Long term effects of neonatal inflammatory pain on the nocifensive behaviors and trans-synaptical transmission in the adult rat spinal cord dorsal horn neurons

• Main Authors: Chih-Peng Lin, 林至芃
• Other Authors: Wei-Zen Sun
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/78766242992586438823

碩士 === 國立臺灣大學 === 臨床醫學研究所 === 93 === Advances in medical technology have significantly improved survival among medically compromised premature babies. However, poorly managed pain during this period will result in long-term physiologic and behavioral consequences. Animal studies reveal that neonatal peripheral inflammatory pain results in long-standing changes on the development of nociceptive neuronal circuitry. Morphologically, fine primary afferent fibers termination area in spinal cord dorsal horn expands longitudinally and sprouts into deeper lamina. Electrophysiologic study shows increase in background activity and responses to noxious and non-noxious stimuli. The distribution and extent of “Fos like immunoreactivity” (a marker of post-synaptic activation) in spinal cord dorsal horn is also increased. Materials and methods: On postnatal day 1 (P1) and day 21 (P21), rat pups were intraplantarlly injected with complete Freund’s adjuvant (CFA). Inflammatory pain was thus induced on the left hind paw and lasted for 5-7 days. Control group (N) rats wer left untreated. All animals were allowed to mature into adulthood without further manipulation. Further assessments were done at 8 weeks of age. We determined the pain thresholds and nocifensive behaviors among all 3 different treatment groups (P1, P21 and N) bilaterally by CO2 pulse laser stimulation. Pain thresholds were determined by serially increases of the laser pulse duration (5 ms increaments) with fixed CO2 pulse laser output energy to 5 watts until leg withdrawal were elicited. Nocifensive behaviors were induced by supra-threshold laser stimulation (5watts, 200ms). Leg-lifting and licking times were recorded. For accessing the trans-synapticall transmission, interactions of nociceptive neuromodulator- substance P and it receptor- NK1 receptor were analysed. P1 rats were intraplantarlly injected bilaterally with 5% fomalin. Rats were sacrificed 8 minutes after the intense pain rechallenge. After transcardially perfusion and fixation, L4-L5 spinal cord were retrieved, cryosectioned and processed for substance P receptor - NK1 Receptor (NK1R) immunofluorescent staining. Relative immunofluorescent density was measured. For analysis of dorsal horn secondary sensory neuron activation, all 3 treatment groups (P1, P21 and N) were intraplantarlly injected bilaterally with 5% fomalin to induce intense pain rechallenge. Five minutes later, rats were sacrificed and transcardially perfused and fixed by 4℃ fixatives. L4-L5 spinal cord were retrieved, cryosectioned and processed for phosphorylated extracellular signal-regulated kinase (pERK) immunohistochemistry staining by ABC method. Numbers of pERK (+) neurons were counted and compared. All data were presented as mean±SEM, Paired T test was performed for side-by-side comparison. ANOVA test was performed for comparison among the 3 treatment groups. P<0.05 was considered statistically significant. All analysis was assisted by statistic software STATA 8.0. Results: (1) Pain thresholds assessed by CO2 pulse laser stimulation showed that thresholds were significantly higher in P1_nonCFA side than in P1_CFA side (71.66±1.47 vs 65.00±1.86 p=0.004). P1_nonCFA side had greater pain threshold than P21 and N groups as well. (ANOVA，F=5.38，p=0.0030). The difference of pain thresholds among P1_CFA side, P21 and N groups were not significant. Nocifensive behaviors induced by supra-threshold pain stimulation was significant greater in P1_CFA than in P1_non CFA, P21 and N groups. (ANOVA, F=10.57, p=0.0001) (2)In both L4 and L5, after 8 minutes of intense pain rechallenge, the NK1R relative immunofluorescent densities were significantly greater in P1_CFA side than in P1_nonCFA side. (3)In both L4 and L5, after intense pain rechallenge, the number of pERK (+) neurons of superficial dorsal horn was significantly greater in P1_CFA side than in P1_nonCFA side and than in P21 and N groups. (P1_CFA > P1_nonCFA > P21=N). Conclusion: Adult rat experienced neonatal inflammatory insult has higher pain threshold and stronger nocifensive behaviors. When suffering from intense peripheral pain rechallenge, it has stronger dorsal horn trans-synaptical transmission at the neonatal insult site. It has more superficial dorsal horn neurons activated by peripheral noxious stimulation, especially at neonatal insult site. Similar responses cannot be elicited in adult rat experienced juvenile inflammatory pain. These long term changes of nociceptive signal transduction happen only when the neonatal insults occurred during “window of vulnerability”.