A cyclic peptide targeted against PSD-95 blocks central sensitization and attenuates thermal hyperalgesia.

Post-synaptic density protein PSD-95 is emerging as a valid target for modulating nociception in animal studies. Based on the key role of PSD-95 in neuronal plasticity and the maintenance of pain behavior, we predicted that CN2097, a peptide-based macrocycle of nine residues that binds to the PSD-95 Discs large, Zona occludens 1 (PDZ) domains of PSD-95, would interfere with physiologic phenomena in the spinal cord related to central sensitization. Furthermore, we tested whether spinal intrathecal injection of CN2097 attenuates thermal hyperalgesia in a rat model of sciatic neuropathy. Results demonstrate that spinal CN2097 reverses hyperexcitability of wide dynamic range (WDR) neurons in the dorsal horn of neuropathic rats and decreases their evoked responses to peripheral stimuli (brush, low caliber von Frey and pressure), whereas CN5125 ("negative control") has no effect. CN2097 also blocks C-fiber long-term potentiation (LTP) in the dorsal horn, which is linked to neuronal plasticity and central sensitization. At a molecular level, CN2097 attenuates the increase in phosphorylated p38 MAPK, a key intracellular signaling pathway in neuropathic pain. Moreover, spinal injection of CN2097 blocks thermal hyperalgesia in neuropathic rats. We conclude that CN2097 is a small molecule peptide with putative anti-nociceptive effects that modulates physiologic phenomena related to central sensitization under conditions of chronic pain.

Activated polymorphonuclear cells promote injury and excitability of dorsal root ganglia neurons.

Therapies aimed at depleting or blocking the migration of polymorphonuclear leukocytes (PMN or neutrophils) are partially successful in the treatment of neuroinflammatory conditions and in attenuating pain following peripheral nerve injury or subcutaneous inflammation. However, the functional effects of PMN on peripheral sensory neurons such as dorsal root ganglia (DRG) neurons are largely unknown. We hypothesized that PMN are detrimental to neuronal viability in culture and increase neuronal activity and excitability. We demonstrate that isolated peripheral PMN are initially in a relatively resting state but undergo internal oxidative burst and activation by an unknown mechanism within 10 min of co-culture with dissociated DRG cells. Co-culture for 24 h decreases neuronal count at a threshold<0.4:1 PMN:DRG cell ratio and increases the number of injured and apoptotic neurons. Within 3 min of PMN addition, fluorometric calcium imaging reveals intracellular calcium transients in small size (<25 microm diam) and large size (>25 microm diam) neurons, as well as in capsaicin-sensitive neurons. Furthermore, small size isolectin B4-labeled neurons undergo hyperexcitability manifested as decreased current threshold and increased firing frequency. Although co-culture of PMN and DRG cells does not perfectly model neuroinflammatory conditions in vivo, these findings suggest that activated PMN can potentially aggravate neuronal injury and cause functional changes to peripheral sensory neurons. Distinguishing the beneficial from the detrimental effects of PMN on neurons may aid in the development of more effective drug therapies for neurological disorders involving neuroinflammation, including painful neuropathies.

Neutrophils invade lumbar dorsal root ganglia after chronic constriction injury of the sciatic nerve.

To test whether neutrophils (PMN) target lumbar dorsal root ganglia (DRG) following axonal injury leading to neuropathic pain, we visualized PMN infiltration in DRG tissue sections and estimated PMN count by flow cytometry following sciatic chronic constriction injury (CCI). Seven days after CCI, results show PMN within DRG where their count increased by three fold ipsilateral to injury compared to contralateral or sham, concomitant with peak neuropathic pain behavior. Superoxide burst in PMN isolated from rats d7 after CCI was elevated by 170% +/-18 compared to naïve and MCP-1 mRNA expression in DRG increased by 8.9+/-2.9 fold, but that of MIP-2, CINC-1, and RANTES did not change. We conclude that CCI causes PMN invasion of the DRG whereby the functional implication of their close proximity to neuronal axon and soma remains unknown.

Cerebellar stimulation modulates the intensity of a visceral nociceptive reflex in the rat.

The cerebellum modulates different nociceptive phenomena and influences visceral functions. This study shows cerebellar modulation of an abdominal reflex elicited by a visceral noxious stimulus (colorectal distension, CRD). The intensity of the reflex was measured by electromyographic (EMG) recording from the rectus abdominus muscle, and the cerebellar cortex (vermis, lobule VIII), the fastigial nucleus, or the dentate nucleus was stimulated using D, L-homocysteic acid (0.1 M, 1 micro l). To release the fastigial nucleus from inhibition by the Purkinje cells, bicuculline (GABA(A) receptor antagonist, 100 micro M, 1 micro l) was used. Stimulation of the cerebellar cortex enhanced, whereas stimulation or disinhibition of the fastigial nucleus decreased, the responses to CRD measured by EMG. Stimulation of the dentate nucleus did not have an obvious effect on the intensity of the reflex. These results are in agreement with the hypothesis that the cerebellum modulates visceral nociceptive functions, whereby the cerebellar cortex and the fastigial nucleus, respectively, play a pro-nociceptive and an anti-nociceptive role.

Stimulation in the rat fastigial nucleus enhances the responses of neurons in the dorsal column nuclei to innocuous stimuli.

The cerebellum was recently proposed to play a role in cognition and sensation in addition to motor phenomena. We have shown that the cerebellum is involved in the processing of sensory nociceptive information. In this study, the activity of neurons in the dorsal column nuclei (DCN) was tested following stimulation in the rat fastigial nucleus. The results showed an enhancement of the extracellularly recorded responses of DCN neurons to somatic non-noxious stimuli following injection of D,L-homocysteic acid (0.1 M, 1 microl) into the area of the fastigial nucleus. We conclude that the cerebellum influences the processing of non-noxious somatosensory information at the level of the DCN, an important relay and a center for the processing of fine tactile and vibratory information. This observation is not yet supported by clinical data.

Nociceptive visceral stimulation modulates the activity of cerebellar Purkinje cells.

The cerebellum is a system with various input and output functions that influence motor, sensory, cognitive, and other processes. In a previous study, we showed that cerebellar cortical stimulation increases spinal neuronal responses to visceral noxious stimulation by colorectal distension (CRD). However, the neuronal network underlying the cerebellar modulation of nociceptive phenomena is largely unknown. Purkinje cells of the cerebellar cortex receive ascending and descending inputs and exert a major inhibitory control over neurons in the underlying cerebellar nuclei that constitute the cerebellar output. Therefore, in this study, we tested the effect of CRD and other somatic stimuli on the firing rate of Purkinje cells using in vivo extracellular recording techniques. The results suggest that Purkinje cells respond to nociceptive visceral and somatic stimulation in the form of early and delayed changes in activity. Based on these and previous findings, we propose a negative feedback circuitry involving the cerebellum for the modulation of peripheral nociceptive events.

Cerebellar cortical stimulation increases spinal visceral nociceptive responses.

The role of the cerebellum in modulating nociceptive phenomena is unclear. In this study, we focus on the effects of cerebellar cortical stimulation on the responses of midline neurons of the lumbosacral spinal cord to graded nonnoxious and noxious visceral (colorectal distension) as well as somatic (brush, pressure, pinch) stimuli. Extracellular recording was used for the isolation and recording of spinal nociceptive neurons, while electrical current pulses and chemical injection of D, L-homocysteic acid were used to stimulate the cortex of the posterior cerebellar vermis. Cerebellar cortical stimulation increased the responses of all isolated cells to colorectal distension, whereas the effect on the responses to somatic stimuli was variable. These findings indicate that the posterior cerebellar vermis may exert a pro-nociceptive effect on spinal visceroceptive neurons.