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On-line version ISSN 1806-907X
Rev. Bras. Anestesiol. vol.54 no.1 Campinas Jan./Feb. 2004
Effects of different spinal morphine doses in rats*
Efectos observados con diferentes dosis de morfina subaracnoidea en ratones
Neuzimar de Souza Freire Silva, M.D.I; Rioko Kimiko Sakata, TSA, M.D.II; Adriana Machado Issy, M.D.II
IPós-Graduando da Disciplina
de Anestesiologia, Dor e Terapia Intensiva da UNIFESP
IIProfessora Adjunta de Anestesiologia da UNIFESP
BACKGROUND AND OBJECTIVES: Spinal morphine
promotes adequate pain relief, but is not free from side effects. This study
aimed at investigating the effects of different spinal morphine doses.
METHODS: Five groups of seven rats were studied, 24 h after spinal catheter insertion via cisterna magna under anesthesia with muscular ketamine and xylazine. G1 received 10 µl saline solution; groups G2, G3, G4 and G5 received respectively 0.1; 0.3; 0.5 and 1 µg morphine in 10 µl saline solution. Animals were submitted to tail immersion test in hot water at M0 (prior to injection), and M15, M30, M60, M120 and M180 minutes after the injection.
RESULTS: Analgesic effects were observed in groups receiving morphine, in different times, as compared to control and to time before morphine injection. In G1 there was paws weakness in 4 animals. Agitation was observed at M15 in G2 and at M15 and M30 in G3. Mandible tremors were observed at moments M5, M15, M30 and M60 in G2; at M5 and M15 in G3, in M5 in G4, and in M5 in G5. Pruritus was observed at M5, M15, M30 and M60 in G2; at M5, M15, M30, M60 and M120 in G3; at M5, M15, M30, M60, and M120 in G4; and at M5, M15, M30, M60 and M120 in G5. Absence of diuresis was observed at M60 in G2; at M15, M30, M60 and M120 in G3; at M30 and M120 in G4, and at M30, and M60 in G5. Sedation was observed at M15, M30 and M60 in groups 2, 3 and 4; and at M15, M30, M60 and M120 in G5. Respiratory changes were observed at M15, M30 and M60 in G2; at M15, M30 and M60 in G3; at M15 and M30 in groups 4 and 5.
CONCLUSIONS: In this study all morphine doses below 1 µg have promoted short duration analgesia; all spinal morphine doses have produced side effects.
Key Words: ANALGESICS, Opioids: morphine; ANIMAL: rat, ANESTHETIC TECHNIQUES, Regional: spinal block
JUSTIFICATIVA Y OBJETIVOS: La morfina
por vía espinal promueve buen efecto analgésico, solamente no
es exenta de efecto colateral. El objetivo de este estudio fue investigar los
efectos observados con diferentes dosis de morfina por via subaracnoidea.
MÉTODO: Fueron estudiados cinco grupos de siete ratones, 24 horas después de la colocación de catéter subaracnoideo vía cisterna magna bajo anestesia con cetamina y xilazina por la vía muscular. El G1 recibió 10 µl de solución fisiológica; los grupos G2, G3, G4, y G5 recibieron respectivamente 0,1; 0,3; 0,5 y 1 µg de morfina en 10 µl de solución fisiológica. Los animales fueron sometidos al teste de inmersión de la cola en agua caliente en el M0 (antes de la inyección), y M15, M30, M60, M120 y M180 minutos después de la inyección.
RESULTADOS: Se observó analgesia en los grupos que recibieron morfina, en diversos momentos, cuando comparados con el grupo control y con el tiempo antes de la inyección de morfina. En el G1 hubo flaqueza de las patas en 4 animales. Ocurrió agitación en M15 en el G2 y en M15 y M30 en el G3. Tremor de mandíbula fue observado en M5, M15, M30, y M60 en el G2; en el G3 fue observado en M5 y M15; en el G4, en M5 y en el G5, en M5. Prurito fue observado en M5, M15, M30 y M60 en el G2; en M5, M15, M30, M60 y M120 en el G3; en M5, M15, M30, M60, y M120 en el G4; en M5, M15, M30, M60 y M120 en el G5. Ausencia de diúresis ocurrió en M60 en el G2; en el M15, M30, M60 y M120 en el G3; en el M60 y M120 en el G4; y en el M30 y M60 en el G5. Sedación ocurrió en M15, M30 y M60 en los grupos 2, 3 y 4; y en M15, M30, M60, y M120 en el G5. Alteración respiratoria fue observada en M15, M30, M60 en el G2; en M15, M30 y M60 en el G3; en M15 y M30 en los grupos 4 y 5.
CONCLUSIONES: En este estudio, todas las dosis de morfina subaracnoideas administradas provocaran efectos colaterales; y dosis menores que 1 µg promovieron analgesia de corta duración.
With the understanding of pharmacological properties of analgesics administered by different routes, it became possible to improve pain control, decreasing complications resulting from different painful syndromes.
Experimental investigations have established the basis for the safe and effective use of opioids in men to control acute and chronic pain. After the discovery of opioid receptors in 1974 1, long duration analgesia was obtained with spinal morphine in rats 2 and men 3. Since then, these spinal drugs have been frequently used to control acute or chronic pain.
Morphine is a hydrophilic opioid promoting deep and long lasting analgesia without sympathetic or motor block. However, it is cranially spread and may promote side effects such as nausea, vomiting and respiratory failure.
To evaluate the possibility of spinal morphine analgesic effects without side effects, different doses were administered and animals were observed in different moments.
This experimental study was performed after the Research Ethics Committee of the Universidade Federal, São Paulo and Universidade Federal, Amazonas approval and involved 35 Wistar, male, healthy rats aged approximately 90 days and weighing 250 to 300 grams. Excluded from the protocol were animals with paws paralysis or flaccidity. Animals were maintained in individual cages receiving 12 hours of light per day, water and feed ad libitum throughout the experiment.
Animals were distributed in 5 groups of 7 and all have received 10 µl spinal solution: G1 (control) has received 10 µl of 0.9% saline solution; G2, has received 0.1 µg morphine, G3 has received 0.3 µg morphine, G4 has received 0.5 µg morphine, and G5 has received 1 µg morphine.
Morphine was prepared through the dilution of 1 mg morphine sulfate salt in 1 ml of 0.9% saline solution (1 mg/ml). For the 0.1 µg/10 µl solution, 50 µl were removed from the stored solution and 4950 µl of 0.9% saline solution were added. For the 0.3 µg/10 µl solution, 100 µl were removed from the stored solution and 3000 µl of 0.9% saline solution were added. For the 0.5 µg/10 µl solution, 100 µl were removed from the stored solution and 2000 µl of 0.9% saline solution were added. For the 1 µg/10 µl solution, 500 µl were removed from the stored solution and 4500 µl of 0.9% saline solution were added.
Animals were submitted to general anesthesia with muscular ketamine (60 µg.g-1) and xylazine (16 µg.g-1) for spinal catheter insertion by the modified Yaksh, Rudy technique (1976).
Free catheter tip was externalized on the skin between the occipital and high cervical spine and was fixed with polymerized acrylic and nylon thread.
Animals were maintained in individual cages during anesthetic recovery and those with neurological deficits were excluded from the study. Animals were submitted to tail immersion test in hot water at a temperature of 50 ± 0.2 ºC. For the test, animals' tails were marked with red ink 5 cm above the tip, for immersion. Animals were placed in a plastic semi-transparent cylindrical tube to prevent free movement. Time for tail removal was recorded and tail was not maintained immersed for more than 15 seconds.
After analgesia test in M0 (before morphine), different morphine concentrations were randomly administered 24 hours after spinal catheter insertion. All animals participating in the study were active, moving, receiving free water and feed and totally recovered from anesthesia.
Catheter injection was performed with 10 µl Hamilton syringe graduated at 1 µl, at a rate of 5 µl per second.
Analgesia was tested at M0 = before morphine; M15 = 15 minutes after morphine; M30 = 30 minutes after morphine; M60 = 60 minutes after morphine; M120 = 120 minutes after morphine and M180 = 180 minutes after morphine.
Possible side effects and complications were recorded at M0, M5, M15, M30, M60, M120 and M180.
Non-parametric tests were used for statistical analysis. Variance Analysis was used for tail immersion test analysis to compare control group in every evaluated time and times within each group. Null hypothesis rejection level for all tests was established as 0.05 or 5% (p < 0.05).
Analgesic effect was not evaluated at M5 in all groups. There have been statistically significant differences in analgesia evaluated by tail removal between animals receiving morphine and control group animals (G1). In G2, time for tail removal was longer as compared to G1 in M15; in G3, in M30, M60 and M120; in G4 in M15, M30, M60 and M120; and in G5 in M15, M30, M60, M120 and M180 (Table I).
G2 presented analgesic effects in 85.72% of rats in M15; and 71.43% in M30 and M60. In G2, 71.43% in M15; 100% in M30 and M60 and 14.28% in M120. In G4, 100% of animals in M15, M30, M60; 71.43% in M120. In G5, 100% in M15, 85.72% in M30 and 100% in M60.
There were no statistically significant differences in mean times for tail removal in M0, M15, M30, M60, M120 and M180 in the control group. In groups 2, 3 and 5, in M15, M30 and M60 they were longer than in M120 and M180, which were similar to M0. In G4, in M15, M30, M60 and M120 they were longer than in M180, which was similar to M0. Variance Analysis, p > 0.05 (Table II).
Side Effects Observed after Injections
There have been no effects in G1, except for M5 where there has been paws weakness in four animals (57.15%).
Agitation was observed in G2 in 14.28% of animals in M15; in G3 in 14.28% in M15 and M30. There has been mandible tremor in G2 in 85.72% of animals in M5; 71.43% in M15 and 14.28% in M30 and M60. This side effect was observed in 100% of G3 animals in M5 and in 14.28% in M15. In G4, it has been observed in 85.72% of animals in M5. In G5 it has been observed in 85.72% of animals in M5.
There has been pruritus in G2 in 57.15% of animals in M5; 71.43% in M15; 57.15% in M30; and 42.86% in M60. In G3, this effect was observed in 42.86% in M5, 85.72% in M15; 100% in M30 and M60 and 57.15% in M120. In G4, it has been observed in 85.72% in M5, 100% of animals in M15, M30 and M60 and 71.43% in M120. No G5, it has been observed 71,43% pruritus in M5, 100% in M15, M30, M60 and 28,58% in M120.
There has been absence of diuresis in G2 in 85.72% of animals in M60. In G3, this effect has been observed in 14.28% in M15 and M30; 71.43% in M60 and 28.58% in M120. In G4, there has been absence of diuresis in 85.72% in M60 and 28.58% in M120. In G5, absence of diuresis has been observed in 14.28% of animals in M30 and 100% in M60.
Sedation was observed in G2 in 100% of animals in M15, 85.72% in M30 and 57.15% in M60. In G3, the same effect has been observed in 57.15% in M15, 85.72% in M30 and 57.15% in M60. In G4, sedation was observed in 100% of animals in M15 and M30 and 71.43% in M60. In G5, sedation has been observed in 100% of animals in M15, and M30, in 57.15% in M60 and 14.28% in M120.
There has been respiratory changes in G2 in 71.43% of animals in M15 and M30, and 42.86% in M60. In G3, there has been respiratory changes in 42.86% of animals in M15, 14.28% in M30 and M60. In G4, this effect has been observed in 100% of animals in M15 and M30. In G5, respiratory changes have been observed in 42.86% in M15 and 57.15% in M30.
Wistar rats were used in this study due to their fast reproduction and small size, making easy their handling and the maintenance of hygiene and food in the cages. They are also very resistant to infections and have low cost. Several authors have also chosen rats for spinal morphine injections, due to the above-described reasons 4,5.
Morphine was the opioid of choice for being a potent spinal analgesic drug, especially to treat acute pain and chronic painful syndromes. For being hydrophilic, it has prolonged action time, but this characteristic is also responsible for longer CSF permanence, as compared to lypophilic opioids. This leads to more cranial spread and opioid binding to brain receptors promoting side effects such as pruritus, nausea, vomiting and respiratory depression. These side effects limit the use of spinal opioids; so, if there could be the possibility of administering a dose producing analgesic effect without side effects its use could be expanded.
First investigations have questioned whether any volume of opioids would reach brain structures immediately after injection. Initially, it was thought that the drug would move in the cranial direction passively following CSF movement within the spinal space. After the administration of different bromphenol blue volumes in the spinal space of rats followed by spinal cord analysis 4, authors have concluded that 10 µl is the most adequate volume. Other authors have used 5 to 20 µl 6, up to 100 µl 7 epidural and spinal volumes. An author, however, has compared different spinal volumes and has concluded that there has been a better spread with 10 µl, even stating that large volumes would promote losses by reflux and by the lateral sides of the catheter 8. Studies have shown no need for high spinal morphine volumes to obtain analgesic effects. As from the above-mentioned studies, we decided for 10 µl for this study, for being a volume with better drug spread uniformity within the spinal space, and for safety reasons.
Doses of 0.1 to 1 µg were chosen because a study has shown that 1 µg spinal morphine produces analgesic effects 8, but also promotes side effects; so, we had to use lower doses to check whether there are analgesic effects without side effects.
Some authors have evaluated extracellular morphine concentrations in spinal cord, peri-aqueduct gray matter, raphe nucleus and lateral hypothalamus after 5 and 10 mg intraperitoneal morphine. In our study, morphine doses have promoted analgesia in several studied moments, however in most moments, morphine CSF concentration was lower than the minimum 200-400 pg/100 µl concentration reported in the literature 9.
We decided for silicone rubber catheters for being flexible, inert and nontoxic, not promoting tissue inflammatory reactions. Such features were confirmed by spinal cord histopathological studies in rats which remained with silicone rubber catheters for four weeks 10. Small catheters were used in this study to prevent spinal cord compression.
Modified Yaksh, Rudy technique 4 was used for catheter insertion, for allowing relatively easy spinal space access via cisterna magna. It also allows for catheter maintenance for prolonged periods because they can be effectively fixed, in addition to being isolated from animal's mouth and paws, thus preventing removal. This method has been successfully used to study the efficacy of spinal agents. However, if strictly reproduced, it demands a very long time for catheter insertion with high CSF collection failure rate.
Initial transmembrane puncture was performed according to authors' recommendations, with puncture in the middle of the atlanto-occipital membrane, but there has been failure in serial CSF collection. With the modification and the catheter more laterally inserted (atlanto-occipital membrane and occipital bone junction) there has been successful catheter insertion and functionality for drug administration and CSF collection. Free catheter tip was occluded with a small metal probe to prevent CSF leakage.
The experiment was performed 24 hours after catheter insertion to eliminate issues on residual anesthetic analgesia, and also to provide the evaluation of no neurological injuries. Studies in the literature were also just performed in awaken animals moving freely without contention 4-6.
Tail immersion in hot water is a test which may be performed with a large number of animals when there is the need for repeating the test along the experiment to evaluate analgesia. It is sensitive, reliable and easy to reproduce. It is fast and does not induce heat injury. Other authors have also used this test 11.
In this study, 5 centimeters of tail were immersed in hot water for a maximum period of 15 seconds to prevent burns, taking into account that the test has been performed several times during the experiment. Water was heated in double boiler at 50 ºC because this temperature is not deleterious to animals and allows for the observation of opioid's effects.
It has been apparent from recent studies that physical immobilization or movement restriction stresses rats and promotes hormonal and central nervous system changes. Endogenous opioid system is activated and changes analgesia tests results 12.
Our study has used plastic tubes with small posterior edge opening to maintain animal's tail externalized. There has been no need for manual contention of animals for the test because they have spontaneously entered the plastic cylinder, thus decreasing stress and preventing interferences in tail immersion test in hot water results.
We have used low morphine doses (0.1, 0.3, 0.5 and 1 µg) to check the possibility of no side effects. Agitation and strong mandible stimulation with fast muzzle hair movements and teeth shivering were earlier observed in some animals.
Paws weakness was observed in 4 animals soon after saline injection; this was not an opioid-induced effect since it was not seen after morphine injection, but could be related to solution-induced pressure 2.
No reference was found in the literature to justify the presence of mandible tremor preceding pruritus. However, mandible tremor was observed in our study in several animals of all groups receiving morphine.
Pruritus was the most common side effect. It has been clinically observed on muzzle and face of a large number of animals for approximately 2 hours and in line with the literature 13. The lower the dose used, the milder the intensity and duration of pruritus.
After labeled lumbar spine morphine in rats with X-rays to identify opioid distribution, highest concentration was observed 14 minutes after, resulting in muzzle and face pruritus clinically observed up to 2 hours after injection 13. These authors have suggested that pruritus would result from high morphine concentration on the trigeminal nerve. After systemic morphine administration, pruritus partially involves the opioid effect on nervous system and may also be a consequence of histamine release 14.
There has been absence of diuresis in our study in a large number of animals for some time and this may be related to urinary retention commonly observed in men 13.
After spinal morphine administration, rats remain quiet, with low response to handling 2. In our study, quietness has been observed in some animals of all groups and was considered mild sedation since they were easily awakened.
There has been no respiratory depression in all studied animals with the doses used in our study. Arterial blood gases analysis has not shown changes, however this was an isolated measurement because blood was collected from one animal of each group only, due to technical difficulties. Respiratory depression has been observed in human healthy volunteers with 2 to 10 mg epidural morphine 5. Our study has observed early respiratory changes characterized by irregular breathing maintained for approximately 30 minutes after morphine injection.
There have been no side effects in the last evaluation moment in all groups and this might be justified by a possible correlation with time elapsed after spinal morphine injection. In addition, side effects were observed even without analgesia.
Based on this experimental study with rats and 0.1, 0.3, 0.5 and 1 µg spinal morphine in 10 µl of 0.9% saline solution, one may conclude that doses below 1 µg promote short duration analgesia and that there is no analgesic dose without side effects.
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Dra. Rioko Kimiko Sakata
Address: Rua Três de Maio 61/51 Vila Clementino
ZIP: 04044-020 City: São Paulo, SP
Submitted for publication February 4, 2003
Accepted for publication May 6, 2003
* Received from Disciplina de Anestesiologia, Dor e Terapia Intensiva da Universidade Federal de São Paulo (UNIFESP), São Paulo, SP