Threshold effect of G9a/Glp on peripheral nerve injury-induced hypersensitivity

Subjects and peri-surgical care

After approval by the institutional ethical committee, a total of 410 male C57BL/6 mice (from Nanjing Medical University, Nanjing, China) weighing 20–25 g, 7–9 weeks of age, were used for behavioral tests in accordance to the Ethical Guidelines for Investigations of Experimental Pain in Conscious Animals. The peri-surgical treatment was reported previously. ⁷ Briefly, animals were housed in a cage on a 12:12 h light/dark cycle with 23 ± 1℃. After randomization, each mouse was placed in a test box with three mirrored sides for a 10-min habituation period, and then the test sessions took place. No food or water was available to the mice during the experiment. After the experiment, a lethal dose of pentobarbital was injected to euthanize each mouse.

Study protocol

First, the mechanical withdrawal behavior was observed for a period of 98 days post SNI evoked with von Frey filaments. Next, various dosages of G9a inhibitor BIX01294 (0.625, 1.25, 2.5, 5.0, 10.0, 20.0, 40.0, and 80.0 µg) or Glp inhibitor UNC0638 (5.0, 10.0, 20.0, 40.0, 80.0, 160.0, 320.0, and 640.0 µg) were, respectively, intrathecally injected through the pre-buried catheter once in bolus at the 14th day post SNI, and nociceptive behavior was detected at the same day. Besides, the effect of a three days’ intrathecal injection of BIX/UNC or their solvent dimethyl sulfoxide (DMSO) on nociceptive behavior in sham mice was observed. Further, various dosages of both BIX01294 and UNC0638 were intrathecally injected for a 14 days’ duration, and withdrawal behaviors were compared for 49 days post SNI. Finally, nociceptive behavior after BIX01294/UNC0638 treatment on the same animal was observed with a crossover intervention that was a threshold dose followed by a lower or higher dose with a 14 days’ interval, as well as an inverse sequence.

G9a and Glp share as high as 80% sequence identity in their respective SET domains, which are also BIX01294 and UNC0638 binding sites. ⁸ BIX01294 and UNC0638 inhibit both G9a and Glp potently and selectively over a wide range of epigenetic as well as nonepigenetic targets, while UNC0638 is considered less toxic to cells compared to BIX01294.^8,9 In our study, BIX01294 and UNC0638 were injected intrathecally using a microsyringe in a volume of 5 µL over 30 s, and then 10 µL saline was used to flush the catheter dead space. For the DMSO-treated sham mice, a total of 15 µL of DMSO only was given.

Intrathecal catheterization

Mice underwent intrathecal catheterization (ALZET Osmotic Pumps, Cupertino, CA) one week before nerve injury as described previously. ¹⁰ In brief, cephalic–cervical area was shaved and sterilized, a midline incision was performed, and then the paravertebral muscles were dissected from the spinous processes. With the help of a surgical microscope, a hole (1 × 1 mm) was drilled through cisternal membrane until the dura was exposed. The catheter was inserted for 2.5 mm, fixed with tissue glue, and further secured on the fascia of paravertebral muscle. Finally, sodium penicillin 10,000 IU was given intramuscularly against infection. The mice would be excluded (∼10%) if neurological deficits were exhibited after catheterization, and in our study, a total of 300 mice underwent intrathecal catheterization, and 25 of them (8%) were excluded from final statistical analysis because of neurological deficits. The intrathecal catheter was not removed until the day of sacrifice.

Spared nerve injury

The animal model of chronic neuropathic pain was established by SNI as described elsewhere. ¹¹ In brief, animals were anesthetized with isoflurane, and the tibial and common peroneal branches of the sciatic nerve were ligated and sectioned distally, but the sural nerve was left intact. For sham-operated ones, the sciatic nerve was merely exposed but not ligated and dissected.

Pain threshold measurement

Pain threshold was assessed using the von Frey filaments (Stoelting Co., Wood Dale, IL) as described previously. ¹² In brief, the filaments were applied to the central surface of the hind paw plantar for a maximal 10 s to detect the stimulus threshold via triggering a withdrawal reaction. The stimulation was initiated from the weakest filament (0.407 g). The stimulus increment was based on the response of the mouse to the current filament: if the paw withdrew, the same filament was again used 60 s later; but if not, the next stronger filament was presented. If with the same filament, the mouse paw withdrew in two consecutive tests, no further testes were need. Withdrawal responses were used to determine the absolute threshold, that is, the 50% withdrawal threshold, by adjusting to a Gaussian integral psychometric function using a maximum likelihood method.

Statistical analysis

Data are presented as the mean ± SEM and analyzed using GraphPad Prism version 5.0 (GraphPad Software Inc., San Diego, CA). When there were two testing groups, Student’s t test was used to analyze the intergroup difference. One-way analysis of variance followed by the Bonferroni post hoc tests for multiple comparisons were used for all other data when necessary. All reported p values are two sided, and a p value of less than 0.05 was considered to be statistically significant.

Characteristic behavioral changes post SNI

Ipsilateral and contralateral mechanical withdrawal threshold was measured for a period of 98 days post SNI. The ipsilateral mechanical withdrawal threshold trajectory showed that the nerve injury induced an immediate, remarkably, and consistent reduction in pain threshold, peaked at the 14th day after nerve injury and progressively recovered thereafter, which was still significantly lower than the sham-operated ones at the end of 98 days’ observational period (Figure 1(a)). However, no withdrawal threshold differences were found in the contralateral injured nerve (Figure 1(b)). OPEN IN VIEWER

Dose–behavior curves of various dosages of G9a/Glp inhibitor

One of the following dosages of G9a/Glp inhibitor BIX01294: 0.625, 1.25, 2.5, 5.0, 10.0, 20.0, 40.0, and 80.0 µg was intrathecally injected, respectively, through the pre-buried catheter once in bolus at the 14th day post SNI, and the nociceptive behaviors were detected at the same day. With the dose of BIX01294 increased from 0.0625 to 10 µg, the mechanical withdrawal threshold progressively decreased; however, with the dose further increased from 10.0 to 80.0 µg, pain threshold gradually increased, showing a prominent “V” shape on the dose–behavior curve (Figure 2(a)). OPEN IN VIEWER

Likewise, various dosages of G9a/Glp inhibitor UNC0638: 5.0, 10.0, 20.0, 40.0, 80.0, 160.0, 320.0, and 640.0 µg were intrathecally injected, and mechanical withdrawal threshold was recorded as well. Similar “V”-shaped dose–behavior curve was observed for UNC0638 with the lowest mechanical threshold at a dose of 80.0 µg (Figure 2(b)).

Behavior changes of BIX01294/UNC0638 in sham-operated mice

To explore the effect of G9a/Glp inhibitors on basal mechanical sensitivity, different doses of BIX01294 (2.5, 10.0, or 50.0 µg), UNC0638 (20.0, 80.0, or 400.0 µg), or equal volume of solvent DMSO were intrathecally injected daily in bolus for three days in sham mice from the seventh day postsurgery, and nociceptive behaviors were compared with those in normal and SNI mice for a 35 days’ period. The behavioral trajectory showed neither BIX (Figure 3(a)) nor UNC (Figure 3(b)) had effect on mechanical withdrawal threshold in sham mice, reflecting their specific for SNI pathologic process. OPEN IN VIEWER

Behavior changes of different dosages of inhibitor-treated animals

According to the dose–behavior curves of BIX01294/UNC0638, low and high doses of BIX01294/UNC0638 might have different effects on nociceptive behaviors. To further understand such differential pain regulation, each of the following dosages of BIX01294: 1.0, 2.5, 5.0, 10.0, 25.0, 50.0, and 100.0 µg, as well as UNC0638: 10.0, 20.0, 40.0, 80.0, 150.0, 300.0, and 600.0 µg, respectively, was intrathecally injected daily in bolus from the seventh post SNI for 14 days, and ipsilateral nociceptive behaviors were observed for a 49 days’ period.

For BIX01294, a dose of 10.0 µg significantly worsen nerve injury-induced allodynia during the 14 days’ treatment period; however, both lower (1.0, 2.5, and 5.0 µg) and higher doses (25.0, 50.0, and 100.0 µg) improved nociceptive behaviors, showing a contrary effect. Further, the pain improvement effect of BIX01294 was dose-dependent and greater with a far lower (1.0 > 2.5 > 5.0 µg) or far higher dose (100.0 > 50.0 > 25.0 µg; Figure 4(a) and (b)). Likewise, UNC0638 has a similar role in affecting nerve injury-induced allodynia with a threshold dose at 80.0 µg to worsen pain. Dose-dependent pain improvement effect of UNC0638 was also observed with a lower (10.0 > 20.0 > 40.0 µg) or higher (600.0 > 300.0 > 150.0 µg) dose, showing a contrasting effect (Figure 4(c) and (d)). These results were in agreement with the findings of Figure 2. OPEN IN VIEWER

Effect of different dosages of inhibitors on nociceptive behavior in the same mice

To further clarify G9a/Glp inhibitors’ potential role in pain change, a crossover intervention was applied to the same mice. A 14-day intrathecal injection of BIX01294 2.5 µg or 50.0 µg increased withdrawal threshold; however, after a 14 days’ washout, BIX01294 10.0 µg always worsened nociceptive behaviors (Figure 5(a) and (d)), the effect which also applied after an inverse treatment sequence (Figure 5(b) and (c)). Similar role was observed with UNC0638; a 14-day intrathecal injection of UNC0638 20.0 µg or 400.0 µg increased while 80.0 µg decreased withdrawal threshold with a 14 days’ interval in the same mice, independent of treatment sequence (Figure 5(e) to (h)). OPEN IN VIEWER