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Inhibitory interneurons are an important component of dorsal horn circuitry where they serve to modulate spinal nociception. There is now considerable evidence indicating that reduced inhibition in the spinal dorsal horn contributes to neuropathic pain. A loss of these inhibitory neurons after nerve injury is one of the mechanisms being proposed to account for reduced inhibition; however, this remains controversial. This is in part because previous studies have focused on global measurements of inhibitory neurons without assessing the number of inhibitory synapses. To address this, we conducted a quantitative analysis of the spatial and temporal changes in the number of inhibitory terminals, as detected by glutamic acid decarboxylase 65 (GAD65) immunoreactivity, in the superficial dorsal horn of the spinal cord following a chronic constriction injury (CCI) to the sciatic nerve in rats. Isolectin B4 (IB4) labelling was used to define the location within the dorsal horn directly affected by the injury to the peripheral nerve. The density of GAD65 inhibitory terminals was reduced in lamina I (LI) and lamina II (LII) of the spinal cord after injury. The loss of GAD65 terminals was greatest in LII with the highest drop occurring around 3–4 weeks and a partial recovery by 56 days. The time course of changes in the number of GAD65 terminals correlated well with both the loss of IB4 labeling and with the altered thresholds to mechanical and thermal stimuli. Our detailed analysis of GAD65+ inhibitory terminals clearly revealed that nerve injury induced a transient loss of GAD65 immunoreactive terminals and suggests a potential involvement for these alterations in the development and amelioration of pain behaviour.

Transgenic mice ectopically expressing nerve growth factor in oligodendrocytes have high levels of nerve growth factor immunoreactivity in the white matter of the spinal cord from birth until 2 months of age. The nerve growth factor over-expression leads to the appearance of ectopic substance P containing sensory fibers in the white matter of the spinal cord that persist throughout the life of the animal. These transgenic mice have been found to display hypersensitivity to a thermal stimulus following a sensitizing pinch stimulus known to release endogenous substance P. Surprisingly, this hypersensitivity is completely reversed following the administration of morphine, to the extent that transgenic mice become less sensitive to pain than the wild type mice given morphine. Endomorphin-2, an endogenous opioid peptide, has been found co-localized with substance P in primary sensory fibers in the spinal cord. In this study, we show that the ectopic fibers also express endomorphin-2, and describe the postnatal development of such expression, as detected by immunocytochemistry. We confirmed that endomorphin-2 expression starts later in the postnatal period than substance P. Surprisingly, transgenic animals had delayed appearance of endomorphin-2 in the superficial dorsal horn, compared with wild type, and expressed particularly high levels of endomorphin-2 immunoreactivity in the ectopic fibers from postnatal days 10-30, coinciding with the peak of nerve growth factor expression in oligodendrocytes. Endomorphin-2 immunoreactivity was still readily detected in ectopic fibers of 120-day-old animals. Furthermore, we detected immunoreactivity for the mu-opioid receptor in the ectopic fibers, where it was co-localized with endomorphin-2 immunoreactivity. In the superficial dorsal horn, there were no apparent differences in the distribution and intensity of mu-opioid receptor immunoreactivity between wild type and transgenic animals. Taken together, these data could provide an explanation for the enhanced effect of opioid analgesics in transgenic mice, when compared with control mice, as well as provide the basis for studies of the postnatal development of the hyperalgesia and allodynia demonstrated by these animals.

Provoked vestibulodynia, the most common form of vulvodynia (unexplained pain of the vulva), is a prevalent, idiopathic pain disorder associated with a history of recurrent candidiasis (yeast infections). It is characterized by vulvar allodynia (painful hypersensitivity to touch) and hyperinnervation. We tested whether repeated, localized exposure of the vulva to a common fungal pathogen can lead to the development of chronic pain. A subset of female mice subjected to recurrent Candida albicans infection developed mechanical allodynia localized to the vulva. The mice with allodynia also exhibited hyperinnervation with peptidergic nociceptor and sympathetic fibers (as indicated by increased protein gene product 9.5, calcitonin gene-related peptide, and vesicular monoamine transporter 2 immunoreactivity in the vaginal epithelium). Long-lasting behavioral allodynia in a subset of mice was also observed after a single, extended Candida infection, as well as after repeated vulvar (but not hind paw) inflammation induced with zymosan, a mixture of fungal antigens. The hypersensitivity and hyperinnervation were both present at least 3 weeks after the resolution of infection and inflammation. Our data show that infection can cause persistent pain long after its resolution and that recurrent yeast infection replicates important features of human provoked vulvodynia in the mouse.

It is a clinical reality that women make up the large majority of chronic pain patients, and there is now consensus from laboratory experiments that when differences are seen, women are more sensitive to pain than men. Research in this field has now begun to concentrate on finding explanations for this sex difference. Although sex differences in sociocultural, psychological, and experiential factors likely play important roles, evidence largely from animal studies has revealed surprisingly robust and often qualitative sex differences at low levels of the neuraxis. Although not yet able to affect clinical practice, the continued study of sex differences in pain may have important implications for the development of new analgesic strategies.

Facial expression is widely used as a measure of pain in infants; whether nonhuman animals display such pain expressions has never been systematically assessed. We developed the mouse grimace scale (MGS), a standardized behavioral coding system with high accuracy and reliability; assays involving noxious stimuli of moderate duration are accompanied by facial expressions of pain. This measure of spontaneously emitted pain may provide insight into the subjective pain experience of mice.

It is a clinical reality that women make up the large majority of chronic pain patients, and there is now consensus from laboratory experiments that when differences are seen, women are more sensitive to pain than men. Research in this field has now begun to concentrate on finding explanations for this sex difference. Although sex differences in sociocultural, psychological, and experiential factors likely play important roles, evidence largely from animal studies has revealed surprisingly robust and often qualitative sex differences at low levels of the neuraxis. Although not yet able to affect clinical practice, the continued study of sex differences in pain may have important implications for the development of new analgesic strategies.

It is well known that following peripheral nerve injury, there are numerous changes in neurotransmitter and neuropeptide expression in the superficial dorsal horn, the dorsal root ganglion and the periphery. Of particular interest are the relative contributions of two sub-types of unmyelinated C-fibers in the initiation and maintenance of chronic pain, the peptidergic, and the non-peptidergic. Evidence gathered in recent years has led researchers to believe that the non-peptidergic nociceptive primary afferents are functionally distinct from their peptidergic counterpart. For our study, we used a well-established animal model of constriction neuropathy (the Kruger model) and studied Wistar rats at 5, 7, 10, 15 and 21 days after nerve lesion caused by the application of a fixed-diameter polyethylene cuff to the left sciatic nerve. Animals were assessed for the onset and evolution of mechanical allodynia using calibrated von Frey filaments and were additionally tested for thermal (heat and cold) hypersensitivity. Immunocytochemical detection of calcitonin gene-related peptide (CGRP) and isolectin B4 (IB4) binding was used to visualize the dorsal horn distribution of the boutons from the peptidergic and non-peptidergic fibers respectively. Using confocal microscopy and image analysis, we detected a significant decrease in the density of IB4-labeled boutons, ipsilateral to the lesion, at seven and 10 days following nerve injury. The density of IB4-labeled varicosities retuned to control levels by 15 days. There were no significant changes in the density of CGRP-labeled varicosities at all time points examined. Applying electron microscopy, we initially detected degenerative changes in the central elements of type I glomeruli and then a considerable reduction in their number followed by recovery at 15 days post-lesion. As the central boutons of type Ia represent varicosities from the fibers which bind IB4, the ultrastructural changes confirmed that there was a bona fide transient loss of varicosities, not simply a loss of IB4 binding. These data indicate that, in this animal model, morphological changes in the nociceptive C-fiber input of the rat dorsal horn are restricted to the non-peptidergic sub-population and are transient in nature. Furthermore, such changes do not correlate with the time-course of the allodynia.