Brain Research 1038 (2005) 238 – 242 www.elsevier.com/locate/brainres
Spinal preprodynorphin mRNA expression in neonatal rats following peripheral inflammation Rui-Xin Zhanga,*, Bing Liua, Lixing Laoa, Jian-Tian Qiaob, M.A. Rudac a
Center for Integrative Medicine, School of Medicine, University of Maryland, Baltimore, MD 21201, USA b Department of Neurobiology, Shanxi Medical University, Taiyuan, Shanxi, 030001, P. R. China c Pain and Neurosensory Mechanisms Branch, NIDCR, NIH, Bethesda, MD 20892, USA Accepted 11 January 2005
Abstract Spinal nociceptive neural circuits undergo considerable changes during the postnatal period. This study showed that neonatal rats exhibited earlier upregulation and faster recovery of spinal preprodynorphin (PPD) mRNA than did the adults during complete Freund’s adjuvant (CFA)-induced peripheral inflammation. These data suggest that the central nervous systems of neonates and adults respond differently to peripheral noxious inputs, a fact that should be considered when selecting pain treatment strategies for neonate populations. D 2005 Elsevier B.V. All rights reserved. Theme: Sensory systems Topic: Pain modulation: anatomy and physiology Keywords: Neonates; Complete Freund’s adjuvant; Hyperalgesia; Spinal dorsal horn; Preprodynorphin mRNA; Rats
Pain is a serious clinical problem in newborn, premature and young infants due to the wide application of invasive and traumatic procedures in neonatal intensive care units . Although it has been shown that the central spinal nociceptive neuronal circuits of neonates undergo postnatal development [4–6], the differences in mechanisms underlying the modulation of spinal nociception between neonates and adults have not been elucidated. In adult rats, spinal dynorphin (DYN) is known to play an important role in the processing of noxious inputs at the spinal level [1,7,14,15]. The injection of complete Freund’s adjuvant (CFA) into the hind paw of an adult rat induces inflammation and hyperalgesia in that paw, as well as a significant up-regulation of spinal preprodynorphin (PPD)
* Corresponding author. Center for Integrative Medicine, 3rd Floor, James Kernan Hospital Mansion, 2200 Kernan Drive, Baltimore, MD 21207, USA. Fax: +1 410 706 1583. E-mail address: [email protected]
(R.-X. Zhang). 0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.01.039
mRNA and DYN [10,13,17]. However, little is known about PPD mRNA expression in the spinal dorsal horn of neonates following persistent noxious stimulation. The aim of the present study was to characterize the PPD mRNA expression and behavioral changes in neonatal rats as compared to adult rats during the CFA-induced hind paw inflammation. Two sets of experiments were conducted to examine spinal PPD mRNA, the first using the reverse transcription polymerase chain reaction (RT-PCR) technique and the second using Northern blot. The first set was performed on four groups (n = 8 each) of Sprague–Dawley rats: a group of adult males aged 60 days, and three neonatal groups, aged 13, 9 and 3 days with day of birth defined as P0. Each neonatal group contained pups from different litters. In each group, 4 animals were used as control and the others received CFA injections. Because previous studies have shown that saline injected into the hind paw of rats does not increase PPD mRNA expression in the spinal cord [10,16], we did not inject vehicle in control groups in the present
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study. In CFA groups, one hind paw of each rat was injected subcutaneously with CFA (Sigma). After CFA injection, only the injected paw exhibits inflammation and hyperalgesia and the CFA does not disperse throughout the whole body. Since hind paw volume was 1.2 ml in adults and 0.45–0.55 ml between days P9–13, we adjusted the CFA solution volumes to make them roughly comparable to the hind paw size in each group. However, because the same dosage of CFA might initiate similar peripheral nociceptive inputs, we kept the amount of CFA constant and injected a 1:1 oil/saline CFA emulsion into the adults and a 9:1 (the highest concentration of CFA that can be made from the commercial product) solution into the P9–13 group. That is, each adult received 81 Al vehicle containing 40.5 Ag of CFA and each P9–13 pup received 45 Al vehicle containing 40.5 Ag of CFA. The hind paws of the P3 pups were about half that of the P13, or 0.27 ml, too small to absorb 40.5 Ag of CFA in the 9:1 solution, so we injected them with only 20 Ag CFA in a 9:1 solution. Twenty-four hours after CFA injection, rats or pups were deeply anesthetized with 50 mg/kg of sodium pentobarbital and exsanguinated by cardiac puncture. An 8-mm portion of the lumbar enlargement of the spinal cord was removed from adults, while a 4-mm portion was removed from pups. Each spinal cord was divided along the midline into CFAinjected and contralateral sides, immediately frozen in dry ice, and stored at 80 8C. Total RNA was extracted from the spinal L4–5 using the RNeasy Mini kit (QIAGEN Sciences, MD) following the procedure recommended by the manufacturer. A 260/280 ratio of the final RNA solution was not less than 1.90. The total RNA extracted from these groups was used for RT-PCR analysis to show potential difference of spinal PPD mRNA expression induced by CFA. For quantitative comparison of RT-PCR product, a standard curve was constructed . The cDNA, synthesized through reverse transcription using 250 ng of total RNA, was subjected to 17–31 cycles of PCR in a 50-Al OneStep RT-PCR reaction mixture (QIAGEN). A volume of 20 Al of each reaction mixture subjected to 17–31 cycles of PCR was electrophoresed on 3% ethidium bromide-stained agarose gel. The PCR products, indicated by intensity of ethidium bromide, were plotted on a logarithmic scale against the PCR cycle number. Similarly, we determined the relationship of PCR product accumulation and the amount of RNA used in the RT-PCR. Synthesized cDNA produced by using 250–1000 ng of total RNA was subjected to 26 cycles of PCR in a 50 Al RT-PCR reaction mixture. The results were plotted on a logarithmic scale against the amount of total RNA used in the RT-PCR reaction. These preliminary analyses ensured that the following PCR reactions were in the linear range for both glyceraldehyde phosphate dehydrogenase (GAPDH) and PPD genes. Thus, in the subsequent experiments, the RT-PCR reaction mixture (50 Al) contained 250 ng of total RNA as template, 5 QIAGEN OneStep RT-PCR buffer providing a
final concentration of 2.5 mM MgCl2, 4 deoxynucleoside triphosphates (0.4 mM each), enzyme mixture (Omnizcript and Sensiscript reverse transcriptase, HotStarTaq DNA ploymerase), 1 AM of each of the 5V and 3V PPD sequencespecific target primers (5V-TGATGAATGATGAAGCCGCAC-3V/5V-ACCGAGTCACCACCTTGAACTG-3V), and 0.6 AM of each of the 5V and 3VGAPDH sequence-specific target primers (5V-TGAAGGTCGGTGTGAACGGATTTGGC-3V/ 5VCATGTAGGCCATGAGGTCCACCAC-3V). The primers were synthesized by the Biopolymer/Genomics Core Facility at University of Maryland. GAPDH was used as an internal control since its expression is not regulated by inflammation or by the manipulations of these experiments [12,17]. PCR was performed after reverse transcription at 50 8C for 30 min and initial denaturation at 95 8C for 15 min. The temperature cycles (Roche) were 94 8C/30 s (denaturing), 55 8C/40 s (annealing), and 72 8C/1 min (extension). A total of 26 cycles and a final 10-min extension at 72 8C were conducted. The PCR amplified fragments were separated on 3% ethidium bromide-stained agarose gel. The PCR gel image was captured and analyzed by a gel documentation system (DigiGenius Syst. DG1T, SYNGENE, Frederick, MD). The positive PCR bands were purified (Wizard DNA Clean-Up kit, Promega) and sequenced, and the resulting sequences were identical to the targeted cDNA sequences. The raw data from 4 individual RT-PCR analysis of each group were used for statistical analysis. The relative PPD mRNA levels on the CFA-injected sides in different groups were illustrated as a percentage of the naive control with the same ages (see Fig. 2). Mock RT-PCR reaction controls included the omitting of reverse transcriptase, primers, or template. No specific PCR product was found in these reactions. In the second set of experiments, Northern blot was used to analyze the time course of spinal PPD mRNA expression following CFA injection in neonatal and adult rats. One hind paw of each P9 (n = 45) and adult (n = 45) rat was injected with CFA as described above. Pups from any single litter were assigned to different time-point groups. Spinal cord tissues were dissected at 4, 8, 24, 48 or 72 h post-CFAinjection (n = 9 for each time point in both neonatal and adult groups). To correlate spinal PPD mRNA changes to changes in nociceptive behavioral activities, the hind paw withdrawal latency (PWL) of both adult rats and P9 pups was examined prior to CFA injection and just before the Northern blot detection conducted 72 h after CFA injection using the method previously described [9,17]. For Northern blot detection, total RNA was purified with a cesium gradient from each side of the spinal cord as described before . Each extraction consisted of tissue from three P9 neonates or three adult rats. Northern blot was performed as described previously . Autoradiographs were produced by exposing the a-32P dCTP-DYN probelabeled membranes to Biomax film at 70 8C. Membranes were then exposed to a phosphor screen and analyzed using a phosphoimaging system for quantification (Molecular
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Dynamics, Sunnyvale, CA). Northern blots were normalized by reprobing the stripped blots with a GAPDH oligonucleotide (Oncogene Science, Cambridge, MA) labeled with a-32P dATP by a DNA-end labeling method. Autoradiographs and phosphoimage for GAPDH were produced as before. Quantification of PPD mRNA expression was made from three different northern blots produced from three different RNA extractions. Values in both P9 and adult rats were expressed as a percentage of the level of PPD mRNA in contralateral spinal cord 72 h post-CFA (set arbitrarily as 100%) in adult rats or P9 pups. See Fig. 3. Data were presented as mean F SEM. Statistical comparisons were made using analysis of variance (ANOVA), and post hoc comparison was conducted by the Fisher’s protected least significant difference test. P b 0.05 was considered significant in all cases. As shown by the standard curve in Fig. 1, the linear range of the amplification for PPD mRNA was 25–31 cycles in the RT-PCR reactions with 250 ng of total RNA. For GAPDH, the range was 21–29 cycles in the same reactions. Fig. 1 also shows that the amplification was in the linear range when 26 cycles of PCR reaction were performed with 250–1000 ng of total RNA. Therefore, our subsequent RTPCR analyses of PPD expression in groups of different ages were performed for 26 cycles using 250 ng of total RNA. These analyses ensured that the quantitative comparison of PPD mRNA expression was appropriate. The results of the first set of experiments were shown in Fig. 2. It was clear that the spinal constitutive PPD mRNA in naive animals of P3, P9, P13 and adult rats was not significantly different. All groups of adult and neonatal rats showed significantly higher levels of PPD mRNA on the ipsilateral spinal sides than on the contralateral sides, as determined by RT-PCR analysis 24 h post-CFA-injection ( P b 0.01). However, the increased expression of PPD
Fig. 2. (A) RT-PCR analysis of spinal PPD mRNA expression examined 24 h post-CFA injection using 250 ng total RNA from one spinal side of different groups, with GAPDH expression as internal control. (B) Relative quantification of spinal PPD mRNA expression in different groups, with setting arbitrarily the level in naive adult control group as 100%. Each bar represents the mean F SEM. The expression of spinal PPD mRNA on the contralateral side was equal to that in the naive spinal cord in each group. Inductive PPD mRNA up-regulation, measured from the spinal cord ipsilateral to the inflamed hind paw, was significantly greater than the contralateral/naive constitutive expression in each group. **P b 0.01 compared to contralateral/naive sides. I and Ipsi: ipsilateral; C and Contra: contralateral; N: naive.
mRNAs in the CFA-injected sides of the dorsal horn was not different between groups with different ages. The PPD mRNA levels on the contralateral spinal sides of all CFAinjected groups were similar to those of naive groups (Fig. 2). Fig. 3 shows the time course of PPD mRNA upregulation in the CFA-injected sides of the spinal cords of P9 and adult rats as revealed by Northern blot analysis. In P9 neonatal
Fig. 1. Standard curve (top) and an example of agarose gel electrophoresis of PCR products (bottom) for RT-PCR analysis of PPD and GAPDH mRNAs. (A) PCR product amplification is represented as a function of the number of PCR cycles for GAPDH (21–31 cycles) and PPD (25–31 cycles). The cDNA template for each reaction was reverse-transcribed from 250 ng of total RNA extracted from a rat spinal cord. (B) PCR product amplification for GAPDH and PPD is represented as a function of the total RNA (250–1000 ng) used in the RT-PCR (26 cycles).
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Fig. 3. Northern blot analysis showing the time courses of PPD mRNA expression in the spinal cord 4–72 h following CFA injection in P9 (A) and adult rats (B). Note that spinal PPD mRNA upregulation was significantly increased at 8 h, peaked at 24 h, and declined obviously by 72 h post-CFA in P9 (C), while it appeared at 8 h, peaked at 24 h, and persisted at a high level even 72 h post-CFA in adult (D). *P b 0.05 and **P b 0.01 compared to the contralateral side of each group, respectively, #P b 0.05 compared to that detected at the same time point in adult group. I and Ipsi: ipsilateral (black bars); C and Contra: contralateral (hatched bars).
rats, the majority of the total PPD mRNA was expressed during the 8–48 h post-CFA; while in adults, most of this mRNA was expressed during the 24–72 h post-CFA (Fig. 3). Eight hours post-CFA, P9 neonates exhibited significantly higher PPD mRNA than did adults, while 72 h post-CFA, the neonate levels were significantly lower than those of the adults ( P b 0.05). These results indicate that CFA-induced upregulation of spinal PPD mRNA exhibits an earlier initiation and a faster recovery to normal levels in neonates than in adults. Fig. 4 shows the baseline PWLs for P9 and adult rats and the changes detected 72 h post-CFA. The data show that PWL of the injected sides was significantly less in adults than in P9 pups 72 h post-CFA, suggesting that hyperalgesia of the injected hind paw was more severe in the adults ( P b 0.05). The present study demonstrates that the upregulation of spinal PPD mRNA expression on the side ipsilateral to CFA-injection in P3–P13 neonates is the same as that in adult rats 24 h after CFA injection, while PPD mRNA levels on the contralateral sides were unchanged in all age groups, being similar to those of naive groups. These results suggest that the ability to express PPD mRNA is fully developed by P3 [10,13,17]. However, the time-course analysis of the present study clearly demonstrates that upregulation of PPD mRNA expression occurs earlier and progresses more rapidly in neonates than in adult rats. (see Fig. 3). It might be argued that the early increase of PPD mRNA was due to the fact that a relatively larger amount of CFA (equal to that given to the adults) was administered to the P9 group, but this is negated by the fact that the increased PPD mRNA declined
earlier in the P9 rats than in the adults. These data imply that although the DYN-containing neurons in the spinal cord of neonates have a similar reaction to CFA-induced inflammation as those of adults, the dynamics of activation of these neurons differ in neonates and adults. We hypothesize that postnatal development of the central nervous system determines the characteristics of spinal hyperalgesic neurochemical responses such as PPD mRNA expression or
Fig. 4. Comparisons of hind paw withdrawal latencies (PWLs) between P9 (n = 9, right side) and adult rats (n = 9, left side) as measured prior to CFA injection and 72 h post-CFA injection. Note that data were presented as percentage changes of the baseline set arbitrarily as 100%. *P b 0.05 as compared to that in P9 measured 72 h post-CFA injection.
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dynorphin content, thus influencing the behavioral nociceptive responses in neonates. This is supported by previous studies reporting that peripheral polymodal nociceptors show completely matured firing frequencies and response patterns at birth  and that the cutaneous receptive fields of the dorsal horn cells are large in newborn pups and gradually decrease in size over the course of the first two postnatal weeks . Because DYN is the main post-translational product of PPD mRNA in rats, the specific dynamics of spinal PPD mRNA expression in neonates should affect the level of spinal dynorphin [13,17], which is considered to be a pronociceptive agent and thus plays a key role in inducing spinal hyperalgesia [1,7,14,15]. Our nociceptive behavioral examinations 72 h post-CFA injection provide the expected results: adult rats exhibited significantly shorter PWLs than did P9 neonates (see Fig. 4), which indicates greater hyperalgesia in the adult rats. This is consistent with the fact that 72 h post-CFA, the PPD mRNA levels in adults were significantly higher than those in neonates. These results suggest that DYN is one of the critical factors responsible for the characteristics of hyperalgesia in neonates. In summary, the present study showed that neonatal rats exhibit an earlier upregulation and faster recovery of spinal PPD mRNA than adult rats following the CFA-induced peripheral inflammation. As an example, our results suggest that the central nervous system in neonates and adults responds differently to persistent peripheral noxious inputs. Thus, strategies designed for managing and controlling persistent pain in neonates should be based on their structural and functional characteristics that still wait to clarify in detail.
Acknowledgments We would like to thank Dr. Ke Ren for his critical reading of the manuscript and Dr. Lyn Lowry for her editorial support.
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