大鼠急性牙髓炎痛敏模型的疼痛效应评价

doi:10.12300/j.issn.1674-5817.2021.171

摘要/Abstract

摘要：

目的采用大鼠上颌磨牙开髓暂封脂多糖处理的方法诱导急性牙髓炎模型，并对造模后大鼠出现的急性疼痛和牙髓炎症状态进行分析和评价，探究急性牙髓炎模型大鼠的痛阈改变。方法将45只成年雄性SD大鼠分为脂多糖（LPS）组、生理盐水（NS）组和空白对照（SHAM）组，每组15只。LPS组大鼠右上第一磨牙开髓后暂封LPS，制备大鼠急性牙髓炎模型；NS组开髓暂封生理盐水；SHAM组仅进行诱导麻醉。在造模前及造模后的2 h、24 h、48 h和72 h，分别检测3组大鼠的疼痛行为学指标，包括自发性疼痛行为评分、足机械刺激后的50%缩足反射阈值（paw withdrawal threshold，PWT）、舌机械刺激后的头部退缩反射阈值（head withdrawal threshold，HWT）、冷刺激后的缩足反射潜伏期（paw withdrawal latency，PWL）以及牙齿冷刺激后的头部退缩反射潜伏期（head withdrawal latency，HWL）；麻醉处死大鼠后，HE染色观察牙髓组织病理学变化，ELISA法测定大鼠血清中白细胞介素1β（interleukin-1β，IL-1β）和肿瘤坏死因子α（tumor necrosis factor-α，TNF-α）水平。结果在自发性疼痛行为方面，造模后LPS组大鼠面部梳理时间明显多于NS组（P＜0.05），且随造模后时间延长而不断增加，而大鼠活动时间未见明显差异。在机械痛敏方面，造模后2 h、24 h及48 h的LPS组HWT明显低于NS组（P＜0.05）；LPS组和NS组大鼠的50% PWT分别较SHAM组明显降低（P＜0.05），但LPS组与NS组间未见明显差异。在冷刺激痛敏方面，LPS组较NS组大鼠的HWL在造模后2 h和48 h明显降低（P＜0.05）；造模后72 h，LPS组的PWL较NS组明显降低（P＜0.05）。LPS及NS组大鼠血清中IL-1β和TNF-α水平在造模后2 h开始升高，并于48 h达到峰值，组间差异明显（P＜0.01或P＜0.05）。病理学结果显示，自造模后24 h至72 h，LPS组中性粒细胞浸润区逐渐由穿髓点依次扩大至冠髓及上段根髓，NS组较LPS组的炎症表现程度较轻。结论基于大鼠行为学观察、牙髓病理学特征改变及血清炎性因子检测，LPS诱导急性牙髓炎模型可导致大鼠出现病理性疼痛和痛阈改变。

关键词: 急性牙髓炎, 痛觉过敏, 脂多糖, 疼痛行为学, SD大鼠

Abstract:

Objective To establish a rat hyperalgesia model of acute pulpitis induced by lipopoly-saccharide (LPS) administration to the maxillary molars. In addition, to analyze and evaluate the acute pain and LPS-induced pulpitis, and to investigate the change of pain threshold in rats with acute pulpitis. Methods Forty-five adult male Sprague-Dawley (SD) rats were equally divided into LPS group, normal saline (NS) group, and blank control (SHAM) group, with 15 rats in each group. Under isoflurance anesthesia, the rats in the LPS group had their right upper molar teeth drilled and LPS was sealed temporarily with Caviton, while those in the NS group had their right upper molar teeth drilled and normal saline was sealed temporarily with Caviton. Rats in the SHAM group, serving as blank control group,were only anesthetized with isoflurane. Pain behavioral indexes, including spontaneous pain behavior scores, 50% paw withdrawal threshold (PWT), head withdrawal thresholds (HWT), paw withdrawal latency (PWL), and head withdrawal latency (HWL) were measured before and 2 h, 24 h, 48 h, and 72 h after surgery. After rats were anesthetized and sacrificed, the pathological status of pulp tissues was confirmed with hematoxylin-eosin staining and pathological examination and serum IL-1β and TNF-α were determined by ELISA. Results Concerning spontaneous pain behavior, the facial grooming time in the LPS group was significantly higher than that in the NS group postoperatively (P＜0.05), and increased gradually with time, although there was no significant difference in activity time. The HWT in the LPS group was significantly lower than that in the NS group at 2 h, 24 h, and 48 h postoperatively (P＜0.05). Additionally, 50% PWT in the LPS and NS groups were significantly lower than those in the SHAM group (P＜0.05). The LPS group had a significantly lower HWL than the NA group at 2 h and 48 h postoperatively (P＜0.05). Likewise, the PWL was significantly lower in the LPS group than the NS group (P＜0.05) at 72 h postoperatively. The IL-1β and TNF-α levels in the rats’ serum were significantly higher in the LPS group than in the NS group (P＜0.01 or P＜0.05). Moreover, the IL-1β and TNF-α levels in all groups began increasing 2 h after the model establishment, reaching a peak at 48 h. The pathological results of the LPS group showed a gradual expansion in the neutrophil infiltration area from the point of perforation to the crown pulp and upper root pulp, in 24-72 h after modeling. The rats in the NS group exhibited milder degree of inflammation than those in the LPS group. Conclusion Based on the observations regarding rat behavior, pathological changes of dental pulp, and detection of inflammatory factors in serum, the LPS induced acute pulpitis model causes pathological pain and pain threshold alterations in rats.

Key words: Acute pulpitis, Hyperalgesia, Lipopolysaccharide, Pain behavior, Sprague-Dawley rats

中图分类号:

R-332

赵斯佳, 何新宇, 景泉, 马林, 郭春岚, 万阔. 大鼠急性牙髓炎痛敏模型的疼痛效应评价[J]. 实验动物与比较医学, 2022, 42(4): 333-341.

Sijia ZHAO, Xinyu HE, Quan JING, Lin MA, Chunlan GUO, Kuo WAN. Evaluation of Pain in Acute Pulpitis Hyperalgesia Model Rats[J]. Laboratory Animal and Comparative Medicine, 2022, 42(4): 333-341.

图/表 4

图1 急性牙髓炎模型大鼠自发性疼痛行为学评价注：A为面部梳理时间；B为直立时间；C为活动时间。SHAM组指仅进行异氟烷麻醉的空白对照组，LPS组指异氟烷麻醉下大鼠右上磨牙开髓并暂封脂多糖组，NS组指异氟烷麻醉下大鼠右上磨牙开髓并暂封生理盐水组；造模前每组15只大鼠，造模后各时间点每组3只大鼠。与SHAM组比较，*P<0.05，**P<0.01；与NS组比较，#P<0.05，P<0.01。

Figure 1 Evaluation of spontaneous pain in acute pulpitis model ratsNote: A, Facial grooming time; B, Upright time; C, Activity time. The rats in the SHAM group, a blank control group, only anesthetized with isoflurane; Under isoflurance anesthesia, the rats in the LPS group had their right upper molar teeth drilled and lipopolysaccharide was sealed temporarily with Caviton, while those in the NS group had their right upper molar teeth drilled and normal saline was sealed temporarily with Caviton. There were 15 rats in each group before modeling and 3 rats in each group at different time points after successful modeling. Compared with the SHAM group, *P<0.05, **P<0.01; Compared with the NS group, #P<0.05, P<0.01.

图2 急性牙髓炎模型大鼠机械刺激及冷刺激下的痛阈变化注：A为足机械刺激后的50%缩足反射阈值；B为舌机械刺激后的头部退缩反射阈值；C为足趾冷刺激后的缩足反射潜伏期；D为牙齿冷刺激后的头部退缩反射潜伏期。SHAM组指仅进行异氟烷麻醉的空白对照组，LPS组指异氟烷麻醉下大鼠右上磨牙开髓并暂封脂多糖组；NS组指异氟烷麻醉下大鼠右上磨牙开髓并暂封生理盐水组，造模前每组15只大鼠，造模后各时间点每组3只大鼠。与SHAM组比较，*P<0.05，**P<0.01；与NS组比较，#P<0.05，P<0.01。

Figure 2 Mechanical and cold pain threshold of acute pulpitis model ratsNote: A, 50% Paw withdrawal threshold (50% PWT); B, Head withdrawal threshold (HWT); C, Paw withdrawal latency (PWL); D, Head withdrawal latency (HWL). The rats in the SHAM group, a blank control group, only anesthetized with isoflurane; Under isoflurance anesthesia, the rats in the LPS group had their right upper molar teeth drilled and lipopolysaccharide was sealed temporarily with Caviton, while those in the NS group had their right upper molar teeth drilled and normal saline was sealed temporarily with Caviton. There were 15 rats in each group before modeling and 3 rats in each group at different time points after successful modeling. Compared with the SHAM group, *P<0.05, **P<0.01; Compared with the NS group, #P<0.05, P<0.01.

图3 LPS和NS诱导大鼠急性牙髓炎的组织病理变化（HE，40倍）注：A～C分别为LPS组造模后24 h、48 和72 h；D为正常对照（SHAM组）；E～G分别为NS组造模后24 h、48 h和72 h。SHAM组指仅进行异氟烷麻醉的空白对照组，LPS组指异氟烷麻醉下大鼠右上磨牙开髓并暂封脂多糖组；NS组指异氟烷麻醉下大鼠右上磨牙开髓并暂封生理盐水组，造模前每组15只大鼠，造模后各时间点每组3只大鼠。

Figure 3 Histopathological changes of LPS and NS induced acute pulpitis in rats （HE，×40）Note: A-C, LPS groups at 24, 48, and 72 h after modeling, respectively; D, Normal control; E-G, NS groups at 24, 48, and 72 h after modeling, respectively. The rats in the SHAM group, a blank control group, only anesthetized with isoflurane; Under isoflurance anesthesia, the rats in the LPS group had their right upper molar teeth drilled and lipopolysaccharide was sealed temporarily with Caviton, while those in the NS group had their right upper molar teeth drilled and normal saline was sealed temporarily with Caviton. There were 15 rats in each group before modeling and 3 rats in each group at different time points after successful modeling.

图4 急性牙髓炎模型大鼠血清炎症因子水平变化注：A为血清IL-1β；B为血清TNF-α。SHAM组指仅进行异氟烷麻醉的空白对照组，LPS组指异氟烷麻醉下大鼠右上磨牙开髓并暂封脂多糖组；NS组指异氟烷麻醉下大鼠右上磨牙开髓并暂封生理盐水组，造模前每组15只大鼠，造模后各时间点每组3只大鼠。与SHAM组比较，*P<0.05，**P<0.01；与NS组比较，#P<0.05，P<0.01。

Figure 4 Changes in serum inflammatory factors in acute pulpitis model ratsNote: A, IL-1β levels in serum; B, TNF-α levels in serum. The rats in the SHAM group, a blank control group, only anesthetized with isoflurane; Under isoflurance anesthesia, the rats in the LPS group had their right upper molar teeth were drilled and lipopolysaccharide was sealed temporarily with Caviton, while those in the NS group had their right upper molar teeth were drilled and normal saline was sealed temporarily with Caviton. There were 15 rats in each group before modeling and 3 rats in each group at different time points after successful modeling. Compared with the SHAM group, *P<0.05, **P<0.01; Compared with the NS group, #P<0.05, P<0.01.

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