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Up-regulation of substance P in the lungs during acute myocardial ischemia and infarction in rats
Regulatory Peptides 160 (2010) 160–167
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Regulatory Peptides j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / r e g p e p
Up-regulation of substance P in the lungs during acute myocardial ischemia and infarction in rats Zheng Guo a,b,⁎, Xiu-Ping Wang a, Jin-Ping Wang a, Ri-Hua Zhou a, Li-Li Wang a, Jie Wu a a b
Department of Anesthesiology, Shanxi Medical University and Second Hospital of Shanxi Medical University, 56 South Xinjian Nan Road, Taiyuan 030001, Shanxi, PR China Key Laboratory of Cellular Physiology, Shanxi Medical University, National Education Commission, PR China
a r t i c l e
i n f o
Article history: Received 30 September 2009 Received in revised form 21 November 2009 Accepted 22 November 2009 Available online 1 December 2009 Keywords: Substance P Lungs Acute myocardial infarction
a b s t r a c t Objective: Previous studies indicate that disturbance of respiratory functions during acute myocardial ischemia and infarction was not always parallel with decline of cardiac functions, indicating that some nonhemodynamic mechanism might be underlying the changes in the lungs. Methods: The current study was designed to investigate the changes in the expression of substance P and its mRNA in the lungs and dorsal root ganglia using assays of immunohistochemistry, enzyme immunoassay, in situ hybridization and real-time quantitative reverse transcription-polymerase chain reaction, with a rodent model of acute myocardial infarction induced by permanent coronary artery occlusion, without and with epidural anesthesia in the upper thoracic segments of the spinal cord. Results: Marked up-regulation of substance P in the lungs and the dorsal root ganglia of upper thoracic segments (T1–T5) was detected at 15, 30, 60, 180 and 360 min of coronary artery occlusion. The peptide was observed in the alveolar epithelium, mainly in type II pneumocytes, epithelium of bronchiole and the vascular walls. The preprotachykinin mRNA was mainly observed in the dendrites of nerve cells in the alveolar wall, the bronchiole and the pulmonary vessels and the basal lamina of the bronchiole. Pre-treatment of the animals with epidural local anesthetic could completely abolish the up-regulation of SP in the lungs and the dorsal root ganglia. Conclusions: The findings may suggest the involvement of substance P in the pathology of cross-talk between the heart and lungs in acute myocardial ischemia and infarction. Neural mechanism may be involved in the upregulation of substance P in the lungs. © 2009 Elsevier B.V. All rights reserved.
1. Introduction The functions of the heart and lungs are closely associated with each other under physiological and pathological conditions. For instance, when the heart fails to maintain the normal cardiac output, the pulmonary blood volume will increase, which may lead to an abnormality of the lungs and pulmonary function. Substantial evidence from clinical and laboratory studies presents the association of respiratory disturbances with acute myocardial infarction in patients [1–3] and experimental animals [4–6]. It was noticed that the incidence of the disturbances was not always parallel with the decline of the functions of left ventricle. However, pathophysiological and molecular profiles of the mechanism underlying the cross-talk between the heart and lungs under the pathologic condition are still unclear.
Abbreviations: CAO, coronary artery occlusion; ECG, electrocardiography; EIA, enzyme immunoassay; IHC, immunohistochemistry; ISH, in situ hybridization; PPT mRNA, preprotachykinin mRNA; qRT-PCR, real-time quantitative reverse transcriptionpolymerase chain reaction. ⁎ Corresponding author. Tel.: +86 351 3365790; fax: +86 351 2024239. E-mail address: [email protected] (Z. Guo). 0167-0115/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2009.11.015
It was found that acute myocardial ischemia and infarction could cause a remarkable increase in the activity of sympathetic nervous system and massive release of catecholamines [7]. Neuronal evidence associated with acute myocardial ischemia was reported as an increase in the activity of spinal neuron during coronary artery occlusion (CAO) by other researchers [8,9] and as increases in neuronal discharges in nociception specific neurons in the thalamus by our group [10]. We found that acute focal myocardial ischemia and infarction could cause a marked increase in the expression of substance P (SP) in the global myocardium, including the remote virgin myocardium [11], which was not directly insulted by acute ischemic injury, and in the upper thoracic dorsal root ganglia (DRG) and spinal cord [12] of the rats during coronary artery occlusion. The findings may imply that neural mechanism may be involved in the pulmonary reactions during acute myocardial ischemia and infarction. Anatomically, the heart and lungs share some common innervations of autonomic efferent and primary sensory afferents [13–21]. Extending the observations based on the knowledge, we hypothesize that the potential cross-talk between the heart and lungs could be mediated through neural mechanism, activating the biochemicals in the lungs, which may be triggered and modulated by the neural signals associated with acute myocardial ischemia and infarction.
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Fig. 1. Protocol of the experiment. The ‘timings’ of ‘intervention’ (CAO, coronary artery occlusion; EA, epidural anesthesia) and the collection of the samples (lungs shown as ‘L’ and ‘DRG’) for the assays are presented. The assays are presented by the circles.
The current study was designed to examine the changes in the expression of SP and its mRNA, preprotachykinin mRNA (PPT mRNA), in the lungs of the animals in acute myocardial infarction induced by permanent coronary artery occlusion in rats. The neural mechanism of the reaction was testified by blockade in the spinal nerves innervating the heart and lungs through pre-treatment of the animals with epidural anesthesia in the thoracic segments of the spinal cord.
Fig. 1 shows the protocol of the study. The experiments were carried to examine the expressions of SP at 15, 30, 60, 180 and 360 min of the CAO in the lungs and the DRG of upper thoracic segments (T1–T5), compared with the time-matched sham surgery controls, in rats. The changes of SP (at 180 min of CAO) and β-PPTA mRNA (at 30 min of CAO) in the lungs and the DRG of the CAO animals without and with pre-treatment of thoracic epidural anesthesia were also investigated.
2. Materials and methods 2.2. Epidural catheterization 2.1. Protocols The experiments were conformed to the guidelines for the care and use of laboratory animals (National Institute of Health Guide for the Care and Use of Laboratory Animals, NIH Publications No. 80-23, revised 1996, http://grants.nih.gov/grants/olaw/olaw.htm) and approved by the Institutional Animal Care and Use Committee of Shanxi Medical University. One hundred and fifty-one adult male Sprague–Dawley rats, weighing 260 g–280 g, supplied by the Experimental Animal Center of Shanxi Medical University, were employed in the study and 147 animals fulfilled the experiment, while three animals died of bleeding and one died of obstruction of airway during the CAO. The animals were housed individually and provided with laboratory chow and water ad libitum in an air-conditioned environment (with 12 h light– dark cycle). Efforts were made to minimize the number of animals used and their suffering in the study.
The epidural catheterization was performed as previously reported [22]. Each rat was placed in prone position under general anesthesia. The occipitoatlantoid articulations were exposed. A small incision was made through the occipitoaxial ligament. A fine tubing of length 3 cm made from PE-10 tubing (0.61 mm outer diameter, with a dead capacity of about 8 µl; BD Intramedic, Franklin Lakes, New Jersey, USA) was inserted caudally into the epidural space, with the tip of the tubing reaching at the level of T2–T3 of the rat. Sutures were made to secure the placement of the catheter and close the wound. The dead capacity of the catheter was flushed with 0.9% physiological saline. The rats were allowed to recover from the surgery and the general anesthesia for 48 h before further experiment. Animals exhibiting any sign of neurological impairment, evidenced by paralysis, abnormal gait, weight loss or negligent grooming, were excluded from the study. Rats were housed individually to ensure catheter patency. A successful epidural catheterization was verified after the animal was
Fig. 2. Quantitation of the purity of the RNA samples. Agilent 2100 Bioanalyzer was used to analyze the concentration and integrity of the RNA samples using the RNA 6000 Pico LabChip kit. The quality of the samples was determined by the 28S/18S rRNA ratio. The histogram represents RNA concentration = 89 ng/μl; the rRNA ratio (28S/18S) = 1.2; RNA integrity number (RIN) = 8.6, verifying high quality RNA.
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immediately recovered from the surgery and anesthesia by presentation of reversible segmental loss of response to noxious stimulation in thoracic dermatomal segments, (T1–T8) without motor and sensory disturbance in the hind limbs following injection of 15 µl of 2% lidocaine through the catheter. In the study, 15 µl of 0.5% ropivacaine was epidurally administered (at a rate of 1 µl/s) 15 min before the CAO. The range of distribution of the drug in the epidural space was examined in three animals (between the arrows, as shown in Supplementary material). After completion of each experiment, the position of catheter was verified for each animal by autopsy. 2.3. Acute myocardial infarction model The animals were anesthetized with sodium pentobarbital (induction with 65 mg kg− 1, i.p., and maintained with 15 mg kg− 1 h− 1, i.v.).
The procedure of coronary artery occlusion and sham surgery was performed as previously reported [11,12]. Briefly, the rat's pericardium was opened through an incision in the left fourth intercostal space for the animals of CAO and sham surgery group under anesthesia. The anterior part of the heart was exposed. Till then the chest was closed for the animals of the sham surgery group. A suture was made around the left anterior descending branch of the coronary artery and ligation of the artery was performed in animals of the CAO group. In the procedure of ligation of the coronary artery, special attention was paid in placing the suture around the artery, assuring that the artery was occluded without re-opening in the maneuver, by exerting strength through the suture, i.e. the artery was ligated under a proper sustained pulling force through the suture. Then the chest was closed under negative-pressure. Spontaneous respiration was restored. The length of time consumed for open-chest maneuver in the animals of the CAO and sham surgery groups was not statistically different (5 ± 1 min).
Fig. 3. Changes in ST segment during CAO. The ECG shows the ST segment in pre-CAO status and during the CAO (for 6 h) in a CAO animal. Significant elevation of ST segment was detected after onset of the CAO, indicating myocardial injury.
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The CAO was confirmed by changes in the ECG during the experiment and by autopsy at the end of each test. The anesthesia was maintained and the ECG and blood pressure (through left femoral artery) were monitored (Multiphysiological signal processing system, RM 6240BD, Chengdu, China) and the respiratory patterns were watched throughout the experiments. At the end of the experiment the animals were euthanized by an overdose of sodium pentobarbital. 2.4. Immunohistochemistry The samples of the median lobe of the right lungs were collected from CAO animals (n = 24, 6 animals for each subgroup) as scheduled at 15 min, 30 min, 60 min, 180 min and 360 min of CAO or sham (n = 24, 6 animals for each subgroup) after perfusion with 0.9% saline (50 ml/5 min) followed by 4% paraformaldehyde (400 ml/40 min) through the aorta. The samples were embedded in OCT (Optimal Cutting Temperature, Bioportfolio, Dorset, UK) and sectioned at 10 µm on a cryostat (Leica CM 1850, Germany). The sections were processed, as previously reported [12]. The primary anti-substance P (rabbit antirat, Serotec Ltd, UK) at the dilution of 1:1000 and the secondary antibody (goat anti-rabbit, Zhongsan Biotechnology Inc, Beijing, China) at the dilution of 1:200 in PBS were used in the experiment.
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The specificity of the antibodies was examined simply by omitting the primary anti-SP with the other procedure the same as described above. 2.5. Enzyme immunoassay The samples of the right lungs and the bilateral DRG of T1–T5 segments were quickly removed from CAO and sham surgery animals at 15 min, 30 min, 60 min, 180 min and 360 min of CAO or the surgery, respectively. The DRG from each animal were pooled and analyzed separately with EIA Kit (Cayman Chemical, Michigan, USA) according to the manufacturer's instruction and presented as pg/ mgTP (pg/per milligram of total protein of the tissues). Effect of epidural anesthesia on the expression of SP was performed in the CAO animals (3 h) without and with thoracic epidural anesthesia pretreatment and in the time-matched sham group (pretreated with epidural injection of 0.9% saline). 2.6. In situ hybridization In situ hybridization (ISH) was performed to investigate the location of β-PPTA mRNA in the lungs in three CAO (30 min) animals,
Fig. 4. Expression of SP-immunoreactive material in the lungs and DRG. SP-immunoreactive material was markedly increased in the alveolar epithelium (B), bronchioles (D) and pulmonary vessels (F) in a CAO (0.5 h) animal, compared with the sham group (A, C and E). The scale bar represents 400 μm in A, B, C, D, E and F.
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using digoxigenin-labelled riboprobes, with a mix of β-PPTA mRNA probe of ISH Kit (rat PPT-A mRNA, Boster Biotechnology Inc, Wuhan, China) according to the manufacturer's instruction. Each staining run included appropriate positive and negative controls. 2.7. Real-time quantitative reverse transcription-polymerase chain reaction The effects of CAO and the thoracic epidural anesthesia on the transcription of β-PPTA mRNA in the lungs and the DRG were studied at 30 min of CAO in the animals. The samples of the right lungs and the bilateral DRG of T1–T5 segments were collected, pooled and analyzed for CAO animals without and with pre-treatment of epidural anesthesia and the sham group (n = 18, 6 rats for each group). Total RNA was isolated from samples using an Absolutely RNA® Miniprep Kit (Stratagene, USA), in accordance with the manufacturer's instructions. An Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) was used to measure RNA concentration and integrity (Fig. 2) to ensure the RNA integrity was adequate for analysis. Synthesis of cDNA was done using a TIANScript RT Kit according to the manufacturer's instructions (TIANGEN BIOTECH CO. LTD., Beijing, China). For PCR, 1 μl of the cDNA was added to a pre-made reaction mixture containing the following components: 11.25 μl of a standard commercial premixed PCR reagent solution (Syber Green 2× PCR Master Mix, TIANGEN BIOTECH CO. LTD., Beijing, China), 2 μl of a solution containing 100 nmol of gene specific forward and reverse primers and 100 nmol of primers specific for the internal standard, and 10.75 μl of water. The primers for the target gene are as follows: β-PPTA: 5′-GAGCCCTTTGAGCATCTTCT-3′ (forward), 5′-ACGCCTTCTTTCGTAGTTCTG-3′ (reverse, amplified fragment = 252 bp) and β-actin: 5′-TGGAATCCTGTGGCATCCAT-3′ (forward), 5′-TAAAACGCAGCTCAGAACA-3′ (reverse, amplified fragment = 349 bp). Reactions were denatured at 95 °C for 10 min and then PCR was carried out on a fluorescent temperature cycler (MX3005P Real-time PCR System; Stratagene, USA) using the following cycling conditions for 40 cycles: 95 °C for 20 s, 61 °C for 30 s and 68 °C for 1 min. A control (‘no template control’) reaction was also run for each primer set to determine the extent, if any, of non-specific amplification occurring in a given reaction. All reactions were run in triplicate and average values were reported.
Fig. 5. Quantitative analysis of expression of SP in the lungs and the DRG. The concentrations of SP in the lungs (A) and DRG (B) of CAO and sham group at 0.5 h, 1 h, 3 h and 6 h were significantly greater in CAO group than the sham (*P b 0.05, ▲P b 0.01, vs. sham).
sham controls (Fig. 4A, C and E). The increase in the expression could be observed at 30 min of the CAO and throughout 6 h of the experiment.
3.2. Enzyme immunoassay The concentration of SP was significantly increased in the lungs (Fig. 5A) at 30 min (P b 0.01) and in the DRG (Fig. 5B) as early as 15 min (P b 0.01) of CAO and throughout the 360 min of the study in the CAO animals, compared with the sham. There was a 100% suppression of the up-regulation of SP in the lungs (Fig. 6A, P b 0.05), and 81.69 ± 11.67% suppression in the DRG (Fig. 6B, P b 0.05) at 180 min of
2.8. Statistical analysis Data are expressed as mean ± SD of the experiments. A factorial ANOVA followed by post hoc Tukey's test was used to compare the changes of SP detected by EIA (data shown in Fig. 4A and B). One-way ANOVA followed by a post hoc Bonferroni's test was performed to analyze the changes of SP or its mRNA (data shown in Figs. 5 and 7). P value less than 0.05 was considered significant. 3. Results The elevation of the ST segment in ECG was detected 5–10 min after the coronary artery occlusion and throughout the 360 min of the observation (Fig. 3), which indicates myocardial injury. There was no obvious variation in artery blood pressures, heart rate and breathing patterns in the animals with coronary artery occluded, when compared with the controls, throughout the 6 h of experiment. 3.1. Immunohistochemistry The immunoreactive material for SP was observed in the lungs of the CAO rats and the sham surgery groups. The immunoreactive material was markedly increased in CAO animals, mainly in the alveolar epithelium, mainly in type II pneumocytes, epithelium of bronchiole and vascular walls (Fig. 4B, D and F) in the lungs, compared with the
Fig. 6. Effect of epidural anesthesia on the changes in SP. Difference in the concentration of SP in the lungs (A) and DRG (B) of CAO (3 h) animals without and with thoracic epidural anesthesia (TEA) was observed and compared with the sham (**P b 0.01, vs. sham; ▲P b 0.05, vs. CAO).
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CAO in epidural anesthesia group, compared with the time-matched CAO animals, given 15 μl of 0.9% saline.
3.3. In situ hybridization The β-PPT mRNA was mainly observed in the dendrites of ganglion nerves and in the alveolar wall, the basal lamina of the bronchiole and the pulmonary vessels of the CAO (30 min) animals (Fig. 7A–E).
3.4. Real-time quantitative reverse transcription-polymerase chain reaction The results showed a significant increase in the number of copies of β-PPTA mRNA in the lungs (Fig. 8A) and the DRG (Fig. 8B) of the CAO (30 min) animals, compared with the sham group (P b 0.01). The up-regulation of β-PPTA mRNA was inhibited in epidural anesthesia group, by 69 ± 7% in the lungs (P b 0.05, vs. CAO group) and 51 ± 11% in the DRG (P b 0.05, vs. CAO group).
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4. Discussion Functionally, the heart and lungs are working in good co-ordination under physiological condition, which is mainly maintained by neural and neuro-humoral regulations. The pulmonary disturbances in acute myocardial ischemia and infarction [1–6] may indicate a link between the pathological changes of the heart and lungs. However, the underlying mechanism could not always be explained by a decline of cardiac functions and remains unknown. Anatomically, the heart and lung share some common innervations of autonomic nerves and primary sensory afferents. Based on the knowledge and extending the observations, we hypothesize that the potential inter-organ activation of biochemicals in the lungs by the neural signals associated with acute myocardial ischemia could happen. SP is an important neurotransmitter of primary sensory afferents of the lungs [13–15,17–20] and could be up-regulated in response to stress [23], which is found associated with some inflammatory disturbances in respiratory system [14,15]. It was firstly documented by Bayliss that stimulation of neurons in dorsal root ganglia resulted in vasodilation [24], suggesting that the afferent (sensory) nerves also had an efferent (motor) function. Substance P and calcitonin
Fig. 7. Location of the PPT mRNA in the lungs and the DRG. PPT mRNA was observed in the dendrites of ganglion cells in the alveolar wall (A, B), the basal lamina of the bronchiole (A, C, D) and the pulmonary vessels (A, E) of the CAO animals. The scale bar represents 200 μm in D and E, 800 μm in B and C and 2000 μm in A.
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Fig. 8. Quantitative analysis of the changes of PPT mRNA in the lungs (A) and the DRG (B). The expression of PPT mRNA was highly up-regulated in CAO animals (**P b 0.01, vs. sham) and attenuated by thoracic epidural anesthesia (TEA, ▲P b 0.05, vs. CAO and *P b 0.05, vs. sham).
gene related peptide are identified as key neurotransmitters of the function. It was found that acute myocardial ischemia and infarction could cause increases in the neuronal activity of spinal and thalamic neurons [8–10] and up-regulation of SP in the neurons of thoracic dorsal root ganglia and spinal cord in laboratory animals [10,25]. The neural response of the nervous system to myocardial ischemia and infarction could be expressed as an increase in chemicals in the DRG cells and release of the bioactive chemicals from their terminals [26]. SP, as a mediator and neurotransmitter of the primary afferent neurons, could participate in the response in two ways [26]. One is the release of SP from the central axon of the DRG cells to the dorsal horn of spinal cord, conveying the neural signals from ischemic myocardium to the central nervous system, as shown in previous studies [11,12], which may further cause neural reaction of supra spinal nervous structures including sympathetic and parasympathetic nervous system. Another way is releasing SP from the peripheral terminals of the DRG cells innervating the organs, such as the lungs. Extending the observations based on the neural anatomic features of the heart and lungs, we proposed that the neural mechanism is one of the main linkages between the up-regulation of SP in the lungs and acute myocardial infarction. The current findings of up-regulation of SP and β-PPT mRNA, in the pulmonary structures and in the thoracic DRG during acute myocardial ischemia and infarction and the facts that changes in SP and its mRNA could be blocked by epidural anesthesia in thoracic spinal segments, support the hypothesis. The up-regulation of SP in the lungs could be the result of an increase in synthesis and release of SP by intrinsic pulmonary cells, which might be under neural regulation of the nerves innervating the lungs, possibly including autonomic and sensory afferent nerves, both of which could be blocked by thoracic epidural anesthesia. Alternatively, the SP-containing afferents innervating the lungs, per se, could possibly increase synthesis and release of SP in the lungs under the modulation of the stressful neural signals evoked by CAO. The combination of the mechanisms may be another option of the mechanisms underlying the changes of SP in the lungs during acute myocardial ischemia and infarction. In addition to neural mechanism, some other factors, for instance, hemodynamic change during CAO may potentially play a role in the alterations of SP in the lungs. However, a slight hemodynamic variation
during CAO was observed in the current and our previous study, with the same model and setting [22], which is unlikely to affect pulmonary status. Another possibility was the humoral mechanism. The relevant mediators could travel from ischemic heart to the lungs via circulation, inducing up-regulation of SP in the lungs. SP could be up-regulated in the non-ischemic myocardium during the CAO, as we found in a previous study [11], which could reach the lungs via circulation and exerts bioactive function in the pulmonary structures. Current experimental setting, i.e. using permanent ligation of coronary artery rather than ischemia and reperfusion model and the results showing blockade of up-regulation of SP and its mRNA in the lungs by thoracic epidural anesthesia, did not suggest that this humoral mechanism, if it exists, was playing a dominant role in the up-regulation of SP in the lungs during the early stage (within 6 h) of CAO. However the profiles of the neural mechanism remain unknown and need to be investigated. However, only primary observations were made in this study but the findings provide strong suggestion that SP may be involved in the cross-talk between the heart and lungs in acute myocardial ischemia and infarction, which could be activated and modulated by some mechanisms, among which neural ingredient may play an important role, which is currently not understood. The iceberg may lead us to a better understanding of the physiological and pathophysiological communication between the heart and lungs, which may be associated with pathological changes or its protection in the lungs in acute myocardial infarction, most of which currently remain unknown. 5. Conclusion The findings of up-regulation of SP in the lungs of the rats suffering from acute myocardial infarction may suggest the involvement of SP, as a mediator, in the cross-talk between the heart and lungs, via neural mechanism, in acute myocardial infarction. Acknowledgements This work has been supported by grants from the National Natural Science Foundation of China (30471656 and 30772083). Appendix A. Supplementary data Figure S1. Note: The supplementary material accompanying this article is available at doi:10.1016/j.regpep.2009.11.015. References [1] Biddle T, Khanna P, Yu P, Hodges M, Shah P. Lung water in patients with acute myocardial infarction. Circulation 1974;49:115–23. [2] Gee M, Gwirtz R, Spath J. Extravascular water content of heart and lungs after acute myocardial ischemia/infarction. J Appl Physiol 1978;45:102–8. [3] Timmis A, Fowler M, Burwood R, Gishen VR, Chamberlain D. Pulmonry edema without critical increase in left atrial pressure in acute myocardial infarction. Brit Med J 1981;283:636–8. [4] Collins J, Harris T, McKeen C, Brigham K. Increased lung lymph transport without heart failure after coronary ligation in sheep. J Appl Physiol 1979;47:792–7. [5] Richeson JG, Paulshock C, Yu P. Non-hydrostatic pulmonary edema after coronary artery ligation in dogs. Protective effects of indomethacin. Circ Res 1982;50:301–9. [6] Slutsky RA, Peck WW, Higgins CB. Pulmonary edema formation with myocardial infarction and left atrial hypertension: intravascular and extravascular pulmonary fluid volumes. Circulation 1983;68:164–9. [7] Schömig A. Catecholamines in myocardial ischemia/infarction: systemic and cardiac release. Circulation 1990;82:II_13–22. [8] Blair RW, Ammons WS, Foreman RD. Responses of thoracic spinothalamic and spinoreticular cells to coronary artery occlusion. J Neurophysiol 1984;51:636–48. [9] Foreman RD, Ohata CA. Effects of coronary artery occlusion on thoracic spinal neurons receiving viscerosomatic inputs. Am J Physiol Heart Circ Physiol 1980;238: H667–74. [10] Guo Z, Yuan DJ. Midazolam inhibits cardiac nociception evoked by coronary artery occlusion in rats. Eur J Anaesthesiol 2008;25:479–84. [11] Zhang JW, Guo Z. Changes in substance P in myocardial and dorsal root ganglion following coronary artery occlusion in rats. Chinese J Crit Care Med 2006;18:201–5.
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Regulatory Peptides j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / r e g p e p
Up-regulation of substance P in the lungs during acute myocardial ischemia and infarction in rats Zheng Guo a,b,⁎, Xiu-Ping Wang a, Jin-Ping Wang a, Ri-Hua Zhou a, Li-Li Wang a, Jie Wu a a b
Department of Anesthesiology, Shanxi Medical University and Second Hospital of Shanxi Medical University, 56 South Xinjian Nan Road, Taiyuan 030001, Shanxi, PR China Key Laboratory of Cellular Physiology, Shanxi Medical University, National Education Commission, PR China
a r t i c l e
i n f o
Article history: Received 30 September 2009 Received in revised form 21 November 2009 Accepted 22 November 2009 Available online 1 December 2009 Keywords: Substance P Lungs Acute myocardial infarction
a b s t r a c t Objective: Previous studies indicate that disturbance of respiratory functions during acute myocardial ischemia and infarction was not always parallel with decline of cardiac functions, indicating that some nonhemodynamic mechanism might be underlying the changes in the lungs. Methods: The current study was designed to investigate the changes in the expression of substance P and its mRNA in the lungs and dorsal root ganglia using assays of immunohistochemistry, enzyme immunoassay, in situ hybridization and real-time quantitative reverse transcription-polymerase chain reaction, with a rodent model of acute myocardial infarction induced by permanent coronary artery occlusion, without and with epidural anesthesia in the upper thoracic segments of the spinal cord. Results: Marked up-regulation of substance P in the lungs and the dorsal root ganglia of upper thoracic segments (T1–T5) was detected at 15, 30, 60, 180 and 360 min of coronary artery occlusion. The peptide was observed in the alveolar epithelium, mainly in type II pneumocytes, epithelium of bronchiole and the vascular walls. The preprotachykinin mRNA was mainly observed in the dendrites of nerve cells in the alveolar wall, the bronchiole and the pulmonary vessels and the basal lamina of the bronchiole. Pre-treatment of the animals with epidural local anesthetic could completely abolish the up-regulation of SP in the lungs and the dorsal root ganglia. Conclusions: The findings may suggest the involvement of substance P in the pathology of cross-talk between the heart and lungs in acute myocardial ischemia and infarction. Neural mechanism may be involved in the upregulation of substance P in the lungs. © 2009 Elsevier B.V. All rights reserved.
1. Introduction The functions of the heart and lungs are closely associated with each other under physiological and pathological conditions. For instance, when the heart fails to maintain the normal cardiac output, the pulmonary blood volume will increase, which may lead to an abnormality of the lungs and pulmonary function. Substantial evidence from clinical and laboratory studies presents the association of respiratory disturbances with acute myocardial infarction in patients [1–3] and experimental animals [4–6]. It was noticed that the incidence of the disturbances was not always parallel with the decline of the functions of left ventricle. However, pathophysiological and molecular profiles of the mechanism underlying the cross-talk between the heart and lungs under the pathologic condition are still unclear.
Abbreviations: CAO, coronary artery occlusion; ECG, electrocardiography; EIA, enzyme immunoassay; IHC, immunohistochemistry; ISH, in situ hybridization; PPT mRNA, preprotachykinin mRNA; qRT-PCR, real-time quantitative reverse transcriptionpolymerase chain reaction. ⁎ Corresponding author. Tel.: +86 351 3365790; fax: +86 351 2024239. E-mail address: [email protected] (Z. Guo). 0167-0115/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2009.11.015
It was found that acute myocardial ischemia and infarction could cause a remarkable increase in the activity of sympathetic nervous system and massive release of catecholamines [7]. Neuronal evidence associated with acute myocardial ischemia was reported as an increase in the activity of spinal neuron during coronary artery occlusion (CAO) by other researchers [8,9] and as increases in neuronal discharges in nociception specific neurons in the thalamus by our group [10]. We found that acute focal myocardial ischemia and infarction could cause a marked increase in the expression of substance P (SP) in the global myocardium, including the remote virgin myocardium [11], which was not directly insulted by acute ischemic injury, and in the upper thoracic dorsal root ganglia (DRG) and spinal cord [12] of the rats during coronary artery occlusion. The findings may imply that neural mechanism may be involved in the pulmonary reactions during acute myocardial ischemia and infarction. Anatomically, the heart and lungs share some common innervations of autonomic efferent and primary sensory afferents [13–21]. Extending the observations based on the knowledge, we hypothesize that the potential cross-talk between the heart and lungs could be mediated through neural mechanism, activating the biochemicals in the lungs, which may be triggered and modulated by the neural signals associated with acute myocardial ischemia and infarction.
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Fig. 1. Protocol of the experiment. The ‘timings’ of ‘intervention’ (CAO, coronary artery occlusion; EA, epidural anesthesia) and the collection of the samples (lungs shown as ‘L’ and ‘DRG’) for the assays are presented. The assays are presented by the circles.
The current study was designed to examine the changes in the expression of SP and its mRNA, preprotachykinin mRNA (PPT mRNA), in the lungs of the animals in acute myocardial infarction induced by permanent coronary artery occlusion in rats. The neural mechanism of the reaction was testified by blockade in the spinal nerves innervating the heart and lungs through pre-treatment of the animals with epidural anesthesia in the thoracic segments of the spinal cord.
Fig. 1 shows the protocol of the study. The experiments were carried to examine the expressions of SP at 15, 30, 60, 180 and 360 min of the CAO in the lungs and the DRG of upper thoracic segments (T1–T5), compared with the time-matched sham surgery controls, in rats. The changes of SP (at 180 min of CAO) and β-PPTA mRNA (at 30 min of CAO) in the lungs and the DRG of the CAO animals without and with pre-treatment of thoracic epidural anesthesia were also investigated.
2. Materials and methods 2.2. Epidural catheterization 2.1. Protocols The experiments were conformed to the guidelines for the care and use of laboratory animals (National Institute of Health Guide for the Care and Use of Laboratory Animals, NIH Publications No. 80-23, revised 1996, http://grants.nih.gov/grants/olaw/olaw.htm) and approved by the Institutional Animal Care and Use Committee of Shanxi Medical University. One hundred and fifty-one adult male Sprague–Dawley rats, weighing 260 g–280 g, supplied by the Experimental Animal Center of Shanxi Medical University, were employed in the study and 147 animals fulfilled the experiment, while three animals died of bleeding and one died of obstruction of airway during the CAO. The animals were housed individually and provided with laboratory chow and water ad libitum in an air-conditioned environment (with 12 h light– dark cycle). Efforts were made to minimize the number of animals used and their suffering in the study.
The epidural catheterization was performed as previously reported [22]. Each rat was placed in prone position under general anesthesia. The occipitoatlantoid articulations were exposed. A small incision was made through the occipitoaxial ligament. A fine tubing of length 3 cm made from PE-10 tubing (0.61 mm outer diameter, with a dead capacity of about 8 µl; BD Intramedic, Franklin Lakes, New Jersey, USA) was inserted caudally into the epidural space, with the tip of the tubing reaching at the level of T2–T3 of the rat. Sutures were made to secure the placement of the catheter and close the wound. The dead capacity of the catheter was flushed with 0.9% physiological saline. The rats were allowed to recover from the surgery and the general anesthesia for 48 h before further experiment. Animals exhibiting any sign of neurological impairment, evidenced by paralysis, abnormal gait, weight loss or negligent grooming, were excluded from the study. Rats were housed individually to ensure catheter patency. A successful epidural catheterization was verified after the animal was
Fig. 2. Quantitation of the purity of the RNA samples. Agilent 2100 Bioanalyzer was used to analyze the concentration and integrity of the RNA samples using the RNA 6000 Pico LabChip kit. The quality of the samples was determined by the 28S/18S rRNA ratio. The histogram represents RNA concentration = 89 ng/μl; the rRNA ratio (28S/18S) = 1.2; RNA integrity number (RIN) = 8.6, verifying high quality RNA.
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immediately recovered from the surgery and anesthesia by presentation of reversible segmental loss of response to noxious stimulation in thoracic dermatomal segments, (T1–T8) without motor and sensory disturbance in the hind limbs following injection of 15 µl of 2% lidocaine through the catheter. In the study, 15 µl of 0.5% ropivacaine was epidurally administered (at a rate of 1 µl/s) 15 min before the CAO. The range of distribution of the drug in the epidural space was examined in three animals (between the arrows, as shown in Supplementary material). After completion of each experiment, the position of catheter was verified for each animal by autopsy. 2.3. Acute myocardial infarction model The animals were anesthetized with sodium pentobarbital (induction with 65 mg kg− 1, i.p., and maintained with 15 mg kg− 1 h− 1, i.v.).
The procedure of coronary artery occlusion and sham surgery was performed as previously reported [11,12]. Briefly, the rat's pericardium was opened through an incision in the left fourth intercostal space for the animals of CAO and sham surgery group under anesthesia. The anterior part of the heart was exposed. Till then the chest was closed for the animals of the sham surgery group. A suture was made around the left anterior descending branch of the coronary artery and ligation of the artery was performed in animals of the CAO group. In the procedure of ligation of the coronary artery, special attention was paid in placing the suture around the artery, assuring that the artery was occluded without re-opening in the maneuver, by exerting strength through the suture, i.e. the artery was ligated under a proper sustained pulling force through the suture. Then the chest was closed under negative-pressure. Spontaneous respiration was restored. The length of time consumed for open-chest maneuver in the animals of the CAO and sham surgery groups was not statistically different (5 ± 1 min).
Fig. 3. Changes in ST segment during CAO. The ECG shows the ST segment in pre-CAO status and during the CAO (for 6 h) in a CAO animal. Significant elevation of ST segment was detected after onset of the CAO, indicating myocardial injury.
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The CAO was confirmed by changes in the ECG during the experiment and by autopsy at the end of each test. The anesthesia was maintained and the ECG and blood pressure (through left femoral artery) were monitored (Multiphysiological signal processing system, RM 6240BD, Chengdu, China) and the respiratory patterns were watched throughout the experiments. At the end of the experiment the animals were euthanized by an overdose of sodium pentobarbital. 2.4. Immunohistochemistry The samples of the median lobe of the right lungs were collected from CAO animals (n = 24, 6 animals for each subgroup) as scheduled at 15 min, 30 min, 60 min, 180 min and 360 min of CAO or sham (n = 24, 6 animals for each subgroup) after perfusion with 0.9% saline (50 ml/5 min) followed by 4% paraformaldehyde (400 ml/40 min) through the aorta. The samples were embedded in OCT (Optimal Cutting Temperature, Bioportfolio, Dorset, UK) and sectioned at 10 µm on a cryostat (Leica CM 1850, Germany). The sections were processed, as previously reported [12]. The primary anti-substance P (rabbit antirat, Serotec Ltd, UK) at the dilution of 1:1000 and the secondary antibody (goat anti-rabbit, Zhongsan Biotechnology Inc, Beijing, China) at the dilution of 1:200 in PBS were used in the experiment.
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The specificity of the antibodies was examined simply by omitting the primary anti-SP with the other procedure the same as described above. 2.5. Enzyme immunoassay The samples of the right lungs and the bilateral DRG of T1–T5 segments were quickly removed from CAO and sham surgery animals at 15 min, 30 min, 60 min, 180 min and 360 min of CAO or the surgery, respectively. The DRG from each animal were pooled and analyzed separately with EIA Kit (Cayman Chemical, Michigan, USA) according to the manufacturer's instruction and presented as pg/ mgTP (pg/per milligram of total protein of the tissues). Effect of epidural anesthesia on the expression of SP was performed in the CAO animals (3 h) without and with thoracic epidural anesthesia pretreatment and in the time-matched sham group (pretreated with epidural injection of 0.9% saline). 2.6. In situ hybridization In situ hybridization (ISH) was performed to investigate the location of β-PPTA mRNA in the lungs in three CAO (30 min) animals,
Fig. 4. Expression of SP-immunoreactive material in the lungs and DRG. SP-immunoreactive material was markedly increased in the alveolar epithelium (B), bronchioles (D) and pulmonary vessels (F) in a CAO (0.5 h) animal, compared with the sham group (A, C and E). The scale bar represents 400 μm in A, B, C, D, E and F.
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using digoxigenin-labelled riboprobes, with a mix of β-PPTA mRNA probe of ISH Kit (rat PPT-A mRNA, Boster Biotechnology Inc, Wuhan, China) according to the manufacturer's instruction. Each staining run included appropriate positive and negative controls. 2.7. Real-time quantitative reverse transcription-polymerase chain reaction The effects of CAO and the thoracic epidural anesthesia on the transcription of β-PPTA mRNA in the lungs and the DRG were studied at 30 min of CAO in the animals. The samples of the right lungs and the bilateral DRG of T1–T5 segments were collected, pooled and analyzed for CAO animals without and with pre-treatment of epidural anesthesia and the sham group (n = 18, 6 rats for each group). Total RNA was isolated from samples using an Absolutely RNA® Miniprep Kit (Stratagene, USA), in accordance with the manufacturer's instructions. An Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) was used to measure RNA concentration and integrity (Fig. 2) to ensure the RNA integrity was adequate for analysis. Synthesis of cDNA was done using a TIANScript RT Kit according to the manufacturer's instructions (TIANGEN BIOTECH CO. LTD., Beijing, China). For PCR, 1 μl of the cDNA was added to a pre-made reaction mixture containing the following components: 11.25 μl of a standard commercial premixed PCR reagent solution (Syber Green 2× PCR Master Mix, TIANGEN BIOTECH CO. LTD., Beijing, China), 2 μl of a solution containing 100 nmol of gene specific forward and reverse primers and 100 nmol of primers specific for the internal standard, and 10.75 μl of water. The primers for the target gene are as follows: β-PPTA: 5′-GAGCCCTTTGAGCATCTTCT-3′ (forward), 5′-ACGCCTTCTTTCGTAGTTCTG-3′ (reverse, amplified fragment = 252 bp) and β-actin: 5′-TGGAATCCTGTGGCATCCAT-3′ (forward), 5′-TAAAACGCAGCTCAGAACA-3′ (reverse, amplified fragment = 349 bp). Reactions were denatured at 95 °C for 10 min and then PCR was carried out on a fluorescent temperature cycler (MX3005P Real-time PCR System; Stratagene, USA) using the following cycling conditions for 40 cycles: 95 °C for 20 s, 61 °C for 30 s and 68 °C for 1 min. A control (‘no template control’) reaction was also run for each primer set to determine the extent, if any, of non-specific amplification occurring in a given reaction. All reactions were run in triplicate and average values were reported.
Fig. 5. Quantitative analysis of expression of SP in the lungs and the DRG. The concentrations of SP in the lungs (A) and DRG (B) of CAO and sham group at 0.5 h, 1 h, 3 h and 6 h were significantly greater in CAO group than the sham (*P b 0.05, ▲P b 0.01, vs. sham).
sham controls (Fig. 4A, C and E). The increase in the expression could be observed at 30 min of the CAO and throughout 6 h of the experiment.
3.2. Enzyme immunoassay The concentration of SP was significantly increased in the lungs (Fig. 5A) at 30 min (P b 0.01) and in the DRG (Fig. 5B) as early as 15 min (P b 0.01) of CAO and throughout the 360 min of the study in the CAO animals, compared with the sham. There was a 100% suppression of the up-regulation of SP in the lungs (Fig. 6A, P b 0.05), and 81.69 ± 11.67% suppression in the DRG (Fig. 6B, P b 0.05) at 180 min of
2.8. Statistical analysis Data are expressed as mean ± SD of the experiments. A factorial ANOVA followed by post hoc Tukey's test was used to compare the changes of SP detected by EIA (data shown in Fig. 4A and B). One-way ANOVA followed by a post hoc Bonferroni's test was performed to analyze the changes of SP or its mRNA (data shown in Figs. 5 and 7). P value less than 0.05 was considered significant. 3. Results The elevation of the ST segment in ECG was detected 5–10 min after the coronary artery occlusion and throughout the 360 min of the observation (Fig. 3), which indicates myocardial injury. There was no obvious variation in artery blood pressures, heart rate and breathing patterns in the animals with coronary artery occluded, when compared with the controls, throughout the 6 h of experiment. 3.1. Immunohistochemistry The immunoreactive material for SP was observed in the lungs of the CAO rats and the sham surgery groups. The immunoreactive material was markedly increased in CAO animals, mainly in the alveolar epithelium, mainly in type II pneumocytes, epithelium of bronchiole and vascular walls (Fig. 4B, D and F) in the lungs, compared with the
Fig. 6. Effect of epidural anesthesia on the changes in SP. Difference in the concentration of SP in the lungs (A) and DRG (B) of CAO (3 h) animals without and with thoracic epidural anesthesia (TEA) was observed and compared with the sham (**P b 0.01, vs. sham; ▲P b 0.05, vs. CAO).
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CAO in epidural anesthesia group, compared with the time-matched CAO animals, given 15 μl of 0.9% saline.
3.3. In situ hybridization The β-PPT mRNA was mainly observed in the dendrites of ganglion nerves and in the alveolar wall, the basal lamina of the bronchiole and the pulmonary vessels of the CAO (30 min) animals (Fig. 7A–E).
3.4. Real-time quantitative reverse transcription-polymerase chain reaction The results showed a significant increase in the number of copies of β-PPTA mRNA in the lungs (Fig. 8A) and the DRG (Fig. 8B) of the CAO (30 min) animals, compared with the sham group (P b 0.01). The up-regulation of β-PPTA mRNA was inhibited in epidural anesthesia group, by 69 ± 7% in the lungs (P b 0.05, vs. CAO group) and 51 ± 11% in the DRG (P b 0.05, vs. CAO group).
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4. Discussion Functionally, the heart and lungs are working in good co-ordination under physiological condition, which is mainly maintained by neural and neuro-humoral regulations. The pulmonary disturbances in acute myocardial ischemia and infarction [1–6] may indicate a link between the pathological changes of the heart and lungs. However, the underlying mechanism could not always be explained by a decline of cardiac functions and remains unknown. Anatomically, the heart and lung share some common innervations of autonomic nerves and primary sensory afferents. Based on the knowledge and extending the observations, we hypothesize that the potential inter-organ activation of biochemicals in the lungs by the neural signals associated with acute myocardial ischemia could happen. SP is an important neurotransmitter of primary sensory afferents of the lungs [13–15,17–20] and could be up-regulated in response to stress [23], which is found associated with some inflammatory disturbances in respiratory system [14,15]. It was firstly documented by Bayliss that stimulation of neurons in dorsal root ganglia resulted in vasodilation [24], suggesting that the afferent (sensory) nerves also had an efferent (motor) function. Substance P and calcitonin
Fig. 7. Location of the PPT mRNA in the lungs and the DRG. PPT mRNA was observed in the dendrites of ganglion cells in the alveolar wall (A, B), the basal lamina of the bronchiole (A, C, D) and the pulmonary vessels (A, E) of the CAO animals. The scale bar represents 200 μm in D and E, 800 μm in B and C and 2000 μm in A.
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Fig. 8. Quantitative analysis of the changes of PPT mRNA in the lungs (A) and the DRG (B). The expression of PPT mRNA was highly up-regulated in CAO animals (**P b 0.01, vs. sham) and attenuated by thoracic epidural anesthesia (TEA, ▲P b 0.05, vs. CAO and *P b 0.05, vs. sham).
gene related peptide are identified as key neurotransmitters of the function. It was found that acute myocardial ischemia and infarction could cause increases in the neuronal activity of spinal and thalamic neurons [8–10] and up-regulation of SP in the neurons of thoracic dorsal root ganglia and spinal cord in laboratory animals [10,25]. The neural response of the nervous system to myocardial ischemia and infarction could be expressed as an increase in chemicals in the DRG cells and release of the bioactive chemicals from their terminals [26]. SP, as a mediator and neurotransmitter of the primary afferent neurons, could participate in the response in two ways [26]. One is the release of SP from the central axon of the DRG cells to the dorsal horn of spinal cord, conveying the neural signals from ischemic myocardium to the central nervous system, as shown in previous studies [11,12], which may further cause neural reaction of supra spinal nervous structures including sympathetic and parasympathetic nervous system. Another way is releasing SP from the peripheral terminals of the DRG cells innervating the organs, such as the lungs. Extending the observations based on the neural anatomic features of the heart and lungs, we proposed that the neural mechanism is one of the main linkages between the up-regulation of SP in the lungs and acute myocardial infarction. The current findings of up-regulation of SP and β-PPT mRNA, in the pulmonary structures and in the thoracic DRG during acute myocardial ischemia and infarction and the facts that changes in SP and its mRNA could be blocked by epidural anesthesia in thoracic spinal segments, support the hypothesis. The up-regulation of SP in the lungs could be the result of an increase in synthesis and release of SP by intrinsic pulmonary cells, which might be under neural regulation of the nerves innervating the lungs, possibly including autonomic and sensory afferent nerves, both of which could be blocked by thoracic epidural anesthesia. Alternatively, the SP-containing afferents innervating the lungs, per se, could possibly increase synthesis and release of SP in the lungs under the modulation of the stressful neural signals evoked by CAO. The combination of the mechanisms may be another option of the mechanisms underlying the changes of SP in the lungs during acute myocardial ischemia and infarction. In addition to neural mechanism, some other factors, for instance, hemodynamic change during CAO may potentially play a role in the alterations of SP in the lungs. However, a slight hemodynamic variation
during CAO was observed in the current and our previous study, with the same model and setting [22], which is unlikely to affect pulmonary status. Another possibility was the humoral mechanism. The relevant mediators could travel from ischemic heart to the lungs via circulation, inducing up-regulation of SP in the lungs. SP could be up-regulated in the non-ischemic myocardium during the CAO, as we found in a previous study [11], which could reach the lungs via circulation and exerts bioactive function in the pulmonary structures. Current experimental setting, i.e. using permanent ligation of coronary artery rather than ischemia and reperfusion model and the results showing blockade of up-regulation of SP and its mRNA in the lungs by thoracic epidural anesthesia, did not suggest that this humoral mechanism, if it exists, was playing a dominant role in the up-regulation of SP in the lungs during the early stage (within 6 h) of CAO. However the profiles of the neural mechanism remain unknown and need to be investigated. However, only primary observations were made in this study but the findings provide strong suggestion that SP may be involved in the cross-talk between the heart and lungs in acute myocardial ischemia and infarction, which could be activated and modulated by some mechanisms, among which neural ingredient may play an important role, which is currently not understood. The iceberg may lead us to a better understanding of the physiological and pathophysiological communication between the heart and lungs, which may be associated with pathological changes or its protection in the lungs in acute myocardial infarction, most of which currently remain unknown. 5. Conclusion The findings of up-regulation of SP in the lungs of the rats suffering from acute myocardial infarction may suggest the involvement of SP, as a mediator, in the cross-talk between the heart and lungs, via neural mechanism, in acute myocardial infarction. Acknowledgements This work has been supported by grants from the National Natural Science Foundation of China (30471656 and 30772083). Appendix A. Supplementary data Figure S1. Note: The supplementary material accompanying this article is available at doi:10.1016/j.regpep.2009.11.015. References [1] Biddle T, Khanna P, Yu P, Hodges M, Shah P. Lung water in patients with acute myocardial infarction. Circulation 1974;49:115–23. [2] Gee M, Gwirtz R, Spath J. Extravascular water content of heart and lungs after acute myocardial ischemia/infarction. J Appl Physiol 1978;45:102–8. [3] Timmis A, Fowler M, Burwood R, Gishen VR, Chamberlain D. Pulmonry edema without critical increase in left atrial pressure in acute myocardial infarction. Brit Med J 1981;283:636–8. [4] Collins J, Harris T, McKeen C, Brigham K. Increased lung lymph transport without heart failure after coronary ligation in sheep. J Appl Physiol 1979;47:792–7. [5] Richeson JG, Paulshock C, Yu P. Non-hydrostatic pulmonary edema after coronary artery ligation in dogs. Protective effects of indomethacin. Circ Res 1982;50:301–9. [6] Slutsky RA, Peck WW, Higgins CB. Pulmonary edema formation with myocardial infarction and left atrial hypertension: intravascular and extravascular pulmonary fluid volumes. Circulation 1983;68:164–9. [7] Schömig A. Catecholamines in myocardial ischemia/infarction: systemic and cardiac release. Circulation 1990;82:II_13–22. [8] Blair RW, Ammons WS, Foreman RD. Responses of thoracic spinothalamic and spinoreticular cells to coronary artery occlusion. J Neurophysiol 1984;51:636–48. [9] Foreman RD, Ohata CA. Effects of coronary artery occlusion on thoracic spinal neurons receiving viscerosomatic inputs. Am J Physiol Heart Circ Physiol 1980;238: H667–74. [10] Guo Z, Yuan DJ. Midazolam inhibits cardiac nociception evoked by coronary artery occlusion in rats. Eur J Anaesthesiol 2008;25:479–84. [11] Zhang JW, Guo Z. Changes in substance P in myocardial and dorsal root ganglion following coronary artery occlusion in rats. Chinese J Crit Care Med 2006;18:201–5.
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