Variable sensitivity to noxious heat is mediated by differential expression of the CGRP gene.

Heat sensitivity shows considerable functional variability in humans and laboratory animals, and is fundamental to inflammatory and possibly neuropathic pain. In the mouse, at least, much of this variability is genetic because inbred strains differ robustly in their behavioral sensitivity to noxious heat. These strain differences are shown here to reflect differential responsiveness of primary afferent thermal nociceptors to heat stimuli. We further present convergent behavioral and electrophysiological evidence that the variable responses to noxious heat are due to strain-dependence of CGRP expression and sensitivity. Strain differences in behavioral response to noxious heat could be abolished by peripheral injection of CGRP, blockade of cutaneous and spinal CGRP receptors, or long-term inactivation of CGRP with a CGRP-binding Spiegelmer. Linkage mapping supports the contention that the genetic variant determining variable heat pain sensitivity across mouse strains affects the expression of the Calca gene that codes for CGRPalpha.

Selective intermediate-/small-conductance calcium-activated potassium channel (KCNN4) blockers are potent and effective therapeutics in experimental brain oedema and traumatic brain injury caused by acute subdural haematoma.

Early deterioration and death after brain injury is often the result of oedema in the injured and peri-lesional tissue. So far, no pharmacotherapy is available that exhibits significant brain oedema-reducing efficacy in patients. We selected two low molecular weight compounds from different chemical classes, a triazole (1-[(2-chlorophenyl)diphenylmethyl]-1,2,3-triazole) and a cyclohexadiene (methyl 4-[4-chloro-3-(trifluoromethyl)phenyl]-6-methyl-3-oxo-1,4,7-tetrahydroisobenzofuran-5-carboxylate) to characterize their pharmacological properties on KCNN4 channels (intermediate/small conductance calcium-activated potassium channel, subfamily N, member 4) in vitro as well as in vivo. In vitro we replaced potassium by rubidium (Rb+) and determined Rb+ fluxes evoked by 10 micro m of the calcium ionophore A23187 on C6BU1 rat glioma cells. Compared with known KCNN4 blockers, such as clotrimazole (IC50=360 +/- 12 nm) and charybdotoxin (IC50=3.3 +/- 1.9 nm), the triazole and cyclohexadiene were considerably more potent than clotrimazole and displayed similar potencies (IC50=12.1 +/- 8.8 and 13.3 +/- 4.7 nm, respectively). In the rat acute subdural haematoma model, both the triazole and cyclohexadiene displayed reduction of brain water content (-26% at 0.3 mg/kg and -24% at 0.01 mg/kg) and reduction of the intracranial pressure (-46% at 0.1 mg/kg and -60% at 0.003 mg/kg) after 24 h when administered as a 4-h infusion immediately after brain injury. When infarct volumes were determined after 7 days, the triazole as well as the cyclohexadiene displayed strong neuroprotective efficacy (-52% infarct volume reduction at 1.2 mg/kg and -43% at 0.04 mg/kg, respectively). It is concluded that blockade of KCNN4 channels is a new pharmacological approach to attenuate acute brain damage caused by traumatic brain injury.

Pharmacological sensitivity and gene expression analysis of the tibial nerve injury model of neuropathic pain.

The tibial nerve injury model is a novel, surgically uncomplicated, rat model of neuropathic pain based on a unilateral transection (neurotomy) of the tibial branch of the sciatic nerve. The aim of the present study was to describe some behavioral and molecular features of the model, and to test its sensitivity to a number of drugs which are currently used for the treatment of neuropathic pain. The model was characterized by a pronounced mechanical allodynia which was present in all subjects and a less robust thermal hyperalgesia. Mechanical allodynia developed within 2 weeks post-surgery and was reliably present for at least 9 weeks. Neurotomized rats showed no autotomy and their body weight developed normally. Gene expression in ipsilateral L5 dorsal root ganglia, analyzed by quantitative polymerase chain reaction (PCR), showed a pronounced up-regulation of galanin and vasointestinal peptide (VIP). This up-regulation developed rapidly (within 1 to 2 days following neurotomy) and remained present for at least 12 days. On the other hand, expression of calcitonin gene-related peptide (CGRP) and substance P mRNA was down-regulated 12 days following neurotomy. Mechanical allodynia was completely reversed by morphine [minimal effective dose (MED): 8 mg/kg, i.p.] and partially reversed by carbamazepine (MED: 64 mg/kg, i.p.), baclofen (MED: 3 mg/kg, i.p.) and amitriptyline (trend for efficacy at 32 mg/kg, i.p.), but not by gabapentin (50-100 mg/kg, i.p.). The finding that the tibial nerve injury model shows a robust and persistent mechanical allodynia which is sensitive to a number of established analgesics, as well as a gene expression profile which is compatible with that obtained in other models of neuropathic pain, further supports its validity as a reliable and surgically uncomplicated model for the study of neuropathic pain.

Binding of two nuclear complexes to a novel regulatory element within the human S100A9 promoter drives the S100A9 gene expression.

S100A9, also referred to as MRP14, is a calcium-binding protein whose expression is tightly regulated during differentiation of myeloid cells. The present study was performed to study the cell type- and differentiation-specific transcriptional regulation of the S100A9 gene. Analysis of the S100A9 promoter in MonoMac-6 cells revealed evidence for a novel regulatory region from position -400 to -374 bp, termed myeloid-related protein regulatory element (MRE). MRE deletion resulted in a 5.2-fold reduction of promoter activity. By electrophoretic mobility shift analysis two nuclear complexes binding to this region were identified and referred to as MRE-binding complex A (MbcA) and MRE-binding complex B (MbcB). By mutagenesis the MRE-binding motif could be narrowed to a 12-bp region. The relevance of MRE is deduced from the observations that the formation of either MRE-binding complex A or MRE-binding complex B strongly correlated with S100A9 gene expression in a cell type-specific, activation- and differentiation-dependent manner. Moreover, DNA affinity chromatography and Western blot studies indicate that a Kruppel-related zinc finger protein and the transcriptional intermediary factor 1beta (TIF1beta) are involved in an MRE-binding complex, thereby regulating the S100A9 gene expression.