In Situ Hybridisation Study of Neuronal Neuropeptides Expression in Models of Mandibular Denervation with or without Inflammation: Injury Dependant Neuropeptide Plasticity.

Neuronal expression of neuropeptides is altered following peripheral tissue injury associated with inflammation or nerve injury. This results in neuropathic pain with or without neurogenic inflammation which is a major health problem regularly seen in trigeminal neuralgia. Activation of the trigeminal system results in the release of vasoactive neuropeptides substance P and Calcitonin Gene-related Peptide (CGRP) which contribute to nociception, pain and neurogenic inflammation in injured tissues. AIM: To study the alterations in the neuronal neuropeptides expressions in models of tissue injury associated with either nerve injury or with inflammation and to determine if denervation would alter the neuronal response to inflammation. MATERIAL AND METHODS: Experiments were performed on rat mandibles to produce three models. Firstly, denervation model by sectioning one of the mandibular nerve branches (inferior alveolar nerve). Secondarily, inflammation model by intra-gingival injection of lipopolysaccharide (LPS). Thirdly, combined denervation and inflammation model by sectioning the nerve with subsequent LPS injection. The animals were sacrificed seven days postoperative. Trigeminal ganglia on the operated sides were processed for in situ hybridisation for neuropeptides; substance P and CGRP mRNAs. Images were analysed for morphological and morphometric analysis using Image J software. RESULTS: substance P and CGRP mRNAs were expressed in small and medium-size primary afferent neurons in the mandibular division of the trigeminal ganglia. Both the denervation and the inflammation models showed alteration in neuropeptides expression in the sensory primary afferent neurons innervating the affected mandibular tissues. While, denervation resulted in a significant (substance P=P<0.04, CGRP=P<0.01) downregulation contrarily, inflammation resulted in a significant (P<0.001) upregulation of neuropeptides' mRNAs. Interestingly, denervation prior to induction of inflammation resulted in insignificant changes in neuropeptides levels. There was a strong correlation (Pearson Correlation=0.8) between substance P and CGRP expression. CONCLUSION: We show that tissue damage associated with nerve injury or inflammation results in alteration of neuropeptides levels in the innervating primary afferent neurons. Tissue destruction associated with chronic inflammatory condition such as arthritis and periodontitis are believed to be due to the production of neuromodulators causing neurogenic inflammation. Here we show that denervation abolishes the neuronal response to inflammation. Therefore, tissues denervation could relieve neurogenic inflammation associated with chronic disorders through regulation of neuronal neuropeptide production. Moreover, the current model that combined denervation and inflammation provides a useful animal model to study the contribution of nerve-related mediators in the pathophysiology of tissue injury.

Inflammation alters somatostatin mRNA expression in sensory neurons in the rat.

Proinflammatory neuropeptides, such as substance P and calcitonin gene-related peptide, are up-regulated in primary afferent neurons in acute and chronic inflammation. While these neuropeptides have been intensively studied, potentially anti-inflammatory and/or anti-nociceptive neuropeptides such as somatostatin (SS) have been less widely investigated. Endogenous somatostatin is thought to exert a tonic antinociceptive effect. Exogenous SS is anti-inflammatory and antinociceptive and is thought to exert these actions through inhibition of proinflammatory neuropeptide release. In this study we have compared the expression of somatostatin in two inflammatory models: arthritis, a condition associated with increased nociception, and periodontitis, in which there is little evidence of altered nociceptive thresholds. In acute arthritis (< 24 h) SS mRNA was down-regulated in ipsilateral dorsal root ganglia (DRG; 52 +/- 7% of control, P < 0.05), and up-regulated in contralateral DRG (134 +/- 10% of control; P < 0.05). In chronic arthritis (14 days) this pattern of mRNA regulation was reversed, with SS being up-regulated ipsilaterally and down-regulated contralaterally. In chronic mandibular periodontitis (7-10 days), SS mRNA was up-regulated in only the mandibular division of the ipsilateral trigeminal ganglion (TG) (day 7, 219 +/- 9% and day 10, 217 +/- 12% of control; P < 0.02) but showed no change in other divisions of the trigeminal ganglion or in the mesencephalic nucleus. These data show that antinociceptive and anti-inflammatory neuropeptides are also regulated in inflammation. It is possible that the degree of inflammation and nociception seen may depend on the balance of pro- and anti-inflammatory and nociceptive peptide expression in a particular condition.

Inflammation alters cation chloride cotransporter expression in sensory neurons.

Cation chloride cotransporters have been proposed to play a role in the modulation of neuronal responses to gamma-aminobutyric acid (GABA). In conditions of neuronal damage, where neuronal excitability is increased, the expression of the KCC2 transporter is decreased. This is also seen in spinal cord in models of neuropathic pain. We have investigated the expression of the Na-K-Cl, and K-Cl cotransporters NKCC1 and KCC2, in dorsal root ganglion (DRG) and spinal sensory neurons during arthritis, a condition in which neuronal excitability is also increased. NKCC1 was expressed in control DRG neurons, and its expression was decreased in arthritis. Both NKCC1 and KCC2 were expressed in sensory neurons in the spinal cord. In acute arthritis, both NKCC1 and KCC2 mRNA increased in superficial but not deep dorsal horn, and this was accompanied by an increase in protein expression. In chronic arthritis, NKCC1 expression remained raised, but KCC2 mRNA and protein expression returned to control levels. Altered KCC2 and NKCC1 expression in arthritis may contribute to the control of spinal cord excitability and may represent novel therapeutic targets in the treatment of inflammatory pain.

Sensory neuropeptide mRNA up-regulation is bilateral in periodontitis in the rat: a possible neurogenic component to symmetrical periodontal disease.

Periodontal disease is a common multifactorial chronic inflammatory disease in humans. In inflammatory conditions that are known to be associated with changes in nociception, such as arthritis, the neuronal expression of the proinflammatory neuropeptides, substance P and calcitonin gene-related peptide is altered. In this study the expression of these neuropeptides' mRNAs has been studied in an inflammatory model that shows no behavioural evidence of altered nociception. Periodontitis was induced in male rats by intragingival injection of lipopolysaccharide adjacent to the second right mandibular molar. The animals were killed at various times after lipopolysaccharide injection and right and left trigeminal ganglia and brain were processed for in situ hybridization for beta-preprotachykinin and alpha-calcitonin gene-related peptide mRNAs. Expression of both neuropeptide mRNAs was significantly increased only in small neurons in the mandibular division of the trigeminal ganglion ipsilateral to the LPS injection from 3 to 10 days postinjection. Neuropeptide mRNA expression was also significantly increased in the contralateral trigeminal ganglion at day 10. No significant changes in neuropeptide mRNA levels were seen in the maxillary and ophthalmic divisions of the trigeminal ganglia or in the trigeminal mesencephalic nucleus. The up-regulation of substance P and CGRP mRNAs in periodontal disease suggests that this is associated with the inflammatory process rather than nociception, as this disease does not appear to result in altered nociception in either rats or humans. The contralateral alteration in neuropeptide mRNA expression suggests a role for neurogenic mechanisms in the development of periodontal disease.