Neuroinflammation in the anterior cingulate cortex: the potential supraspinal mechanism underlying the mirror-image pain following motor fiber injury.

BACKGROUND: Peripheral nerve inflammation or lesion can affect contralateral healthy structures, and thus result in mirror-image pain. Supraspinal structures play important roles in the occurrence of mirror pain. The anterior cingulate cortex (ACC) is a first-order cortical region that responds to painful stimuli. In the present study, we systematically investigate and compare the neuroimmune changes in the bilateral ACC region using unilateral- (spared nerve injury, SNI) and mirror-(L5 ventral root transection, L5-VRT) pain models, aiming to explore the potential supraspinal neuroimmune mechanism underlying the mirror-image pain. METHODS: The up-and-down method with von Frey hairs was used to measure the mechanical allodynia. Viral injections for the designer receptors exclusively activated by designer drugs (DREADD) were used to modulate ACC glutamatergic neurons. Immunohistochemistry, immunofluorescence, western blotting, protein microarray were used to detect the regulation of inflammatory signaling. RESULTS: Increased expressions of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6) and chemokine CX3CL1 in ACC induced by unilateral nerve injury were observed on the contralateral side in the SNI group but on the bilateral side in the L5-VRT group, representing a stronger immune response to L5-VRT surgery. In remote ACC, both SNI and L5-VRT induced robust bilateral increase in the protein level of Nav1.6 (SCN8A), a major voltage-gated sodium channel (VGSC) that regulates neuronal activity in the mammalian nervous system. However, the L5-VRT-induced Nav1.6 response occurred at PO 3d, earlier than the SNI-induced one, 7 days after surgery. Modulating ACC glutamatergic neurons via DREADD-Gq or DREADD-Gi greatly changed the ACC CX3CL1 levels and the mechanical paw withdrawal threshold. Neutralization of endogenous ACC CX3CL1 by contralateral anti-CX3CL1 antibody attenuated the induction and the maintenance of mechanical allodynia and eliminated the upregulation of CX3CL1, TNF-α and Nav1.6 protein levels in ACC induced by SNI. Furthermore, contralateral ACC anti-CX3CL1 also inhibited the expression of ipsilateral spinal c-Fos, Iba1, CD11b, TNF-α and IL-6. CONCLUSIONS: The descending facilitation function mediated by CX3CL1 and its downstream cascade may play a pivotal role, leading to enhanced pain sensitization and even mirror-image pain. Strategies that target chemokine-mediated ACC hyperexcitability may lead to novel therapies for the treatment of neuropathic pain.

Clinical Characteristics of Hydrocephalus Following the Treatment of Pyogenic Ventriculitis Caused by Multi/Extensive Drug-Resistant Gram-Negative Bacilli, Acinetobacter Baumannii, and Klebsiella Pneumoniae.

Objective: Hydrocephalus is common after ventriculitis. This study explores hydrocephalus's clinical characteristics following pyogenic ventriculitis due to multidrug-resistant and extensively drug-resistant Acinetobacter baumannii and Klebsiella pneumoniae. Patients and Methods: We retrospectively reviewed patients with post-neurosurgical pyogenic ventriculitis due to multidrug-resistant and extensively drug-resistant A. baumannii and K. pneumoniae in our department between January 2014 and June 2020. Once diagnosed, patients received intraventricular lavage followed by daily intraventricular administration of Colistin (polymyxin-E). The patient's clinical/radiographic findings were analyzed and evaluated 6 months after discharge. Results: In total, 48 cases were included in this study, and 25% were female. The median age was 45 (SD ± 15) years old. Median intraventricular Colistin administration to acquire sterile cerebrospinal fluid (CSF) was 20 days. Forty-one patients developed hydrocephalus; among them, 18 (43%) had multiloculated hydrocephalus (MLH), 23 (56%) had uni/non-loculated hydrocephalus (ULH/NLH), and 7 (17%) did not develop hydrocephalus. The patients with MLH had (15 days) delayed initiation of intraventricular irrigation (p < 0.022). They had (32 days) longer intraventricular Colistin (p < 0.003) and showed worse outcomes in terms of Glasgow outcome score (GOS) at 6 months follow-up than those without hydrocephalus. The mean score of the MLH group was 1.67 (SD1.23), and ULH/NLH was 2.61 (SD1.4) at p < 0.008. Conclusion: Multiloculated hydrocephalus is common in patients receiving delayed intraventricular administration of Colistin and required a longer duration on intraventricular Colistin to treat the pyogenic ventriculitis caused by multidrug/extensive drug-resistant A. baumannii and K. pneumoniae. It is associated with worse clinical outcomes.

Chronic Oral Administration of Magnesium-L-Threonate Prevents Oxaliplatin-Induced Memory and Emotional Deficits by Normalization of TNF-α/NF-κB Signaling in Rats.

Antineoplastic drugs such as oxaliplatin (OXA) often induce memory and emotional deficits. At present, the mechanisms underlying these side-effects are not fully understood, and no effective treatment is available. Here, we show that the short-term memory deficits and anxiety-like and depression-like behaviors induced by intraperitoneal injections of OXA (4 mg/kg per day for 5 consecutive days) were accompanied by synaptic dysfunction and downregulation of the NR2B subunit of N-methyl-D-aspartate receptors in the hippocampus, which is critically involved in memory and emotion. The OXA-induced behavioral and synaptic changes were prevented by chronic oral administration of magnesium-L-threonate (L-TAMS, 604 mg/kg per day, from 2 days before until the end of experiments). We found that OXA injections significantly reduced the free Mg2+ in serum and cerebrospinal fluid (from ~ 0.8 mmol/L to ~ 0.6 mmol/L). The Mg2+ deficiency (0.6 mmol/L) upregulated tumor necrosis factor (TNF-α) and phospho-p65 (p-p65), an active form of nuclear factor-kappaB (NF-κB), and downregulated the NR2B subunit in cultured hippocampal slices. Oral L-TAMS prevented the OXA-induced upregulation of TNF-α and p-p65, as well as microglial activation in the hippocampus and the medial prefrontal cortex. Finally, similar to oral L-TAMS, intracerebroventricular injection of PDTC, an NF-κB inhibitor, also prevented the OXA-induced memory/emotional deficits and the changes in TNF-α, p-p65, and microglia. Taken together, the activation of TNF-α/NF-κB signaling resulting from reduced brain Mg2+ is responsible for the memory/emotional deficits induced by OXA. Chronic oral L-TAMS may be a novel approach to treating chemotherapy-induced memory/emotional deficits.

Upregulation of tumor necrosis factor-alpha in the anterior cingulate cortex contributes to neuropathic pain and pain-associated aversion.

Injury associated pain involves subjective perception and emotional experience. The anterior cingulate cortex (ACC) is a key area involved in the affective component of pain processing. However, the neuroimmune mechanisms underlying enhanced ACC excitability following peripheral nerve injury are still not fully understood. Our previous work has shown that tumor necrosis factor-alpha (TNF-α) overexpression leads to peripheral afferent hyperexcitability and synaptic transmission potentiation in spinal cord. Here, we aimed to reveal the potential role of ACC TNF-α in ACC hyperexcitability and neuropathic pain. c-Fos, a widely used neuronal activity marker, was induced especially in contralateral ACC early [postoperative (PO) 1 h] and later (PO day 7 and 10) during the development of neuropathic pain. Spared nerve injury (SNI) elevated TNF-α level in contralateral ACC from PO day 5 to 14, delayed relative to decreased ipsilateral paw withdrawal threshold apparent from PO day 1 to 14. Microinjection of anti-TNF-α antibody into the ACC completely eliminated c-Fos overexpression and greatly attenuated pain aversion and mechanical allodynia induced by SNI, suggesting an important role of ACC TNF-α in the pain aversiveness and pain maintenance. Furthermore, modulating ACC pyramidal neurons via a Gi-coupled human M4 muscarinic receptor (hM4Di) or a Gq-coupled human M3 muscarinic receptor (hM3Dq), a type of designer receptors exclusively activated by designer drugs (DREADD), greatly changed the ACC TNF-α level and the mechanical paw withdrawal threshold. The positive interactions between TNF-α and ACC neurons might modulate the cytokine microenvironment thus contribute to the neuropathic pain.

Calpain-2 Regulates TNF-α Expression Associated with Neuropathic Pain Following Motor Nerve Injury.

Both calpain-2 (CALP2) and tumor necrosis factor-α (TNF-α) contribute to persistent bilateral hypersensitivity in animals subjected to L5 ventral root transection (L5-VRT), a model of selective motor fiber injury without sensory nerve damage. However, specific upstream mechanisms regulating TNF-α overexpression and possible relationships linking CALP2 and TNF-α have not yet been investigated in this model. We examined changes in CALP2 and TNF-α protein levels and alterations in bilateral mechanical threshold within 24 h following L5-VRT model injury. We observed robust elevation of CALP2 and TNF-α in bilateral dorsal root ganglias (DRGs) and bilateral spinal cord neurons. CALP2 and TNF-α protein induction by L5-VRT were significantly inhibited by pretreatment using the calpain inhibitor MDL28170. Administration of CALP2 to rats without nerve injury further supported a role of CALP2 in the regulation of TNF-α expression. Although clinical trials of calpain inhibition therapy for alleviation of neuropathic pain induced by motor nerve injury have not yet shown success, our observations linking CALP2 and TNF-α provide a framework of a systems' approach based perspective for treating neuropathic pain.

Early CALP2 expression and microglial activation are potential inducers of spinal IL-6 up-regulation and bilateral pain following motor nerve injury.

Previous work from our laboratory showed that motor nerve injury by lumbar 5 ventral root transection (L5-VRT) led to interleukin-6 (IL-6) over-expression in bilateral spinal cord, and that intrathecal administration of IL-6 neutralizing antibody delayed the induction of mechanical allodynia in bilateral hind paws. However, early events and upstream mechanisms underlying spinal IL-6 expression following L5-VRT require elucidation. The model of L5-VRT was used to induce neuropathic pain, which was assessed with von Frey hairs and the plantar tester in adult male Sprague-Dawley rats. Calpain-2 (CALP2, a calcium-dependent protease) knockdown or over-expression and microglia depletion were conducted intrathecally. Western blots and immunohistochemistry were performed to explore the possible mechanisms. Here, we provide the first evidence that both IL-6 and CALP2 levels are increased in lumbar spinal cord within 30 min following L5-VRT. IL-6 and CALP2 co-localized in both spinal dorsal horn (SDH) and spinal ventral horn. Post-operative (PO) increase in CALP2 in ipsilateral SDH was evident at 10 min PO, preceding increased IL-6 at 20 min PO. Knockdown of spinal CALP2 by intrathecal CALP2-shRNA administration prevented VRT-induced IL-6 overproduction in ipsilateral spinal cord and alleviated bilateral mechanical allodynia. Spinal microglia activation also played a role in early IL-6 up-regulation. Macrophage/microglia markers ED1/Iba1 were increased at 30 min PO, while glial fibrillary acidic protein (astrocyte) and CNPase (oligodendrocyte) markers were not. Increased Iba1 was detected as early as 20 min PO and peaked at 3 days. Morphology changed from a small soma with fine processes in resting cells to an activated ameboid shape. Depletion of microglia using Mac-1-saporin partially prevented IL-6 up-regulation and attenuated VRT-induced bilateral mechanical allodynia. Taken together, our findings provide evidence that increased spinal cord CALP2 and microglia cell activation may have early causative roles in IL-6 over-expression following motor nerve injury. Agents that inhibit CALP2 and/or microglia activation may therefore prove valuable for treating neuropathic pain.