Agent Blue: Pre Emptive Concept

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Food and water were freely available. All experimental procedures were performed in a blind fashion. A 1cm-long incision was made along the gingivo-buccal margin. The incision was begun proximal to the first molar. Approximately 0. The incision was sutured at 2 points, using silk. The sham operation was performed without ligation of the infraorbital nerve. All behavioral tests were conducted between and hours. The withdrawal behavioral responses produced by 10 successive trials of a ramp of air-puff pressure 4 sec of duration, 10 sec of interval were examined as described previously [ 17 , 18 , 19 , 20 ].

The evoked receptive field of air-puff stimuli was limited because a gauge metal tube was used for application. The cut-off pressure for the air puff was 40 psi, as described previously [ 21 , 22 , 23 , 24 ]. A significant decrease in the threshold for an air-puff response, compared with the pre-operative values, was defined as mechanical allodynia.

Following incubation, tissue sections were washed and incubated for 2 h at room temperature with an Alexa conjugated rabbit IgG antibody ; Invitrogen, Carlsbad, CA. Co-localization analysis of immunofluorescence images was performed using a confocal laser scanning microscope LSM ; Carl Zeiss, Jena, Germany. Twelves sections three sections from each trigeminal ganglion were examined in each group using a fluorescence microscope to quantify ATF3-immunoreactivity.

The number of ATF3-immunoreactive neurons was counted at X magnification, using a counting grid. The threshold level for defining cells as immunopositive was set at 15 grey levels in images with gray levels. The trigeminal ganglion was immediately removed from each animal and quickly frozen in liquid nitrogen. Samples were sonicated with Biorupture Cosmo Bio. The blots were subsequently incubated with secondary antibody horseradish peroxidase for 1 hour at room temperature. Image J software NIH was used to quantify specific bands.

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To confirm inflammation of the infraorbital nerve induced by the CCI-ION, we measured the extravasated concentration of Evans blue dye bound to the plasma protein, as described previously [ 26 , 27 ]. For 15 min following the injection of the dye, each rat was perfused with heparinized normal saline. The tissues were incubated overnight in a mixture of acetone and 0. After incubation, the samples were centrifuged at rpm for 10 min and the supernatant was separated. The amount of dye present in each sample was analyzed by spectrophotometric measurement of the absorbance at nm.

The standard curve was generated from a series of the same extraction solution mixed with known amounts of Evans blue dye.

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Changes in air-puff thresholds were then measured. Air-puff thresholds were then measured. Changes in air-puff thresholds were then examined. To investigate the mechanisms involved, we examined whether QXinduced preemptive analgesia affects the infraorbital nerve inflammation produced by CCI-ION.

The concentration of extravasated Evans blue dye was measured on POD 7 in rats that had received immediate double application of QX To investigate a role of Navs in the antinociceptive effects of QXinduced preemptive analgesia, we analyzed the expression of Nav1. QX was prepared in sterile saline. The western blotting data were analyzed by one-way analysis of variance followed by Holm-Sidak post hoc analysis.

Treatment with QX significant boosted early recovery from mechanical allodynia to the basal level compared with the mechanical allodynia observed following treatment with vehicle alone. Therefore, subsequent experiments were performed using QX without capsaicin. Combined treatment with capsaicin 1 mg produced attenuation of neuropathic mechanical allodynia.

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However, the anti-allodynic effects following treatment with QX in combination with capsaicin did not differ from these effects following treatment with QX alone. Arrow, single application of QX There were 8 animals in each group. Double application of QX dramatically alleviated mechanical allodynia in the 10 days following injury of the infraorbital nerve. Perineural double application of QX immediately following CCI-ION significantly attenuated neuropathic mechanical allodynia compared with the effects of a single application of QX Arrow, double application of QX QX single -treated group.

QX double -treated group. Single or double application of QX did not affect neuropathic mechanical allodynia when pain was already established.

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The group that underwent CCI-ION showed a significant increase in the extravasated Evans blue dye concentration compared with the results in the sham group However, QXinduced preemptive analgesia did not affect the extravasated Evans blue dye concentration. There were 6 animals in each group. The upregulations of the area density of GFAP and p-p38 immunoreactivity were significantly decreased following treatment with QX, respectively Fig.

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The sham operation did not affect the expression of Nav isoforms. QXinduced preemptive analgesia significantly reduced the Nav1. GAPDH was used as an internal control. The present study demonstrates that preemptive application of QX reduced neuropathic mechanical allodynia in rats following CCI-ION through the inhibition of Nav isoform expression in the trigeminal ganglion. In addition, preemptive application of QX significantly reduced the upregulated expression of Nav1. These results provide a potential preemptive therapeutic strategy for the treatment of neuropathic pain following nerve injury.

Preemptive analgesia may decrease post-operative pain in the clinic.

In previous clinical studies, preemptive administration of an analgesic agent has been shown to improve postoperative pain control in case of laparoscopic radical prostatectomy [ 28 ], thoracotomy [ 29 ], and third molar extraction [ 30 , 31 ]. In animal studies, pretreatment with intravenous ketamine inhibits inflammatory pain behavior and c-fos expression following formalin injection in rats as a preemptive analgesia [ 33 ] and intraperitoneal administration of diclofenac has preemptive analgesic effects on acute thermal pain and formalin-induced inflammatory pain in rats [ 34 ].

These results suggest that pretreatment with analgesics plays an important role in preemptive analgesia. In contrast to the beneficial role of preemptive analgesia, no beneficial effects of preemptive analgesia have been reported in postoperative pain control. Thoracic extradural block with bupivacaine does not produce an early preemptive effect following thoracotomy [ 35 ]. In an animal model, preincisional treatment is not more beneficial than postincisional treatment for postoperative pain relief [ 36 ].

The lack of effects of preemptive analgesia in these cases may be caused by central sensitization which is responsible for the secondary development of pain, including neuronal plasticity [ 37 ]. These results suggest that preemptive or early pain control is a beneficial strategy for the treatment of neuropathic pain following nerve injury. QX was selected to be used as a preemptive analgesic in this study because it produces long-lasting analgesia via co-administration with capsaicin, a TRPV1 agonist [ 8 , 9 , 10 ].

The rationale behind this combined treatment method is that the activation of TRPV1, which is peripherally expressed in primary afferent nociceptors [ 38 , 39 ], by capsaicin allows QX to enter neurons. This suggests that, under our experimental conditions, the application of QX alone serves as a preemptive analgesic agent without capsaicin. Therefore, we used QX alone as a preemptive analgesic agent for all of our subsequent experiments. We also carried out experiments blocking the transmission of nerve signaling through the application of lidocaine, as an alternative preemptive analgesics.

However, preemptive application of lidocaine did not affect the development of trigeminal neuropathic pain. Preemptive application of QX, which has long lasting sensory blocking, produced a significant inhibition of the development of trigeminal neuropathic pain.

Thus, our data suggest that long-lasting blockade of pain transmission immediately following nerve injury produces significant attenuation of the development of trigeminal neuropathic pain. Our present study is the first to demonstrate the usefulness of QX as a preemptive analgesic agent in neuropathic pain control.

These results suggest that QXinduced analgesia attenuates the development of neuropathic pain only when pain has not been established. Satellite glial cells, the main type of glial cell in the sensory ganglia, respond to damage or inflammation of a sensory neuron by proliferating and expressing GFAP, similar to the glial responses observed in the spinal cord [ 40 ].

Nerve injury or inflammation leads to proliferation and hypertrophy of satellite glial cells. The participation of satellite cells in nerve injury or damage in the orofacial area has previously been reported. These results, together with our present data, suggest that modulation of satellite cell activity in the trigeminal ganglion plays an important role in the attenuation of neuropathic pain in QXinduced preemptive analgesia. However, the underlying mechanism through which satellite cells are involved in preemptive analgesia should be investigated in through further study.

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In addition, the p-p38 immunoreactivity was localized to neurons in the trigeminal ganglion. In previous reports, p-p38 immunoreactivity has been shown to be elevated in dorsal root ganglion nociceptor neurons following spinal nerve ligation [ 44 , 45 ] and chronic constriction injury of the sciatic nerve [ 46 ]. Treatment with a p38 inhibitor has been shown to reduce neuropathic mechanical allodynia [ 44 , 45 ].

The sources distinguish between tactical and strategic weapons, but do not place them in a hierarchy or sequence. There is no universally accepted definition of tactical as opposed to strategic nuclear weapons, but the terms are most often used to describe counterforce strikes on the battlefield, in contrast to countervalue strikes against civilian targets.

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