In vivo imaging of cathepsin B in activated glia in the brain after orofacial formalin test.

PURPOSE Cathepsin B (Cat B) is a cysteine lysosomal protease that is upregulated in many inflammatory diseases and widely expressed in the brain. Here, we used a Cat B activatable near-infrared (NIR) imaging probe to measure glial activation in vivo in the formalin test, a standard orofacial inflammatory pain model. The probe's efficacy was quantified with immunohistochemical analysis of the somatosensory cortex. PROCEDURES Three different concentrations of Cat B imaging probe (30, 50, 100 pmol/200 g bodyweight) were injected intracisternally into the foramen magnum of rats under anesthesia. Four hours later formalin (1.5%, 50 µl) was injected into the upper lip and the animal's behaviors recorded for 45 min. Subsequently, animals were repeatedly scanned using the IVIS Spectrum (8, 10, and 28 h post imaging probe injection) to measure extracellular Cat B activity. Aldehyde fixed brain sections were immunostained with antibodies against microglial marker Iba1 or astrocytic GFAP and detected with fluorescently labeled secondary antibodies to quantify co-localization with the fluorescent probe. RESULTS The Cat B imaging probe only slightly altered the formalin test results. Nocifensive behavior was only reduced in phase 1 in the 100 pmol group. In vivo measured fluorescence efficiency was highest in the 100 pmol group 28 h post imaging probe injection. Post-mortem immunohistochemical analysis of the somatosensory cortex detected the greatest amount of NIR fluorescence localized on microglia and astrocytes in the 100 pmol imaging probe group. Sensory neuron neuropeptide and cell injury marker expression in ipsilateral trigeminal ganglia was not altered by the presence of fluorescent probe. CONCLUSIONS These data demonstrate a concentration- and time-dependent visualization of extracellular Cat B in activated glia in the formalin test using a NIR imaging probe. Intracisternal injections are well suited for extracellular CNS proteinase detection in conditions when the blood-brain barrier is intact.

Tyrosine Kinase Inhibitors Reduce NMDA NR1 Subunit Expression, Nuclear Translocation, and Behavioral Pain Measures in Experimental Arthritis.

In the lumbar spinal cord dorsal horn, release of afferent nerve glutamate activates the neurons that relay information about injury pain. Here, we examined the effects of protein tyrosine kinase (PTK) inhibition on NMDA receptor NR1 subunit protein expression and subcellular localization in an acute experimental arthritis model. PTK inhibitors genistein and lavendustin A reduced cellular histological translocation of NMDA NR1 in the spinal cord occurring after the inflammatory insult and the nociceptive behavioral responses to heat. The PTK inhibitors were administered into lumbar spinal cord by microdialysis, and secondary heat hyperalgesia was determined using the Hargreaves test. NMDA NR1 cellular protein expression and nuclear translocation were determined by immunocytochemical localization with light and electron microscopy, as well as with Western blot analysis utilizing both C- and N-terminal antibodies. Genistein and lavendustin A (but not inactive lavendustin B or diadzein) effectively reduced (i) pain related behavior, (ii) NMDA NR1 subunit expression increases in spinal cord, and (iii) the shift of NR1 from a cell membrane to a nuclear localization. Genistein pre-treatment reduced these events that occur in vivo within 4 h after inflammatory insult to the knee joint with kaolin and carrageenan (k/c). Cycloheximide reduced glutamate activated upregulation of NR1 content confirming synthesis of new protein in response to the inflammatory insult. In addition to this in vivo data, genistein or staurosporin inhibited upregulation of NMDA NR1 protein and nuclear translocation in vitro after treatment of human neuroblastoma clonal cell cultures (SH-SY5Y) with glutamate or NMDA (4 h). These studies provide evidence that inflammatory activation of peripheral nerves initiates increase in NMDA NR1 in the spinal cord coincident with development of pain related behaviors through glutamate non-receptor, PTK dependent cascades.

Punctate Midline Myelotomy Reduces Pain Responses in a Rat Model of Lumbar Spine Pain: Evidence that the Postsynaptic Dorsal Column Pathway Conveys Pain from the Axial Spine.

Punctate midline myelotomy (PMM) has been successfully applied clinically in humans for the relief of intractable visceral pain. The operation is thought to work by interrupting the postsynaptic dorsal column pathway (PSDC) of the spinal cord. In fact, PMM was developed specifically for that purpose after it was demonstrated in rats that the PSDC conveyed about 90% of the visceral pain information to the thalamus. The application of PMM also to the problem of severe intractable back or spine pain was never tested, and it has never been established whether the PSDC pathway relates only to visceral pain or whether there may be a broader involvement with pain affecting structures of embryological midline origin, perhaps including the spine. Retrospective analyses of decades of results from various attempted myelotomy procedures in man for the relief of pain are consistent with the notion that the common element crucial to the successful midline or visceral pain relief was the interruption--even incomplete--of the PSDC pathway. Herein, we present evidence from a rat model of lumbar facet pain that interruption of the PSDC significantly reduces pain responses. The implications for the possible treatment of severe intractable spine pain in man are discussed.

Prevalence of Pancreatitis in Female and Male Pediatric Patients in Eastern Kentucky in the United States.

BACKGROUND & AIMS: Studies in the past decade report worldwide increase of pediatric pancreatitis. The present study focuses on aUnited States region where the first genes associated with hereditary pancreatitis were identified. Aim of the study was to investigate incidences of acute pancreatitis, recurrent acute pancreatitis, and chronic pancreatitis, collecting demographics, etiologies, and comorbid conditions using charted ICD-9-CM codes. METHODS: Retrospective chart review was performed on de-identified patient records of hospitalizations at University of Kentucky hospitals between 2005 and 2013. RESULTS: Of 234 children diagnosed during the 9 year time period, 69.2% (n=162) had a single episode of acute, 27.8% (65) recurrent acute, and 16.2% (38) chronic pancreatitis. Surprisingly, the annual incidence for first time diagnosis of acute pancreatitis was significantly higher for female patients (16.1, 95% CI: 13.5-18.7 per 100,000, P<0.005) compared to males (9.1, 95% CI: 6.8-11.4). Comorbid conditions varied widely depending on patients' age. Between 33.3-46.2% presented with digestive system symptoms, 12.8-26.3% with diseases of stomach and duodenum, and 10.6-31.6% with systemic diseases. Biliary disease was the most common etiology for single acute (28.4% of cases) and recurrent acute pancreatitis (16.9% of cases). Nineteen of 65 patients with recurrent acute pancreatitis developed chronic pancreatitis (29.2%), while only 3 of 162 with a single bout of acute pancreatitis returned with chronic pancreatitis (P<0.0001). CONCLUSIONS: These findings identify a prevalent disease progression from recurrent acute pancreatitis to chronic pancreatitis in the Kentucky pediatric patient population that could be due to hereditary predisposition and other geographically relevant health factors.

The ADP receptor P2Y1 is necessary for normal thermal sensitivity in cutaneous polymodal nociceptors.

BACKGROUND: P2Y1 is a member of the P2Y family of G protein-coupled nucleotide receptors expressed in peripheral sensory neurons. Using ratiometric calcium imaging of isolated dorsal root ganglion neurons, we found that the majority of neurons responding to adenosine diphosphate, the preferred endogenous ligand, bound the lectin IB4 and expressed the ATP-gated ion channel P2X3. These neurons represent the majority of epidermal afferents in hairy skin, and are predominantly C-fiber polymodal nociceptors (CPMs), responding to mechanical stimulation, heat and in some cases cold. RESULTS: To characterize the function of P2Y1 in cutaneous afferents, intracellular recordings from sensory neuron somata were made using an ex vivo preparation in which the hindlimb skin, saphenous nerve, DRG and spinal cord were dissected in continuum, and cutaneous receptive fields characterized using digitally-controlled mechanical and thermal stimuli in male wild type mice. In P2Y1-/- mice, CPMs showed a striking increase in mean heat threshold and a decrease in mean peak firing rate during a thermal ramp from 31-52°C. A similar change in mean cold threshold was also observed. Interestingly, mechanical testing of CPMs revealed no significant differences between P2Y1-/- and WT mice. CONCLUSIONS: These results strongly suggest that P2Y1 is required for normal thermal signaling in cutaneous sensory afferents. Furthermore, they suggest that nucleotides released from peripheral tissues play a critical role in the transduction of thermal stimuli in some fiber types.