Understanding the roles of estrogen receptors (ERs) in the pathogenesis of traumatic brain injury (TBI) is an important topic of putative value for therapeutic approaches. The present study aimed to investigate the effects of a non-selective ER-α/β antagonist ICI182.780 (ICI) on a rat model of TBI. The TBI model was induced by the weight-drop injury and assessed by the Morris water maze (MWM). ICI was dissolved by DMSO and intracerebroventricularly administered before TBI. Adult male rats were randomly divided into three groups: sham + veh, TBI + veh, and TBI + ICI. The level of central estrogen in the cerebral spinal fluid (CSF) was assessed by ELISA. The expressions of aromatase cytochrome P-450 (AROM) and synaptophysin (SYN) as well as glial fibrillary acidic protein (GFAP) were evaluated by Western blotting and immunofluorescence analyses, respectively. Compared with the sham + veh group, the TBI + veh one showed longer escape latency and less number of platform crossings in the MWM test, an elevated level of endogenous estrogen in the CSF, and increased expressions of AROM and SYN as well as GFAP in the injured hippocampus. The TBI + ICI group displayed longer escape latency and less number of platform crossings in the MWM test and more expressions of GFAP in the hippocampus than that of the TBI + veh one. In conclusion, single-dose intracerebroventricular administration of ICI may increase the cognitive deficits and hippocampal astrocytic activation after TBI, but not for the central aromatization and hippocampal synaptogenesis.

The central medial nucleus (CM), a prominent cell group of the intralaminar nuclei (ILN) of the thalamus, and the ventrolateral periaqueductal gray matter (vlPAG) are two major components of the medial pain system. Whether vlPAG and CM are input sources of nociceptive information to the basolateral amygdala (BLA) and whether they are involved in neuropathic pain regulation remain unclear. Clarifying the hierarchical organization of these subcortical nuclei (vlPAG, CM, and BLA) can enhance our understanding on the neural circuits for pain regulation. Behavioral test results showed that a CM lesion made by kainic acid (KA) injection could effectively alleviate mechanical hyperalgesia 4, 6, and 8 days after spared nerve injury (SNI) surgery, with the symptoms returning after 10 days. Morphological studies revealed that: (1) the CM received afferents from vlPAG and sent efferents to BLA, indicating that an indirect vlPAG-CM-BLA pathway exists; (2) such CM-BLA projections were primarily excitatory glutamatergic neurons as revealed by fluorescencein situhybridization; (3) the fibers originated from the CM-formed close contacts with both excitatory and inhibitory neurons in the BLA; and (4) BLA-projecting CM neurons expressed Fos induced by SNI and formed close contacts with fibers from vlPAG, suggesting that the vlPAG-CM-BLA indirect pathway was activated in neuropathic pain conditions. Finally, the vlPAG-CM-BLA indirect pathway was further confirmed using anterograde and monosynaptic virus tracing investigation. In summary, our present results provide behavioral and morphological evidence that the indirect vlPAG-CM-BLA pathway might be a novel pain pathway involved in neuropathic pain regulation.

BACKGROUND Central sensitization plays a pivotal role in the maintenance of chronic pain induced by chronic pancreatitis (CP). We hypothesized that the nucleus tractus solitarius (NTS), a primary central site that integrates pancreatic afferents apart from the thoracic spinal dorsal horn, plays a key role in the pathogenesis of visceral hypersensitivity in a rat model of CP. AIM To investigate the role of the NTS in the visceral hypersensitivity induced by chronic pancreatitis. METHODS CP was induced by the intraductal injection of trinitrobenzene sulfonic acid (TNBS) in rats. Pancreatic hyperalgesia was assessed by referred somatic pain via von Frey filament assay. Neural activation of the NTS was indicated by immunohistochemical staining for Fos. Basic synaptic transmission within the NTS was assessed by electrophysiological recordings. Expression of vesicular glutamate transporters (VGluTs), N-methyl-D-aspartate receptor subtype 2B (NR2B), and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subtype 1 (GluR1) was analyzed by immunoblotting. Membrane insertion of NR2B and GluR1 was evaluated by electron microscopy. The regulatory role of the NTS in visceral hypersensitivity was detected via pharmacological approach and chemogenetics in CP rats. RESULTS TNBS treatment significantly increased the number of Fos-expressing neurons within the caudal NTS. The excitatory synaptic transmission was substantially potentiated within the caudal NTS in CP rats (frequency: 5.87 ± 1.12 Hz in CP rats vs 2.55 ± 0.44 Hz in sham rats, P < 0.01; amplitude: 19.60 ± 1.39 pA in CP rats vs 14.71 ± 1.07 pA in sham rats; P < 0.01). CP rats showed upregulated expression of VGluT2, and increased phosphorylation and postsynaptic trafficking of NR2B and GluR1 within the caudal NTS. Blocking excitatory synaptic transmission via the AMPAR antagonist CNQX and the NMDAR antagonist AP-5 microinjection reversed visceral hypersensitivity in CP rats (abdominal withdraw threshold: 7.00 ± 1.02 g in CNQX group, 8.00 ± 0.81 g in AP-5 group and 1.10 ± 0.27 g in saline group, P < 0.001). Inhibiting the excitability of NTS neurons via chemogenetics also significantly attenuated pancreatic hyperalgesia (abdominal withdraw threshold: 13.67 ± 2.55 g in Gi group, 2.00 ± 1.37 g in Gq group, and 2.36 ± 0.67 g in mCherry group, P < 0.01). CONCLUSION Our findings suggest that enhanced excitatory transmission within the caudal NTS contributes to pancreatic pain and emphasize the NTS as a pivotal hub for the processing of pancreatic afferents, which provide novel insights into the central sensitization of painful CP.

Previous studies have demonstrated that both dorsal raphe nucleus (DR) and insular cortex (IC) are critical for somatic sensory information transmission and regulation, especially for pain, and neurons in the DR send projection fibers to the IC. However, whether these ascending connections are involved in the processing of itch sensation remains unknown. In order to provide evidence for that, fluoro-gold (FG) retrograde tracing combined with immunofluorescent histochemical staining was performed for revealing the chemical nature of the projection neurons and the FOS expression induced by acute itch stimulation via intradermal histamine or chloroquine injection in the mouse. Both FOS- and p-ERK-containing neurons were increased in the DR and IC in the acute itch mice compared to those in the sham group. After FG was injected into the IC, FG-labeled retrograde neuronal cell bodies were observed in the whole extent of the brainstem, especially in the DR. About 81% of the total number of FG-labeled neurons in the DR showed serotonin (5-HT)-immunopositive staining. About 32% FG-labeled 5-HT-ergic neurons within DR expressed FOS in chroloquine-induced acute itch, whereas only 6% FG-labeled 5-HT-ergic neurons within DR expressed FOS in histamine-induced acute itch. These results provide morphological evidence for that there are 5-HT-ergic projections from the DR to IC which might be involved in the sensory information processing of acute itch. These results are helpful for understanding functional roles of 5-HT-ergic ascending projection under the condition of acute itch.

Medullary dorsal horn (MDH), the homolog of spinal dorsal horn, plays essential roles in processing of nociceptive signals from orofacial region toward higher centers, such as the ventral posteromedial thalamic nucleus (VPM) and parafascicular thalamic nucleus (Pf), which belong to the sensory-discriminative and affective aspects of pain transmission systems at the thalamic level, respectively. In the present study, in order to provide morphological evidence for whether neurons in the MDH send collateral projections to the VPM and Pf, a retrograde double tracing method combined with immunofluorescence staining for substance P (SP), SP receptor (SPR) and Fos protein was used. Fluoro-gold (FG) was injected into the VPM and the tetramethylrhodamine-dextran (TMR) was injected into the Pf. The result revealed that both FG- and TMR-labeled projection neurons were observed throughout the entire extent of the MDH, while the FG/TMR double-labeled neurons were mainly located in laminae I and III. It was also found that some of the FG/TMR double-labeled neurons within lamina I expressed SPR and were in close contact with SP-immunoreactive (SP-ir) terminals. After formalin injection into the orofacial region, 41.4% and 34.3% of the FG/TMR double-labeled neurons expressed Fos protein in laminae I and III, respectively. The present results provided morphological evidence for that some SPR-expressing neurons within the MDH send collateral projections to both VPM and Pf and might be involved in sensory-discriminative and affective aspects of acute orofacial nociceptive information transmission.