Changes and prognosis of calcitonin gene-related peptide and neuronal calcium overload after spinal cord injury in rats

doi:10.3877/cma.j.issn.2095-9141.2024.04.002

Abstract

Abstract:

Objective

To study the changes in the concentrations of calcium ion (Ca²⁺)in neurons and calcitonin-gene-related peptide (CGRP) in plasma and their relationship with the severity of spinal cord injury (SCI).

Methods

A total of 100 male Wistar rats were randomly divided into experimental group(75 rats)and control group (25 rats)in a ratio of 3∶1 according to the random number table method.The experimental group was made into pathological models of SCI rats with light, medium and heavy injuries according to the principle of free fall injury by Feeney method, and divided into mild group(25 rats), moderate group (25 rats) and severe group (25 rats) according to the severity of injuries. The control group only underwent surgery, and did not cause SCI. At each time period (0.5, 6, 12, 24, 48 and 72 h) after the establishment of the model, 10 rats in each group were randomly selected to take blood from the eye canthus, and the CGRP concentration in plasma was determined by radioimmunoassay,among which 3 rats were randomly selected (including 3 rats randomly selected from 10 rats with blood taken from the canthus at 72 h after modeling)to be killed by neck-breaking,and the spinal cord (T9-11)was taken out, and the neurons were isolated and the purity was detected, the intracellular Ca²⁺concentration of neurons was measured by laser scanning confocal microscopy.

Results

The intracellular Ca²⁺ fluorescence pixel values of the experimental group rats rapidly increased after injury, reaching the highest value at 24 h and gradually decreased thereafter.Ca²⁺fluorescence pixel values at each time point were significantly higher than those of the control group, with severe group＞moderate group＞mild group,and the differences were statistically significant (P＜0.05).The concentration of CGRP in the plasma of the experimental group rats rapidly increased at 0.5 h after injury,significantly higher than that of the control group, and the difference was statistically significant (P＜0.05); The concentration of CGRP in the plasma began to decrease at 6 h after injury and gradually increased at 48 h, the concentrations of CGRP in the plasma at 6, 12, 24, and 48 h after injury were all lower than those in the control group, and the differences were statistically significant (P＜0.05). In the experimental group, there was no statistically significant difference in the concentration of CGRP in the plasma of the three groups of rats at 0.5 h after injury (P＞0.05); The plasma concentration of CGRP was compared at 12, 24, and 48 h after injury, with the severe grouP＜moderate grouP＜mild group, and the differences were statistically significant (P＜0.05).Correlation analysis showed a negative correlation between Ca²⁺concentration and CGRP concentration in the moderate and severe groups (moderate group: r=-0.513,P＜0.001; severe group: r=-0.391, P=0.013).

Conclusion

In the early stage of SCI, there may be intracellular calcium overload and abnormal CGRP concentration in neurons, which are negatively correlated and may be closely related to the transmission of primary injury signals and secondary cascade reactions. Maintaining the steady state of intracellular Ca²⁺ concentration and CGRP concentration in plasma as soon as possible after SCI is beneficial for the treatment and improvement of prognosis of SCI patients.

Key words: Nerve injury and repair, Spinal cord injury, Calcium overload, Calcitonin-generelated peptide

Wen Xue, Zhuo Liu, Weihua Jia, Xiaoyi Zhang, Jin Liu, Aiguo Wang, Zhigang Feng, Xin Yang, Qi Tian, Hubin Duan. Changes and prognosis of calcitonin gene-related peptide and neuronal calcium overload after spinal cord injury in rats[J]. Chinese Journal of Neurotraumatic Surgery(Electronic Edition), 2024, 10(04): 198-205.

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Figures/Tables 6

Fig.1 Schematic diagram of spinal cord injury modeling device

Fig.2 Fluorescence identification staining of spinal cord neurons

Fig.3 Purity test of spinal cord neurons in two groups of rats(10×)

Fig.4 Ca²⁺fluorescence ion imaging 24 h after spinal cord injury in each group of rats

Tab.1 Comparison of Ca²⁺fluorescence pixel values between experimental group and control group at different time

组别	只数	0.5 h（ $\bar{x}$ ±s）	6 h（ $\bar{x}$ ±s）	12 h[M（Q₁，Q₃）]	24 h[M（Q₁，Q₃）]	48 h（ $\bar{x}$ ±s）	72 h[M（Q₁，Q₃）]
对照组	10	86.56±7.74	85.37±5.84	88.59（84.00，89.89）	85.89（81.78，87.70）	85.80±4.25	85.81（78.90，88.83）
轻度组	10	95.51±4.83^a	106.13±8.55^a	113.14（111.77，114.63）^a	160.64（153.73，162.83）^a	135.69±4.55^a	116.37（110.11，125.16）^a
中度组	10	101.68±6.28^ab	110.70±1.84^ab	122.59（121.17，123.83）^ab	177.50（171.76，181.61）^ab	146.22±4.32^ab	137.43（131.88，139.49）^ab
重度组	10	103.51±5.06^abc	121.04±4.39^abc	132.38（129.23，136.76）^abc	194.01（186.82，203.19）^abc	159.34±4.20^abc	142.43（140.53，151.11）^abc
F/H值		15.225	64.817	157.024	497.836	511.836	93.155
P值		＜0.001	＜0.001	＜0.001	＜0.001	＜0.001	＜0.001

Tab.1 Comparison of Ca²⁺fluorescence pixel values between experimental group and control group at different time

组别	只数	0.5 h（ $\bar{x}$ ±s）	6 h（ $\bar{x}$ ±s）	12 h[M（Q₁，Q₃）]	24 h[M（Q₁，Q₃）]	48 h（ $\bar{x}$ ±s）	72 h[M（Q₁，Q₃）]
对照组	10	86.56±7.74	85.37±5.84	88.59（84.00，89.89）	85.89（81.78，87.70）	85.80±4.25	85.81（78.90，88.83）
轻度组	10	95.51±4.83^a	106.13±8.55^a	113.14（111.77，114.63）^a	160.64（153.73，162.83）^a	135.69±4.55^a	116.37（110.11，125.16）^a
中度组	10	101.68±6.28^ab	110.70±1.84^ab	122.59（121.17，123.83）^ab	177.50（171.76，181.61）^ab	146.22±4.32^ab	137.43（131.88，139.49）^ab
重度组	10	103.51±5.06^abc	121.04±4.39^abc	132.38（129.23，136.76）^abc	194.01（186.82，203.19）^abc	159.34±4.20^abc	142.43（140.53，151.11）^abc
F/H值		15.225	64.817	157.024	497.836	511.836	93.155
P值		＜0.001	＜0.001	＜0.001	＜0.001	＜0.001	＜0.001

Tab.2 Comparison of CGRP concentration between experimental group and control group at different time (µg/mL)

组别	只数	0.5 h（ $\bar{x}$ ±s）	6 h（ $\bar{x}$ ±s）	12 h[M（Q₁，Q₃）]	24 h（ $\bar{x}$ ±s）	48 h（ $\bar{x}$ ±s）	72 h（ $\bar{x}$ ±s）
对照组	10	3.22±0.35	3.03±0.29	3.43（3.11，3.81）	3.14±0.32	3.18±0.46	3.25±0.38
轻度组	10	6.55±0.29^a	2.67±0.60	2.33（1.97，2.73）^a	1.80±0.21^a	2.67±0.35^a	3.68±0.72
中度组	10	6.73±0.63^a	2.96±0.33	1.47（1.34，1.61）^ab	1.21±0.23^ab	1.88±0.19^ab	3.06±0.64
重度组	10	6.08±0.37^a	1.48±0.23^abc	1.12（1.05，1.32）^abc	0.65±0.10^abc	1.48±0.40^abc	1.94±0.62^abc
F/H值		129.109	31.014	128.067	195.703	39.607	13.407
P值		＜0.001	0.013	＜0.001	＜0.001	＜0.001	＜0.001

Tab.2 Comparison of CGRP concentration between experimental group and control group at different time (µg/mL)

组别	只数	0.5 h（ $\bar{x}$ ±s）	6 h（ $\bar{x}$ ±s）	12 h[M（Q₁，Q₃）]	24 h（ $\bar{x}$ ±s）	48 h（ $\bar{x}$ ±s）	72 h（ $\bar{x}$ ±s）
对照组	10	3.22±0.35	3.03±0.29	3.43（3.11，3.81）	3.14±0.32	3.18±0.46	3.25±0.38
轻度组	10	6.55±0.29^a	2.67±0.60	2.33（1.97，2.73）^a	1.80±0.21^a	2.67±0.35^a	3.68±0.72
中度组	10	6.73±0.63^a	2.96±0.33	1.47（1.34，1.61）^ab	1.21±0.23^ab	1.88±0.19^ab	3.06±0.64
重度组	10	6.08±0.37^a	1.48±0.23^abc	1.12（1.05，1.32）^abc	0.65±0.10^abc	1.48±0.40^abc	1.94±0.62^abc
F/H值		129.109	31.014	128.067	195.703	39.607	13.407
P值		＜0.001	0.013	＜0.001	＜0.001	＜0.001	＜0.001

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