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

Abstract

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.

Keywords: Denervation; Inflammation; Neurogenic inflammation; Neuropeptides; Periodontitis.

Figure 1

A representative photomicrographs of the trigeminal ganglia showing signal of the in situ hybridization staining and the negative control to validate the technique: A) Silver grains (black dots) are seen overlaying the neurons expressing mRNAs. B) Negative sections in which the probe was omitted showing a complete absence of the silver grains. Scale bars: A, B=50 μ. C-E) A representative photomicrograph demonstrating image J analysis of in situ hybridisation signals. A) Original image. Silver grains are seen as black dots overlaying the neurons which contain the mRNA. B) Converted image, in situ hybridisation signals, were converted into a distinct colour (red) that the software can score. C) Showing the scored neurons (analysed neurons=outlined structures) with exclusion of the background. The analysis provides information about the number of neurons analysed and the levels of the mRNAs per neuron.

Figure 2

A representative photographs showing substance P mRNA Expression in the mandibular division of the trigeminal ganglia: A) Control trigeminal ganglia showing substance P mRNA expression in neurons only. The positive neuron shows silver grain overlaying the cells (black arrows). B) A higher magnification showing, a variable degree of expression ranging from weak (red arrows) to mild (black arrow). C) Trigeminal ganglia from animals with denervated mandible showing a marked reduction in substance P mRNA in the neurons. Most of the neurons show weak expression. D) Higher magnification showing discrete silver grain overlaying the positive neurons, indicating the low expression (red arrows). E) Trigeminal ganglia from animal with inflamed mandible showing apparent increase in substance P mRNA expression, there is increase in the number of the positive neurons and increase in the expression per neuron. F) A higher magnification showing the coalescence of the silver grains masking the nuclei indicating the marked increase in substance P expression per neuron. Scale bars: A, D, F=50 μ; B=20 μ, C=100 μ, E=400 μ.

Figure 3

A representative photographs showing CGRP mRNA in the mandibular division of the trigeminal ganglia: A, B) Control trigeminal ganglia, showing CGRP expression in neurons only (arrows). There is wider distribution than substance P as seen in the increase in number of positive neurons. C, D) Trigeminal ganglia from animals with Denervated mandible showing marked reduction in the substance P mRNA in the neurons (arrows). D) Higher magnification showing discrete silver grain distribution overlaying the positive neurons indicating the low expression, the expression was almost limited to the sub membranal cytoplasmic compartment with depletion from the perinuclear cytoplasmic compartment (arrows). E) Trigeminal ganglia from animal with inflamed mandible showing marked increase in CGRP mRNA expression, there is an increase in the number of the positive neurons. F) Higher magnification showing the coalescence of the silver grains masking the nuclei (arrows) indicating the marked increase in CGRP expression per neuron. Scale bars: A=200 μ, B=50 μ, C, D=100 μ, E=300 μ, F=200 μ.

Figure 4

A,B Graphs showing mRNA levels of substance P and CGRP mRNAs in the three groups: Denervated group shows a significant (substance P. P<0.04; CGRP, P<0.01) downregulation by comparison to the control sham group. Contrarily, inflammation group shows a significant (P<0.001) upregulation by comparison to the vehicle control group. Interestingly, denervation of the mandible prior to induction of inflammation abolished the stimulatory effect of LPS on neuropeptides mRNAs. C) A graph showing a strong correlation between substance P and CGRP mRNAs expression (Pearson Correlation=0.8). D) A table summarizing the correlation data between substance P and CGRP.