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Anesth Analg 2001;93:424-429
© 2001 International Anesthesia Research Society

PAIN MEDICINE

The Residual Effects of Hemorrhagic Shock on Pain Reaction and C-Fos Expression in Rats

Taeko Fukuda, MD, Chikako Nishimoto, MD, Masayuki Miyabe, MD, and Hidenori Toyooka, MD

Department of Anesthesiology, Institute of Clinical Medicine, Tsukuba University, Tsukuba, Ibaraki, Japan

Address correspondence and reprint requests to Taeko Fukuda, MD, Department of Anesthesiology, Institute of Clinical Medicine, Tsukuba University, Tsukuba, Ibaraki, 305-8575, Japan. Address e-mail to e-mail: taekof{at}md.tsukuba.ac.jp


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
To investigate the residual effects of hemorrhagic shock on pain reaction and c-fos expression, we performed formalin tests after hemorrhage and reinfusion in rats. Twenty adult male Sprague-Dawley rats were divided into Control (n = 10) and Postshock (n = 10) groups. The mean blood pressure of the Control group was 100–120 mm Hg, and that of the Postshock group was kept at 50–60 mm Hg for 30 min by draining blood. After 15 min of returning mean blood pressure to normal levels in the Postshock group, 10% formalin (3.7% formaldehyde solution, 100 µL) was injected into the left rear paw of both groups. Nociceptive behaviors were observed for 1 h after the formalin injection. The rats were killed at 2 h after the formalin injection, and the lumbar spinal cord was then stained for c-fos immunohistochemistry by using the avidin-biotin-peroxidase method. Animals in the Postshock group showed considerably less nociceptive behavior than those in the Control group. C-fos expression in the deep layer (IV–VI) of the spinal cord was significantly less in the Postshock group. In conclusion, decreases of nociceptive behaviors and c-fos expression were observed under normotensive conditions after hemorrhagic shock. The mechanisms governing these reactions remain unclear.

IMPLICATIONS: Formalin tests were performed after hemorrhage and reinfusion in rats. A stress-induced analgesia was observed under normotensive conditions after hemorrhagic shock. The mechanisms remain unclear.


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Decreases in pain sensitivity/reactivity occur after exposure to a variety of stressful events, such as electric shock, cold swims, and presentation of a natural predator. This phenomenon has been labeled stress-induced analgesia (SIA), and its mechanisms have been investigated (1–3).

Extensive blood loss often occurs in accidents, disasters, and during surgery, and the consequent hypovolemia decreases the requirements for IV and inhaled anesthetics. Some authors have investigated this phenomenon during hypovolemic shock (4–6). However, no report has described pain reactivity under normotensive conditions after hemorrhagic shock. Hemorrhagic shock is a kind of acute stress and might induce SIA. However, many mediators, such as proinflammatory cytokines and nitric oxide, are increased after hemorrhage (7). Because some of these mediators enhance nociception at peripheral inflammatory tissues (8–10), it is also possible that pain reactivity might increase after hemorrhagic shock.

On the other hand, c-fos expression of the spinal dorsal horn was reported to reflect the degree of noxious stimulation and function of the endogenous pain control mechanisms. For example, c-fos expression in the dorsal horn was less in the intact side than in the dorsolateral funiculus transected side during formalin-induced noxious stimulation (11). Placing the tail in 50°C water before hind-paw pinch reduced c-fos expression in the dorsal horn (12).

In this study, therefore, we performed a formalin test after recovery from hemorrhagic shock in rats to investigate the effects of hemorrhagic shock on pain reactivity and c-fos expression in the spinal dorsal horn.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
All experimental methods were approved by our institutional animal care committee. Twenty adult male Sprague-Dawley rats weighing 300–350 g were divided into two experimental groups: a Postshock group (n = 10) and a Control group (n = 10). In both groups, a chronic arterial catheter was placed in the right common carotid artery 2 days before the experiment. We confirmed by means of a preliminary study (n = 20) that the common carotid artery ligation had no effect on either the rats’ behavior or the c-fos expression during the formalin test. After measurements of basal mean blood pressure (mBP) and heart rate (HR), 8–14 mL of blood was drained, and the mBP was kept between 50 and 60 mm Hg for 30 min in the Postshock group, whereas mBP and HR were checked for 30 min in the Control group. The drained blood was reinfused into each rat, each rat was checked for neurologic abnormalities, and arterial blood gas was analyzed 15 min after reinfusion. Ten percent formalin (3.7% formaldehyde solution, 100 µL) was injected subcutaneously with a 26-gauge needle into the plantar surface of the left rear paw of the rats in both groups after the arterial blood gas analysis. All rats were restrained manually and received 100 µL of saline into the right hind paw. The saline was injected for evaluating the effects of needle stimulations and hemorrhagic shock per se on c-fos expression.

The observation of the formalin test was started immediately after the formalin and saline injections. The rats were put in a clear plastic chamber installed with a mirror to allow an unobstructed view of the paws. Behavior was rated for 1 h, 6 times for each 5-min period, by using the modified behaviors criteria described by Dubuisson and Dennis (13): 1 = normal weight bearing on the injected paw; 2 = limping during locomotion or resting the paw lightly on the floor; and 3 = elevation of the injected paw. The licking and flinching responses were counted simultaneously.

Numeric ratings were calculated from the following formula:

equation


where T1, T2, and T3 are the durations (s) spent in categories 2 and 3 or licking and flinching responses, respectively, during each 300 s block. All testing was conducted between 9:00 AM and 5:00 PM. The room temperature was kept at 24°C ± 2°C. After observation of the formalin test, arterial blood gas was checked again. Because the body temperature of the rats did not change more than 0.1°C in our preliminary study, we did not measure the temperature in each rat for minimizing stress.

The rats were deeply anesthetized with pentobarbital (60 mg/kg intraperitoneally) and killed 2 h after the formalin injection. The animals were perfused through the ascending aorta with 500 mL phosphate-buffered saline (PBS) (pH 7.4), followed by 500 mL of 4% paraformaldehyde fixative in a 0.1 M phosphate buffer. After perfusion, the lumbar spinal cord was removed and postfixed in the same fixative for 2 h, after which it was cryoprotected overnight in 20% glycerol in a 0.1 M phosphate buffer. Twelve sections (40 µm) were taken at 400-µm intervals from the whole lumbar spinal cord and processed for fos immunohistochemistry through the use of the avidin-biotin-peroxidase method described by Hsu et al. (14). The tissue sections were washed with a solution of 0.05 M PBS with 3% peroxidase and 0.2% Triton-X (Sigma, St. Louis, MO) and then incubated for 1 h at room temperature in a blocking solution of 3% normal goat serum in 0.05 M PBS with 0.2% Triton-X. The sections were incubated overnight at 4°C in PBS containing a polyclonal primary antibody to Fos (Ab-2, 1:1000 dilution; Oncogene Research Products, San Diego, CA) and 0.2% Triton X-100. They were then processed according to the usual protocol for the avidin-biotin-peroxidase method (Vectastain kit; Vector Laboratories, Burlingame, CA), with diaminobenzidine hydrochloride as a chromogen. Every tissue was reacted for the same period and with the same reagents. We quantified the effect of the hemorrhagic shock on the Fos staining by counting all the labeled cells plotted in the surface (laminae I–III) and deep (laminae IV–VI) dorsal horn. Throughout the data collection phase, the investigators were blinded to the animals’ conditions.

Data were presented as mean ± SD. Statistical analyses were performed by using a two-way analysis of variance for licking behavior, and immunoprecipitation study and the Mann-Whitney U-test were used to determine the pain intensity score. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
In the preliminary study, there was no significant difference of nociceptive behavior among Control and Right and Left Common Carotid Artery Ligation groups (Fig. 1). C-fos-positive cell numbers (lamina I–III) in 12 sections were 301 ± 99, 272 ± 32, and 215 ± 88 in the Control, Right Ligation, and Left Ligation groups (P = 0.23).



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  		Figure 1. Time course of the licking time (upper graph) and licking duration (lower graph) after injection of formalin in the preliminary test. Each data point represents the amount of time the animals licked the injected paw during a 5-min observation period. • = Control group (n = 6); {circ} = Left Carotid Artery Ligation group (n = 7); {square} = Right Carotid Artery Ligation group (n = 7). There was no significant difference among the three groups.

 
There was no significant difference of mBP and HR between Postshock and Control groups before and after hypovolemic shock. During hemorrhagic shock, the mBP of the Postshock group was kept between 50 and 60 mm Hg (Fig. 2). Because some rats showed tachycardia and some rats showed bradycardia during hypovolemic shock, mean HR showed no significant difference between the two groups. The base excess and pH of the Postshock group were significantly less than those of the Control group before the formalin test; however, they were within normal limit. Blood gas analysis after the formalin test showed no significant differences (Table 1). No rat showed neurologic abnormality.



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  		Figure 2. Time course of this study. The upper graph is mean blood pressure (mBP), and the lower graph is heart rate (HR). • = Postshock group; {circ} = Control group; BGA = blood gas analysis. *P < 0.05 versus the Control group.

 

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  		Table 1. Blood Gas Analysis Before and After the Formalin Test
 
Rats of the Postshock group struggled to the same degree as the Control group when the formalin was injected into their paws. However, nociceptive behavior was significantly less marked in the Postshock group as compared with the Control group at 0–5 min and 20–25 min (Fig. 3). The pain score of Dubuisson and Dennis (13) showed a significant reduction at every point, because many rats of the Postshock group touched their paws on the floor (Fig. 4).



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  		Figure 3. Time course of the licking time (upper graph) and licking duration (lower graph) after injection of formalin. Each data point represents the amount of time the animals licked the injected paw during a 5-min observation period. • = Postshock group; {circ} = Control group. *P < 0.05 versus the Control group.

 


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  		Figure 4. Time course of the pain intensity score after injection of formalin. • = Postshock group; {circ} = Control group. *P < 0.05 versus the Control group.

 
C-fos expression in the ipsilateral deep lamina of the Postshock group was significantly less than that of the Control group (P < 0.01). The number of c-fos-positive cells of the contralateral side was a few in both groups (Fig. 5). Figure 6 shows the typical c-fos expression of the Control group (photograph A) and the Postshock group (photograph B).



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  		Figure 5. Quantification of the effect of shock on the number of c-fos immunoreactive neurons in 12 sections. Solid columns = Postshock group; open columns = Control group. **P < 0.01 versus the Control group.

 


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  		Figure 6. These photomicrographs illustrate c-fos immunoreactivity in the ipsilateral side of lumbar dorsal horn. A = Control group; B = Postshock group. Bar = 400 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
During hemorrhagic shock, the increase of analgesic effect of exogenous agents has been ascribed to alterations in both pharmacokinetics and end organ sensitivity. For example, hypovolemia reduces hepatic blood flow and renal function, and this might decrease drug clearances and increase plasma concentration. Distribution volume decreases because of the decreased circulating blood volume and cardiac output with the redistribution of blood flow away from less vital organs. Brain blood flow and drug distribution to the brain are probably preserved during shock because of autoregulation. In this study, however, no anesthetics were used, and the decrease of nociceptive behaviors was observed under normotensive conditions. We speculated that the decrease of pain reactivity was caused by some changes of pain or pain control systems.

Some combinations of pain, stress, and conditioning can turn on the endogenous pain control mechanisms (15). A noxious stimulus at one site on the body can reduce the perceived intensity of pain produced by another concomitant noxious stimulus delivered elsewhere on the body surface. This phenomenon is known as diffuse noxious inhibitory controls, which are triggered by only noxious stimuli (16). It is mediated by endogenous opioidergic systems and involves subnucleus reticularis dorsalis in the caudal medulla (17). A wide variety of stressful events produce an analgesia that has been labeled SIA. This analgesia is mediated by opioid and nonopioid mechanisms (2). It involves the ventral tegmental area in the midbrain (1), the rostral ventral medulla, the spinal cord dorsolateral funiculus, and other areas (18). Analgesia can be conditioned by pairing an innoxious stimulus (e.g., light or tone) with an analgesia-producing stressor, such as foot shock. After several sessions, analgesia is produced by the conditioning stimulus alone. The conditioned analgesia involves spinal cord dorsolateral funiculus and nucleus raphe magnus and is mediated by endogenous opioidergic systems (19).

The possible factors that might affect hemorrhagic shock-induced analgesia are suggested as follows: the base excess of the Postshock group was significantly less than that of the Control group before the formalin test, although it was within the normal limit. Acidemia and residual microcirculation impairment might decrease the nociceptive behaviors; however, the magnitude was likely to be small in this study. Hypothermia might also be a factor in the reduction of nociceptive behaviors. Low ambient temperature is reported to decrease the licking response in the formalin test (20). However, there was no significant change of body temperature (±0.1°C) before and after the drainage and reinfusion of blood in our preliminary study. Therefore, we speculated that hypothermia was not a major determinant. The endogenous opioid or descending pain inhibitory system might be involved in these mechanisms (15). The c-fos reduction of the Postshock group was more remarkable in the deep layer of the spinal cord than in the surface layer. C-fos expression of the surface layer and deep layer might indicate activity of pain system (primary afferent fibers) and involvement of pain inhibiting systems, respectively. Williams et al. (21) reported that c-fos expression in the deeper laminae was reduced by general anesthesia, presumably by activating inhibitory mechanisms in the spinal cord, but local anesthetic blockade of the peripheral nerve after stimulation did not reduce the c-fos expression. The activation of neurons in the deeper laminae is unlikely to be the result of a monosynaptic event. We speculated that hemorrhagic shock might affect some modified pain systems of the spinal cord, descending pain inhibitory system, or both of these. Proinflammatory cytokines may also be involved. For example, interleukin-1 induces hyperalgesia at small doses and analgesia at large doses in the brain (22,23). Some cytokines enhance nociception and others reduce it. Furthermore, cytokine actions are site and dose dependent. Further studies involving the preinjection of naloxone or measurements of proinflammatory cytokines will be needed.

The first phase of the formalin test is caused predominantly by C-fiber activation caused by the peripheral stimulus, whereas the second phase seems to be dependent on the combination of an inflammatory reaction in the peripheral tissue and functional changes in the dorsal horn of the spinal cord (24). Because inflammatory mediators and the spinal cord functions were speculated to change after the hemorrhagic shock, we expected that reactions of the first phase were kept and those of the second phase were suppressed strongly. It is unknown why the first phase reactions were strongly suppressed. Rats of the Postshock group struggled to the same degree as the Control group when the formalin was injected into their paws. We interpreted this reaction as indicating the same degree of C-fiber activation. However, it might indicate the same degree of fear. In this study, the formalin test was started 15 minutes after the correction of hemorrhagic shock. The SIA might have occurred at the starting time of the formalin test.

In this study, the nociceptive behavior might have decreased under restricted conditions, for example, the magnitude and duration of the hypovolemia, method of pain sensitivity measurement, and the interval of the stress and analgesia testing. The degree of hemorrhage we imposed was extreme. Its survival rate is 0%–35% at two hours without reinfusing the blood (25). Severe hemorrhagic shock per se induces c-fos in liver and kidney (26). In this study, however, the c-fos-positive cell numbers of the contralateral side were few and almost the same in both groups, and the expression of the ipsilateral side was low in the Postshock group rather than in Control group. Therefore, we speculated that hemorrhagic shock per se did not induce c-fos in the spinal cord and that c-fos was a neural marker of pain in this study. Although many studies use tail-flick to radiant heat as the measure of pain sensitivity, we selected the formalin test because it is regarded as a more adequate model of naturally occurring tissue damage and injury than hot-plate and tail-flick tests (27). We started the formalin test 15 minutes after the correction of hemorrhagic shock. Fanselow (28) reported that the time lag between electric shock and analgesia testing did not reduce analgesia. However, the time lag might be related to the decrease of nociceptive behavior.

In conclusion, a SIA was demonstrated under normotensive conditions after hemorrhagic shock. The mechanisms governing this reaction remain to be elucidated.


   Acknowledgments
 
The authors thank Yumi Isaka for her technical assistance.


   References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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Accepted for publication April 10, 2001.





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Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins with the assistance of Stanford University Libraries' HighWire Press®. Copyright 2006 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press