Diabetes Metab J.  2019 Apr;43(2):222-235. 10.4093/dmj.2018.0020.

Increased Nociceptive Responses in Streptozotocin-Induced Diabetic Rats and the Related Expression of Spinal NR2B Subunit of N-Methyl-D-Aspartate Receptors

• Ismail CA
• Suppian R
• Abd Aziz C
• Haris K
• Long I

• KMID: 2442774
• DOI: http://doi.org/10.4093/dmj.2018.0020

Abstract

BACKGROUND
This study investigated the role of NR2B in a modulated pain process in the painful diabetic neuropathy (PDN) rat using various pain stimuli.
METHODS
Thirty-two Sprague-Dawley male rats were randomly allocated into four groups (n=8): control, diabetes mellitus (DM) rats and diabetic rats treated with ifenprodil at a lower dose (0.5 µg/day) (I 0.5) or higher dose (1.0 µg/day) (I 1.0). DM was induced by a single injection of streptozotocin at 60 mg/kg on day 0 of experimentation. Diabetic status was assessed on day 3 of the experimentation. The responses on both tactile and thermal stimuli were assessed on day 0 (baseline), day 14 (pre-intervention), and day 22 (post-intervention). Ifenprodil was given intrathecally for 7 days from day 15 until day 21. On day 23, 5% formalin was injected into the rats' hind paw and the nociceptive responses were recorded for 1 hour. The rats were sacrificed 72 hours post-formalin injection and an analysis of the spinal NR2B expression was performed.
RESULTS
DM rats showed a significant reduction in pain threshold in response to the tactile and thermal stimuli and higher nociceptive response during the formalin test accompanied by the higher expression of phosphorylated spinal NR2B in both sides of the spinal cord. Ifenprodil treatment for both doses showed anti-allodynic and anti-nociceptive effects with lower expression of phosphorylated and total spinal NR2B.
CONCLUSION
We suggest that the pain process in the streptozotocin-induced diabetic rat that has been modulated is associated with the higher phosphorylation of the spinal NR2B expression in the development of PDN, which is similar to other models of neuropathic rats.

Keyword

Diabetic neuropathies; Hyperalgesia; Ifenprodil; NR2B NMDA receptor; Pain measurement

MeSH Terms

Animals
Diabetes Mellitus
Diabetic Neuropathies
Formaldehyde
Humans
Hyperalgesia
Male
N-Methylaspartate*
Pain Measurement
Pain Threshold
Phosphorylation
Rats*
Rats, Sprague-Dawley
Receptors, N-Methyl-D-Aspartate*
Spinal Cord
Streptozocin
Formaldehyde
N-Methylaspartate
Receptors, N-Methyl-D-Aspartate
Streptozocin

Figure

• 

  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. 2 Duration 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. 3 Chemical 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. 4 Immunohistochemistry 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. 5 Immunohistochemistry 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.

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