Fig. 1(A) Tactile allodynia represented by von Frey test between the groups on day 0 (baseline), day 14 (pre-intervention), and day 22 (post-intervention) on the left hind paw (n=8). (B) Tactile allodynia represented by von Frey test between the groups on day 0 (baseline), day 14 (pre-intervention), and day 22 (post-intervention) on the right hind paw (n=8). DM, diabetes mellitus. aP<0.001 significant compared to control group, bP<0.001 significant compared to DM group, cP<0.05 significant compared to DM group.
Fig. 2Duration of paw licking or jumping by hot-plate test between the groups (n=8). Values are expressed as mean±standard error of the mean. No significant differences were detected between groups. DM, diabetes mellitus.
Fig. 3Chemical hyperalgesia represented by nociceptive response between the groups (mean±standard error of the mean) (n=8). DM, diabetes mellitus. aP<0.05 significant compared to control group, bP<0.05 compared to DM group, cP<0.001 significant compared to DM and control groups.
Fig. 4Immunohistochemistry image showed the total expression of total NR2B subunit positive neurons (tNR2B) in laminae I-II on ipsilateral (i) and contralateral (c) sides of rat spinal cord at 40× magnification. (A) The positive control for NR2B subunit in the rat's brain (thalamus) whilst. (B) The negative control in the rat's spinal cord region. (C) Control group. (D) Diabetes mellitus group. (E) (I 0.5) group. (F) (I 1.0) group. Arrows indicate the dark staining of tNR2B subunit positive neuron expression.
Fig. 5Immunohistochemistry image showed the expression of phosphorylated-NR2B subunit positive neurons in laminae I-II on ipsilateral (i) and contralateral (c) sides of rat spinal cord at 40× magnification. (A) The positive control for phosphorylated-NR2B subunit in the rat's brain (thalamic region) whilst. (B) The negative control in the rat's spinal cord region. (C) Control group. (D) Diabetes mellitus group. (E) (I 0.5) group. (F) (I 1.0) group. Arrows indicate the dark staining of phosphorylated-NR2B subunit positive neurons.
Fig. 6(A) A representative example of Western blot results for total NR2B subunit on the ipsilateral side between all groups with quantification analysis of the integrated density value. (B) Columns represent the mean relative total NR2B subunit protein level±standard error of the mean for eight separate experiments. The mean relative total NR2B subunit protein level (fold change) represents comparative levels of the total NR2B subunit protein in the experimental groups (diabetes mellitus [DM], DM group treated with ifenprodil at lower dose, I 0.5 and DM group treated with ifenprodil at higher dose, I 1.0) over the calibrator group (control group) after normalization by its loading control (housekeeping protein, β-actin protein) (n=8 for each group). aP<0.05 compared to control group, bP<0.001 compared to DM group.
Fig. 7(A) A representative example of Western blot results for total NR2B subunit on the contralateral side between all groups with quantification analysis of the integrated density value. (B) Columns represent the mean relative total NR2B subunit protein level±standard error of the mean for eight separate experiments. The mean relative total NR2B subunit protein level (fold change) represents comparative levels of the total NR2B subunit protein in the experimental groups (diabetes mellitus [DM], DM group treated with ifenprodil at lower dose, I 0.5 and DM group treated with ifenprodil at higher dose, I 1.0) over the calibrator group (control group) after normalization by its loading control (housekeeping protein, β-actin protein) (n=8 for each group). aP<0.05 compared to control group, bP<0.001 compared to DM group.
Fig. 8(A) A representative example of Western blot results for phosphorylated NR2B (pNR2B) subunit on the ipsilateral side between all groups with quantification analysis of the integrated density value. (B) Columns represent the mean relative pNR2B subunit protein level±standard error of the mean for eight separate experiments. The mean relative pNR2B subunit protein level (fold change) represents comparative levels of the pNR2B subunit protein in the experimental groups (diabetes mellitus [DM], DM group treated with ifenprodil at a lower dose, I 0.5 and painful diabetic neuropathy group treated with ifenprodil at a higher dose, I 1.0) over the calibrator group (control group) after normalization by its loading control (housekeeping protein, β-actin protein) (n=8 for each group). aP<0.001 compared to control group, bP<0.05 compared to control group, cP<0.001 compared to DM group, dP<0.001 compared to DM group and control groups.
Fig. 9(A) A representative example of Western blot results for phosphorylated NR2B (pNR2B) subunit on the contralateral side between all groups with quantification analysis of the integrated density value. (B) Columns represent the mean relative pNR2B subunit protein level±standard error of the mean for eight separate experiments. The mean relative pNR2B subunit protein level (fold change) represents comparative levels of the pNR2B subunit protein in the experimental groups (diabetes mellitus [DM], DM group treated with Ifenprodil at a lower dose, I 0.5 and DM group treated with Ifenprodil at a higher dose, I 1.0) over the calibrator group (control group) after normalization by its loading control (housekeeping protein, β-actin protein) (n=8 for each group). aP<0.05 compared to control group, bP<0.001 significant compared to DM and control groups.
Table 1Percentage of change of body weight and blood glucose level between the groups (n=8)
Group |
% Body weight gain, g |
Blood glucose level, mg/dL |
Baseline (day 0) |
Day 3 |
Pre-intervention (day 14) |
Post-intervention (day 22) |
Control |
68.34±5.15 |
4.52±0.42 |
120.00±18.55 |
109.09±12.73 |
107.27±18.00 |
DM |
−17.7±3.4a
|
4.43±0.52 |
568.64±75.27a
|
571.64±62.00a
|
590.91±45.09a
|
(I 0.5) |
4.15±3.8a,b,c
|
4.76±0.61 |
520.00±70.91b
|
543.64±71.82b
|
563.09±68.18b
|
(I 1.0) |
36.6±9.6a,d
|
4.99±0.61 |
465.45±103.64a,d
|
529.09±61.82a,d
|
510.91±47.27a,d
|
Table 2Total number of tNR2B and pNR2B positive neurons on ipsilateral and contralateral sides for all groups 3 days after formalin injection (n=8)
Group |
Control |
DM |
(I 0.5) |
(I 1.0) |
tNR2B |
|
|
|
|
Ipsilateral |
1,151±152.5 |
1,849±144a
|
846±243b,c
|
402±116d,e
|
Contralateral |
1,032±190.1 |
1,622±274.1a
|
708±180.5b,c
|
339±93.2d,f
|
pNR2B |
|
|
|
|
Ipsilateral |
1,401±177.9 |
1,676±236.4b
|
560±112.4d
|
436±87.5d
|
Contralateral |
1,255±236.4 |
1,375±251.4b
|
464±115.9d
|
399±90.9d
|