Korean J Pain.  2022 Jul;35(3):291-302. 10.3344/kjp.2022.35.3.291.

Intrathecal administration of naringenin improves motor dysfunction and neuropathic pain following compression spinal cord injury in rats: relevance to its antioxidant and anti-inflammatory activities

• Fakhri S
• Sabouri S
• Kiani A
• Farzaei MH
• Rashidi K
• Mohammadi-Farani A
• Mohammadi-Noori E
• Abbaszadeh F

• KMID: 2530984
• DOI: http://doi.org/10.3344/kjp.2022.35.3.291

Abstract

Background
Spinal cord injury (SCI) is one of the most debilitating disorders throughout the world, causing persistent sensory-motor dysfunction, with no effective treatment. Oxidative stress and inflammatory responses play key roles in the secondary phase of SCI. Naringenin (NAR) is a natural flavonoid with known antiinflammatory and antioxidative properties. This study aims at evaluating the effects of intrathecal NAR administration on sensory-motor disability after SCI.
Methods
Animals underwent a severe compression injury using an aneurysm clip. About 30 minutes after surgery, NAR was injected intrathecally at the doses of 5, 10, and 15 mM in 20 µL volumes. For the assessment of neuropathic pain and locomotor function, acetone drop, hot plate, inclined plane, and Basso, Beattie, Bresnahan tests were carried out weekly till day 28 post-SCI. Effects of NAR on matrix metalloproteinase (MMP)-2 and MMP-9 activity was appraised by gelatin zymography. Also, histopathological analyses and serum levels of glutathione (GSH), catalase and nitrite were measured in different groups.
Results
NAR reduced neuropathic pain, improved locomotor function, and also attenuated SCI-induced weight loss weekly till day 28 post-SCI. Zymography analysis showed that NAR suppressed MMP-9 activity, whereas it increased that of MMP-2, indicating its anti-neuroinflammatory effects. Also, intrathecal NAR modified oxidative stress related markers GSH, catalase, and nitrite levels. Besides, the neuroprotective effect of NAR was corroborated through increased survival of sensory and motor neurons after SCI.
Conclusions
These results suggest intrathecal NAR as a promising candidate for medical therapeutics for SCI-induced sensory and motor dysfunction.

Keyword

Anti-Inflammatory Agents; Catalase; Glutathione; Inflammation; Matrix Metalloproteinase; Motor Disorders; Naringenin; Neuralgia; Nitrites; Oxidative Stress; Pain; Spinal Cord Injuries

Figure

• 

  Fig. 1 Effects of naringenin (NAR) on heat hyperalgesia and cold allodynia following compression spinal cord injury (SCI) in Wistar rats using (A) acetone drop, and (B) hot plate tests. The data are reported as mean ± standard error of the mean (n = 7). Repeated measures two-way analysis of variance followed by Bonferroni post-hoc tests were used for data analysis. ***P < 0.001 vs. sham group. +P < 0.05, ++P < 0.01, and +++P < 0.001, respectively in the comparisons between NAR groups with the SCI group. #P < 0.05 vs. NAR 15 mM.

• 

  Fig. 2 Effects of naringenin (NAR) on improving motor dysfunction following compression spinal cord injury (SCI) in rats as assessed by (A) Basso, Beattie, Bresnahan (BBB), and (B) inclined plane test. The data are reported as mean ± standard error of the mean (n = 7). Repeated measures two-way analysis of variance followed by Bonferroni post-hoc tests were used for data analysis. ***P < 0.001 vs. sham group. +P < 0.05, ++P < 0.01, and +++P < 0.001, respectively in the comparisons between NAR groups with the SCI group. #P < 0.05 vs. NAR 15 mM.

• 

  Fig. 3 Effects of naringenin (NAR) on matrix metalloproteinase (MMP)-2 and MMP-9 gelatinase activities following compression spinal cord injury (SCI) in rats. (A) and (B) illustrate the gelatin zymograms of MMP-2 and MMP-9, respectively. The data presented in (C) and (D) are expressed as mean ± standard error of the mean (n = 3). Repeated measures one-way analysis of variance followed by Tukey post-hoc tests were used for data analysis. **P < 0.01 and ***P < 0.001 vs. sham group. +P < 0.05, ++P < 0.01, and +++P < 0.001, respectively vs. the SCI group. #P < 0.05 in the comparisons between different NAR groups.

• 

  Fig. 4 Effects of naringenin (NAR) on serum glutathione (GSH) (A), catalase (B), and nitrite (C) levels on day 28 post-spinal cord injury (SCI). Treatment with NAR (15 mM) was associated with significantly decreased serum catalase, GSH and nitrite compared to the SCI group. The data are expressed as mean ± standard error of the mean (n = 3). Repeated measures one-way analysis of variance followed by Tukey post-hoc tests were used for data analysis. ***P < 0.001 vs. sham group. +P < 0.05 and ++P < 0.01, respectively vs. the SCI group.

• 

  Fig. 5 (A) Effects of naringenin (NAR) on H & E staining of dorsal and ventral transverse sections of spinal cord on day 28 post-spinal cord injury (SCI) in rats (40× magnification). (B) and (C) represent the mean numbers of survived neurons at dorsal and ventral horns, respectively. The data are expressed as mean ± standard error of the mean (n = 6). Repeated measures one-way analysis of variance followed by Tukey post-hoc tests were used for data analysis. ***P < 0.001 vs. sham group. +P < 0.05, ++P < 0.01, and +++P < 0.001, respectively vs. the SCI group. #P < 0.05 in the comparisons between different NAR groups.

• 

  Fig. 6 Effects of naringenin (NAR) on body weight changes of rats over time after clip compression spinal cord injury (SCI). The data are expressed as mean ± standard error of the mean (n = 7). Repeated measures two-way analysis of variance followed by Bonferroni post-hoc tests were used for data analysis. ***P < 0.001 vs. sham group. +P < 0.05, ++P < 0.01, and +++P < 0.001, respectively vs. the SCI group.

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