition of primary afferent release or a direct effect on glia.
The most likely sources of BDNF in the spinal cord after peripheral nerve injury include the terminals of primary afferents and microglia. 4, 11, 16, 34
In normal and injured spinal cord, astrocytes can also produce BDNF 6, 7, 19
. We and others have shown that peripheral nerve injury increases BDNF content in the primary sensory neurons and the spinal dorsal horn 11, 16, 26, 34
. Previous studies demonstrated that depletion of noradrenaline itself does not alter BDNF content or expression in the brain 18, 40
, consistent with the lack of effect of noradrenaline depletion on glial activity in the brain in the absence of nerve injury 22
. Although the current study did not test whether NA depletion alters BDNF content in the spinal dorsal horn in the absence of injury, these previous observations in the brain suggest that NA depletion itself is unlikely to affect spinal BDNF content. In the current study, DβH-saporin treated SNL animals showed higher BDNF content in the spinal dorsal horn compared to saporin treated SNL animals, consistent with enhanced glial activation in DβH-saporin treated spinal dorsal horn. Although the contribution of primary sensory afferents to the increase in BDNF content in the spinal dorsal horn after nerve injury is possible, these results suggest that depletion of endogenous spinal noradrenaline induces more BDNF production in the spinal dorsal horn likely via enhancement of glial activation after nerve injury.
BDNF plays important roles in survival and axonal growth in cholinergic neurons and also shifts action of α2-adrenoceptors with those neurons from Gi/o-protein mediated inhibition to Gs-protein-mediated facilitation of acetylcholine release 15
. Previous studies demonstrated that spinal infusion of BDNF or over-expression of BDNF by gene-transfer increases survival and axonal growth of cholinergic neurons in rats after spinal cord injury20, 42
, and that intracerebroventricular infusion of BDNF prevents loss of cholinergic neurons in the brainstem after hypoglossal nerve transection 36
. We have recently demonstrated that blockade of BDNF-trkB signaling by spinal infusion of BDNF antibody or repeated intrathecal injection of K252a reduced ChAT-immunoreactivity in the spinal dorsal horn and blocked the shift in α2-adrenoceptor-mediated facilitatory effect on acetylcholine release in the spinal cord 15, 17
. The current study confirmed the α2-adrenoceptor-mediated facilitation of acetylcholine release after nerve injury and further demonstrated that DβH-saporin treatment increased cholinergic immunoreactivity in the spinal dorsal horn, consistent with enhancement of BDNF up-regulation after nerve injury by DβH-saporin treatment. Interestingly, in the current study, spinal BDNF level in DβH-saporin treated animals was higher than the saporin treated and normal animals at 3–7 days and returned to normal level at 14 days after surgery, suggesting that initial enhancement of BDNF production is important to increase cholinergic immunoreactivity in the spinal dorsal horn. These results should, however, be qualified, since the methodology employed did not allow us to determine whether increased ChAT-immunoreactivity was associated with increased quantitative release of acetylcholine release, as only fractional release was studied. As such, other interpretations of increased ChAT-immunoreactivity, such as increased antigen availability to the antibody without change in fiber density, are possible.
It may appear paradoxical that clonidine’s behavioral effect was enhanced after nerve injury in animals receiving DβH-saporin, consistent with denervation supersensitivity or increased receptor expression, but α2-adrenoceptor facilitated release of acetylcholine in synaptosomes was not increased. Previous studies showed that depletion of spinal noradrenaline increases α2-adrenoceptor number in the spinal cord or affinity to clonidine in normal animals 21, 32
, while peripheral nerve injury itself does not alter neither number nor affinity of α2-adrenoceptors 1
. Although the current study does not address whether expression or affinity of α2-adrenoceptors alters in cholinergic neurons by DβH-saporin treatment, this plasticity in α2-adrenoceptors by depletion of spinal noradrenaline may contribute to enhanced antihypersensitive effect of clonidine after nerve injury. As regards acetylcholine release, if one assumes that the increased ChAT immunostaining reflects increased capacity for acetylcholine release, then the same fractional release of acetylcholine release observed with dexmedetomidine in synaptosomal preparations after DβH-saporin as control would quantitatively release more acetylcholine. Neither the relationship between ChAT immunostaining and acetylcholine content nor the quantitative release of acetylcholine were examined in the current study.
In summary, depletion of spinal noradrenergic fibers does not prevent α2-adrenoceptor analgesia and associated plasticity of its interaction with cholinergic terminals, suggesting that tonic activity of these fibers does not drive these plastic responses. NA fiber depletion is associated with increased expression of a markers of glial activation, consistent with acute pharmacologic studies demonstrated glial inhibition by NA receptor activation.