Organophosphate poisoning is associated with adverse effects on the central nervous system such as seizure/convulsive activity and long term changes in neuronal networks. This study report an investigation designed to assess the consequences of Soman, a highly toxic organophosphorus compound, exposure on regional blood flow in the rat brain and peripheral organs. We performed repeated blood flow measurements in the same animal, using the microspheres technique, to characterize changes in regional blood flow at different times after Soman intoxication. In addition, the cardiopulmonary effects of Soman were followed during the intoxication. Administration of Soman (1 LD50; 90
Organophosphorus compounds (OP) irreversibly inhibit the enzyme acetylcholinesterase (AChE), which is responsible for terminating the neurotransmitter action of acetylcholine (ACh) at the various cholinergic nerve endings. This results in the accumulation of cholinergic receptor sites producing continuous stimulation of cholinergic fibers throughout the central and peripheral nervous systems [
Soman is a highly toxic organophosphorus compound that is rapidly distributed throughout the body after its administration [
Exposure to Soman causes a variety of signs of poisoning involving the cholinergic system. The inhibition of AChE causes an accumulation of ACh in the synaptic cleft, which generates frequent activation of ACh receptors [
In the present experimental set-up, the integrated physiological response to Soman (1 LD50; 90
Our aim of this investigation was to evaluate how the cardiorespiratory system and cerebral and regional blood flow are affected by Soman intoxication. The role of cerebral blood flow (CBF) in Soman-induced convulsions may lead to improved treatment of Soman intoxication and a better understanding of the role of CBF in other forms of seizures, including human epilepsy.
The experiments were performed on 16 male, Wistar rats (Möllegaard, Denmark), with body weight (BW) ranging between 325 and 399 g. The animals were acclimatized in the animal department for at least 1 week prior to the experiments. The room temperature was 21–24°C and humidity
Soman (pinacolyl methylphosphonofluoridate; >95% pure) was synthesized at the Department of Chemistry, FOI CBRN-Defense and Security, Sweden, and diluted to its final concentration with sterile water on the day of the experiment.
At the day of microsphere studies, the animals were anaesthetized by injecting thiobutabarbital (Inactin, Byk-Gulden, Konstanz, West Germany) 120 mg kg−1 intraperitoneally (i.p.) and tracheotomized for spontaneous ventilation. Body temperature was kept at about 37.5°C by a rectal thermistor and servocontrolled heating pad (Atew, Sweden). To replace fluid losses during the experiment, a polyethylene catheter was inserted into a femoral vein for the administration of a Ringer solution, 0.5 mL h−1 100 g BW−1. For microsphere injections, a catheter was retrogradely introduced in the left ventricle via the right carotid artery. The position was confirmed by pressure measurement. Both femoral arteries were cannulated. The left femoral artery was used for arterial blood sampling and the right artery for continuous measurements of heart rate (HR) and mean arterial pressure (MAP) with a Gould P2310 transducer (Gould Inc., CA, USA) and a ABB SE 120 recorder (ABB Goerz AG, Vienna, Austria). To assure free urine flow, the bladder was catheterized through a suprapubic incision. Arterial pO2, pCO2, and pH were determined at intervals with an ABL 520 acid-base analyzer (Radiometer, Copenhagen, Denmark). Heparin (Løvens kemiske Fabrik, Ballerup, Denmark) at 500 I.U. kg−1 i.v. was administered as an anticoagulant. For registration of the respiratory rate (RR), a probe was connected around the chest of the animals.
Microspheres (15 ± 3
An identical protocol was used for both Soman intoxicated animals (
Flow chart of the experiment.
Actions | AChE + acid-base | 141Ce | Soman or saline | AChE + acid-base | 113Sn | AChE + acid-base | 103Ru |
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Time (min) | −2 | 0 | 10 | 13 | 15 | 38 | 40 |
Blood samples (0.4 mL) were drawn from the femoral artery in heparinized syringes 2 min before each injection of microspheres. Part of each sample (0.3 mL) was used for measurements of pO2, pCO2, and pH; the other 0.1 mL was used for determination of AChE activity. The acetylcholinesterase activity was measured in the blood using a modified method of Augustinsson et al. [
The animals were observed at regular intervals throughout the experiment with respect to muscle tremors, seizures, salivation, respiratory rate, heart rate, and mean arterial pressure. The animals were classified into two groups: no signs: no clinical signs of poisoning; signs: marked respiratory depression and salivation.
For comparisons between animal groups, Student’s unpaired
Our results show that when male Wistar rats are intoxicated with 1 LD50 Soman, two distinguished groups are obtained: one with clear signs of poisoning and another group without any symptoms. In the group with significant decrease in respiration rate, total cerebral blood flow was increased by about 290%, while no change in cerebral blood flow could be seen in the rats showing no signs of poisoning. Remarkably, blood AChE activity is depressed in all animals intoxicated with Soman.
Each animal served as its own control and a control period of at least 15 min was recorded prior to administration of saline (control group) or Soman (1 LD50). Signs of cardiovascular and respiratory impact were evaluated throughout the course of intoxication
The influence of the sphere procedure was evaluated by giving saline instead of Soman in separate experiments in control rats (
As can be seen in Table
The absolute baseline values for mean arterial pressure (MAP), heart rate (HR), respiratory rate (RR), and partial pressure of carbon dioxide in the blood (pCO2). No significant difference was seen between the three groups studied.
MAP (mmHg) | HR (beats/minute) | RR (breath/minute) | pCO2 (mmHg) | |
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Control ( |
125 ± 5.9 | 410 ± 15.5 | 92 ± 6.3 | 6 ± 0.3 |
No signs ( |
129 ± 6.1 | 422 ± 24.5 | 94 ± 9.2 | 6 ± 0.2 |
Signs ( |
127 ± 5.1 | 395 ± 9.3 | 89 ± 3.9 | 6 ± 0.1 |
In four of ten animals intoxicated with Soman, the respiratory rate remained stable despite the decrease in blood AChE activity. In six out of ten animals, the respiratory rate decreased to about 40% of baseline after 25 minutes and remained stable during the rest of the experimental period (Figure
Physiological parameters presented as percent of baseline value (100%) in control rats (
Parameter | Symptoms | −10 min | 0 min | 5 min | 20 min | 30 min | 35 min |
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% MAP | Control | 100 ± 0 | 87 ± 5.6 | 92 ± 3.5 | 90 ± 5.5 | 82 ± 7.0 | 83 ± 6.6 |
No signs | 100 ± 0 | 91 ± 5.8 | 84 ± 8.8 | 78 ± 5.6 | 80 ± 3.6 | 78 ± 6.9 | |
Signs | 100 ± 0 | 98 ± 2.6 | 93 ± 1.9 | 109 ± 7.8 | 135 ± 7.3 |
130 ± 4.8 |
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% RR | Control | 100 ± 0 | 103 ± 2.5 | 103 ± 3.9 | 100 ± 2.1 | 103 ± 3.5 | 109 ± 3.4 |
No signs | 100 ± 0 | 97 ± 6.6 | 101 ± 3.6 | 97 ± 7.4 | 86 ± 5.5 | 91 ± 4.5 | |
Signs | 100 ± 0 | 107 ± 3.0 | 105 ± 2.0 | 93 ± 5.9 | 46 ± 5.1 |
42 ± 8.7 |
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% CBF | Control | 100 ± 0 | 109 ± 4.5 | 98 ± 7.9 | |||
No signs | 100 ± 0 | 99 ± 5.0 | 80 ± 9.1 | ||||
Signs | 100 ± 0 | 111 ± 8.0 | 290 ± 43.0 |
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% AChE | Control | 100 ± 0 | 100 ± 0 | 97 ± 4.1 | 95 ± 2.8 | 93 ± 4.6 | |
No signs | 100 ± 0 | 100 ± 0 | 57 ± 22.1 |
17 ± 8.9 |
8 ± 3.6 |
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Signs | 100 ± 0 | 100 ± 0 | 67 ± 10.4 |
6 ± 2.6 |
7 ± 1.4 |
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% HR | Control | 100 ± 0 | 97 ± 2.4 | 98 ± 2.0 | 99 ± 2.6 | 97 ± 2.6 | 97 ± 2.8 |
No signs | 100 ± 0 | 93 ± 3.8 | 92 ± 4.3 | 93 ± 3.2 | 94 ± 2.8 | 91 ± 2.9 | |
Signs | 100 ± 0 | 100 ± 2.0 | 102 ± 1.9 | 105 ± 1.4 | 107 ± 3.2 | 102 ± 3.3 | |
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% pCO2 | Control | 100 ± 0 | 100 ± 0 | 93 ± 0.6 | 90 ± 1.4 | 89 ± 1.8 | |
No signs | 100 ± 0 | 100 ± 0 | 96 ± 2.9 | 96 ± 3.4 | 92 ± 4.7 | ||
Signs | 100 ± 0 | 100 ± 0 | 94 ± 2.0 | 98 ± 2.9 | 105 ± 3.7 |
Physiological parameters presented as percent of baseline value (100%) in control rats (
As seen in Figure
The mean changes in MAP, HR, RR, AChE activity, pCO2, and CBF from baseline values are depicted in Figure
The change in blood cholinesterase activity was the same for all rats intoxicated with Soman (Figure
Cerebral blood flow increased to
The effect of Soman on regional blood flow to different peripheral organs is shown in Figure
Vascular resistance (VR) presented as percent of baseline value (100%) in control rats (
Organ | Symptoms | −10 min | 5 min | 35 min |
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Brain | Control | 100 ± 0 | 91 ± 5.0 | 94 ± 7.1 |
No signs | 100 ± 0 | 91 ± 5.4 | 108 ± 13.3 | |
Signs | 100 ± 0 | 93 ± 6.4 | 51 ± 6.2 |
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Diaphragm | Control | 100 ± 0 | 123 ± 19.2 | 128 ± 13.2 |
No signs | 100 ± 0 | 85 ± 9.2 | 96 ± 16.1 | |
Signs | 100 ± 0 | 92 ± 3.3 | 172 ± 33.4 |
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Facial skin | Control | 100 ± 0 | 87 ± 8.4 | 64 ± 11.2 |
No signs | 100 ± 0 | 132 ± 8.5 | 248 ± 87.6 |
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Signs | 100 ± 0 | 194 ± 27.7 | 602 ± 212.4 |
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Abdominal skin | Control | 100 ± 0 | 95 ± 8.6 | 60 ± 6.5 |
No signs | 100 ± 0 | 102 ± 8.0 | 151 ± 31.5 |
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Signs | 100 ± 0 | 133 ± 25.7 | 235 ± 50.0 |
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Spleen | Control | 100 ± 0 | 118 ± 26.1 | 62 ± 13.0 |
No signs | 100 ± 0 | 115 ± 11.7 | 138 ± 19.1 | |
Signs | 100 ± 0 | 116 ± 16.0 | 1181 ± 442.8 |
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Pancreas | Control | 100 ± 0 | 99 ± 12.6 | 68 ± 11.3 |
No signs | 100 ± 0 | 121 ± 7.1 | 138 ± 7.4 | |
Signs | 100 ± 0 | 141 ± 23.6 | 1212 ± 509.8 |
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Kidney | Control | 100 ± 0 | 101 ± 8.3 | 86 ± 6.4 |
No signs | 100 ± 0 | 88 ± 10.9 | 93 ± 6.5 | |
Signs | 100 ± 0 | 91 ± 5.4 | 136 ± 17.3 |
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Cardiac muscle | Control | 100 ± 0 | 105 ± 13.6 | 104 ± 12.1 |
No signs | 100 ± 0 | 92 ± 12.4 | 80 ± 18.5 | |
Signs | 100 ± 0 | 88 ± 6.8 | 41 ± 6.7 |
Vascular resistant (VR) presented as percent of baseline value (100%) in control rats (
A significant increase in vascular resistance was also observed in the facial and abdominal skin in animals intoxicated with Soman showing no signs of symptoms, but not to the same extent as for animals showing signs of poisoning.
The time dependence of Soman (1 LD50, s.c.) and saline (control) administration is shown in Figure
Our results demonstrate that although administration of 1 LD50 Soman to anaesthetized rats produced a significant decrease in blood AChE activity in all animals tested, only six out of ten rats showed signs of poisoning like a decrease in respiratory rate. The results also show that it was only in the animals with significant signs of poisoning that an increase in cerebral blood flow occurred.
Since all the intoxicated rats showed the same degree of inhibition of AChE in the blood, measurement of blood AChE activity is not a good candidate as a biomarker to confirm that Soman intoxication will prevail. The results demonstrate the importance to distinguish between animals showing signs of symptoms and animals not showing signs of symptoms. A mean in the whole group does not give the same result as if you divide them into two groups. As can be seen in Figure
It has been reported by others [
That intoxication with Soman does not always lead to seizure and signs of poisoning despite a decrease in blood AChE activity which has earlier been reported [
In an earlier study we have shown that signs of poisoning correlate positively to acetylcholinesterase inhibition in the brain and demonstrated that the more severe convulsions, the more inhibition of AChE in the brain [
Prolonged centrally mediated convulsions are one of the major signs that occur following poisoning with organophosphorus anticholinesterase nerve agents such as Soman [
Acetylcholine is an important regulator of CBF in man and in many other species [
The effect of Soman on regional blood flow to different peripheral organs has, to our knowledge, never been published. Our results demonstrate that the vascular resistance decreases significantly in the brain and cardiac muscle in animals showing signs of symptoms. Concomitantly, these animals show a significant increase in vascular resistance in the diaphragm, facial skin, abdominal skin, spleen, pancreas, and the kidney. This can explain the huge increase in cerebral blood flow that was observed at the expense of a decrease in blood flow to these peripheral organs.
A significant increase in vascular resistance was also observed in the facial and abdominal skin in animals intoxicated with Soman showing no signs of symptoms. That this did not affect the cerebral blood flow has to be further elucidated.
Why the rats responded differently to the same dose of Soman has to be further elucidated. Other enzymes like butyrylcholinesterase (BuChE) and carboxylesterase (CarbE) might be involved [
It has been stated that respiratory paralysis following exposure to Soman is the result of a direct action of the agent on certain cholinergic synapses (inhibitory synaptic sites) of the respiratory centers in the brainstem [
The huge increase in CBF demonstrated in the present study did probably lead to a higher concentration of Soman in the brain and as a direct effect decreased the respiration rate centrally.
During the past decades, there has been a continuous discussion on the mechanism of respiratory failure in Soman poisoning [
In conclusion, our results demonstrate thatwhen Wistar rats are intoxicated with 1 LD50 Soman, two distinguished groups are obtained: one with clear signs of poisoning and another group without any symptoms. Remarkably, AChE activity is depressed in all animals intoxicated with Soman. These results show that it is not advisable to rely on AChE activity in the peripheral blood at assessment of the severity of Soman intoxication. We conclude that it is of great importance to treat all data individually. An overall mean can easily be misinterpreted and conceal important effects.
The authors declare no conflict of interests.
The authors are grateful to Mrs. Mona Koch for skillful laboratory support.