Correlation between CSF concentration and side effects after spinal morphine injection in rats

Abstracts

JUSTIFICATIVA E OBJETIVOS: A morfina por via subaracnóidea promove bom efeito analgésico, mas não é isenta de efeito colateral. O objetivo deste estudo foi verificar se há correlação entre a concentração de morfina no líquor e os efeitos colaterais após injeção de morfina por via subaracnóidea. MÉTODO: Foram estudados 28 ratos, em quatro grupos, 24 horas após a colocação de cateter subaracnóideo via cisterna magna. Os grupos G1, G2, G3 e G4 receberam respectivamente 0,1; 0,3; 0,5 e 1 µg de morfina em 10 µl de solução fisiológica a 0,9%. Foram coletadas amostras de líquor e anotados os efeitos colaterais nos momentos M15, M30, M60, M120 e M180 minutos após a injeção. RESULTADOS: Os efeitos colaterais observados foram tremor mandibular, agitação, prurido, ausência de diurese, sedação e alteração respiratória. Houve maior incidência de efeitos colaterais nas avaliações precoces, diminuindo progressivamente com o tempo. As concentrações médias de morfina no líquor no G1 variou de 72,84 a 1,13 pg; no G2, de 114,26 a 5,68 pg; no G3, de 151,18 a 13,62 pg; e no G4, de 561,37 a 18,61 pg. CONCLUSÕES: Não houve correlação entre concentração de morfina no líquor e efeitos colaterais.

ANALGÉSICOS, Opióides; ANIMAL; TÉCNICAS ANESTÉSICAS, Regional

SCIENTIFIC ARTICLE

Correlation between CSF concentration and side effects after spinal morphine injection in rats^*

Correlación entre concentración liquórica y efectos colaterales después de inyección de morfina por vía 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

^{Correspondence}

SUMMARY

BACKGROUND AND OBJECTIVES: Spinal morphine promotes good pain relief, but is not free from side effects. This study aimed at verifying the correlation between CSF morphine concentration and side effects.

METHODS: This study involved 4 groups of 7 rats, which were studied 24 hours after spinal catheter insertion via cisterna magna. Groups G1, G2, G3 and G4 received respectively 0.1; 0.3; 0.5 and 1 µg morphine in 10 µl of 0.9% saline solution. CSF samples were collected and side effects were recorded at M₁₅, M₃₀, M₆₀, M₁₂₀ and M₁₈₀ minutes after injection.

RESULTS: Side effects observed were: mandible tremors, agitation, pruritus, absence of diuresis, sedation, and respiratory changes. The incidence of side effects was higher during early evaluations and progressively decreased with time. Mean CSF morphine concentrations in G1 varied from 72.84 to 1.13 pg; in G2 from 114.26 to 5.68 pg; in G3 from 151.18 to 13.62 pg and in G4, from 561.37 to 18.61 pg.

CONCLUSIONS: There has been no correlation between CSF morphine concentration and side effects.

Key Words: ANALGESICS, Opioids: morphine, ANESTHETIC TECHNIQUES, Regional: spinal block; ANIMAL: rat

RESUMEN

JUSTIFICATIVA Y OBJETIVOS: La morfina por vía subaracnoidea promueve buen efecto analgésico, solamente no es exenta de efecto colateral. El objetivo de este estudio fue verificar si hay correlación entre la concentración de morfina en el líquor y los efectos colaterales después de inyección de morfina por vía subaracnoidea.

MÉTODO: Fueron estudiados 28 ratones, en cuatro grupos, 24 horas después de colocación de catéter subaracnoideo vía cisterna magna. Los grupos G1, G2, G3, y G4 recibieron respectivamente 0,1; 0,3; 0,5 y 1 µg de morfina en 10 µl de solución fisiológica a 0,9%. Fueron colectadas muestras de líquor y anotados los efectos colaterales en los momentos M₁₅, M₃₀, M₆₀, M₁₂₀ y M₁₈₀ minutos después de la inyección.

RESULTADOS: Fueron observados efectos colaterales: tremor de mandíbula, agitación, prurito, ausencia de diuresis, sedación y alteración respiratoria. Hubo mayor incidencia de efectos colaterales en las evaluaciones precoces, diminuyendo progresivamente con el tiempo. Las concentraciones medias de morfina en el líquor en el G1 varió de 72,84 a 1,13 pg; en el G2, de 114,26 a 5,68 pg; en el G3, de 151,18 a 13,62 pg; y en el G4, de 561,37 a 18,61 pg.

CONCLUSIONES: No hubo correlación entre concentración de morfina en el líquor y efectos colaterales.

INTRODUCTION

Morphine has been one of the first opioids used for postoperative analgesia and to control chronic pain. It is an opioid with low lipid solubility, moderate receptor affinity, moderate efficacy, low rate of receptor dissociation and prolonged duration. The discovery of opioid receptors in 1974 ¹ and the identification of endogenous opioids in 1975 ² have represented advances in pain control. Spinal morphine, for its low liposolubility, has long onset and duration. In the spinal space, morphine is cranially spread and may promote side effects such as pruritus, nausea, vomiting and respiratory failure, being the latter the most feared side effect due to its late manifestation.

We have not found in the literature any correlation between CSF morphine concentration and side effects after spinal injection. In an attempt to establish this correlation, this study was performed by dosing CSF concentration after different morphine doses in rats, and observing side effects in different moments.

METHODS

This experimental study was performed after the Universidade Federal, São Paulo and Universidade Federal, Amazonas Ethics Committees approval and involved 28 Wistar healthy, male 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 4 groups of seven and all have received 10 µl spinal saline solution: G1 has received 0.1 µg morphine; G2 has received 0.3 µg morphine; G3 has received 0.5 µg morphine; and G4 has received 1 µg morphine.

Morphine was prepared through dilution of 1 mg morphine sulfate salt in 1 ml of 0.9% saline solution (1 mg/ml). Remaining doses (0.1; 0.3; 0.5 and 1 µg morphine in 10 µl) were prepared by dilution in 0.9% saline solution and maintained at 4 ºC until analysis. 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) and xylazine (16 µg/g) for spinal catheter insertion by the modified Yaksh, Rudy technique ³, with the catheter more laterally inserted to help drug administration and CSF collection. Catheter has reached high thoracic region spinal space approximately at T₃-T₄. Free catheter tip was closed with a small metal probe.

Animals were maintained in individual cages during anesthetic recovery and those with neurological deficits were excluded from the study.

Different morphine concentrations were randomly administered to animals 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.

Injection was performed with 10 µl Hamilton's syringe graduated every 1 µl, at a rate of 5 µl per second.

CSF collection (0.2 ml) was performed in the following moments: 15 (M₁₅), 30 (M₃₀), 60 (M₆₀), 120 (M₁₂₀) and 180 (M₁₈₀) minutes after morphine administration. Samples were centrifuged and stored at -8 ºC. CSF morphine was quantified by high-efficiency chromatography.

Arterial blood (0.3 ml) was collected between 15 and 30 minutes after morphine injection, from one animal of each group.

Possible side-effects and complications were observed and recorded in moments M₀, M₅, M₁₅, M₃₀, M₆₀, M₁₂₀ and M₁₈₀, and were correlated to CSF morphine concentration.

Non-parametric tests were used for statistical analysis. Null hypothesis rejection level was established in 0.05 or 5% (p < 0.05) for all tests.

RESULTS

CSF Morphine Concentration

Mean CSF morphine concentrations in G1 have progressively decreased and were significantly lower as compared to other groups in M₁₅, M₃₀, M₆₀ and M₁₂₀; G1 mean concentration in M₁₈₀ was similar to G2 and lower than G3 and G4. Decrease, followed by increase and again decrease was observed in G2 and G3; increase followed by decrease was also observed in G4 (Table I and Figure 1).

Brazilian Journal of Anesthesiology, 2004; 54: 1: 53 - 59

Correlation between CSF concentration and side effects after spinal morphine injection in rats

Neuzimar de Souza Freire Silva; Rioko Kimiko Sakata; Adriana Machado Issy

Brazilian Journal of Anesthesiology, 2004; 54: 1: 53 - 59

Correlation between CSF concentration and side effects after spinal morphine injection in rats

Neuzimar de Souza Freire Silva; Rioko Kimiko Sakata; Adriana Machado Issy

Correlation between CSF Morphine Concentration and Side Effects

G1, in M₁₅ (72.84 pg/100 µl), has presented: mandible tremor, agitation, pruritus, sedation and respiratory changes; in M₃₀ (19.01 pg/100 µl), mandible tremor, pruritus, sedation and respiratory changes; in M₆₀ (7.38 pg/100 µl) mandible tremor, pruritus, absence of diuresis, sedation and respiratory changes; both in M₁₂₀ (3.25 pg/100 µl) and M₁₈₀ (1.13 pg/100 µl) there were no effects.

G2, in M₁₅ (106.64 pg/100 µl), has presented: mandible tremor, agitation, pruritus, absence of diuresis, sedation and respiratory changes; in M₃₀ (96.91 pg/100 µl), agitation, pruritus, absence of diuresis, sedation and respiratory changes; in M₆₀ (114.26 pg/100 µl) pruritus, absence of diuresis, sedation and respiratory changes; in M₁₂₀ (37.81 pg/100 µl), pruritus and agitation; and in M₁₈₀ (5.68 pg/100 µl) there were no effects.

G3, in M₁₅ (144.77 pg/100 µl), has presented: pruritus, sedation and respiratory changes; in M₃₀ (121.47 pg/100 µl), pruritus, sedation and respiratory changes; in M₆₀ (151.18 pg/100 µl) pruritus, absence of diuresis and sedation; in M₁₂₀ (42.57 pg/100 µl), pruritus and absence of diuresis; and in M₁₈₀ (13.62 pg/100 µl) there were no effects.

G4, in M₁₅ (179.18 pg/100 µl), has presented: pruritus, sedation and respiratory changes; in M₃₀ (386.14 pg/100 µl), pruritus, absence of diuresis, sedation and respiratory changes; in M₆₀ (561.37 pg/100 µl) pruritus, absence of diuresis and sedation; in M₁₂₀ (74.86 pg/100 µl), pruritus and sedation; and in M₁₈₀ (18.61 pg/100 µl) there were no effects (Table II).

Brazilian Journal of Anesthesiology, 2004; 54: 1: 53 - 59

Correlation between CSF concentration and side effects after spinal morphine injection in rats

Neuzimar de Souza Freire Silva; Rioko Kimiko Sakata; Adriana Machado Issy

DISCUSSION

Wistar rats were chosen for this study because they are easy to acquire and handle, and to keep food and hygiene in the cages, in addition to their high organic resistance to infection and low cost. Several authors have also chosen rats for spinal morphine injections, for the above-mentioned reasons ^3-5.

Modified Yaksh, Rudy surgical technique ⁵ was used for catheter insertion, for allowing relatively easy spinal space access through the cisterna magna. It also enables catheter maintenance for prolonged periods because it provides its effective fixation in addition to isolating it from animal's mouth and paws, thus preventing its removal. However, if strictly followed, this technique demands lots of time for catheter insertion with a high CSF collection failure rate.

The experiment was performed 24 hours after catheter insertion to prevent issues on residual anesthetic analgesia and also to enable the evaluation of absence of neurological injury. A longer time was not waited because there could be catheter tip obstruction, preventing solution diffusion and also serial CSF collection. This study, as well as others in the literature, was performed solely with awaken and freely moving animals, without contention ^3-5.

Morphine was the opioid of choice because, for being hydrophilic, it has a prolonged action. However, this characteristic is also responsible for the longer permanence of the drug in the CSF, as compared to lipophylic opioids. So, there is more cranial spread and binding of the opioid to encephalic receptors, promoting side effects such as pruritus, nausea, vomiting and respiratory depression. These side effects limit the use of spinal opioids.

We have found no study on the correlation of CSF morphine concentration and investigated side effects. To check whether this correlation exists, 0.1 to 1 µg doses were used. A study has shown that 1 µg spinal morphine has analgesic and side effect ⁶.

In our study, CSF morphine concentration was lower than minimum concentration of 200 to 400 pg/100 µl reported in the literature ⁷, only reaching this concentration in G4, during M₃₀ and M₆₀.

After intravenous administration, morphine is found in serum, CSF and brain of rats, within 5 minutes. In the brain, there is concentration decrease 30 minutes after, and it is maintained stable until 60 minutes. In CSF there is no concentration change 30 minutes after; however, 60 minutes after intravenous administration there is major increase, suggesting that morphine could have been retained in brain tissue. CSF could be an important pathway to eliminate morphine from the central nervous system, however its metabolites are not found there after single dose injection ⁸.

Our study has found high CSF morphine concentration at 15 minutes. At 30 and 60 minutes, concentration changes were different among groups, either increasing or decreasing. At 120 and 180 minutes, there has been CSF morphine concentration decrease in all groups.

We have found high morphine concentrations in M₁₅ in all groups, probably because CSF samples were collected at the injection site. There has been morphine concentration decrease in M₃₀ in three groups, probably due to drug spread to CSF and spinal cord, to adjacent tissues and brain. There has been increased concentration in G4, probably due to tissue morphine spread to CSF; there has also been increase in Groups 2, 3 and 4, in M₆₀. G1 continued to present decrease, probably because the dose was low and even with spread from tissue to CSF the increase was lower than the transfer of the drug from CSF to tissues.

There has been CSF morphine concentration decrease in M₁₂₀ and M₁₈₀ in all groups, probably because most drug had already been excreted; very low concentrations were detected in M₁₈₀.

Our study has observed side effects with low morphine concentrations, while in some moments when CSF morphine concentration was higher, no effect has been observed.

There has been no correlation between CSF morphine concentration and side effects. It seems that side-effects resolution is correlated to time after spinal morphine injection.

Based on the results of this experimental study with rats, with 0.1, 0.3, 0.5 and 1 µg spinal morphine in 10 µl saline solution, one may conclude that there is no correlation between CSF morphine concentration and side effects.

REFERENCES

Submitted for publication February 4, 2003

Accepted for publication May 6, 2003

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Correspondence to

Dra. Rioko Kimiko Sakata

Address: Rua Três de Maio 61/51 Vila Clementino

ZIP: 04044-020 City: São Paulo, Brazil

*

Received from Disciplina de Anestesiologia, Dor e Terapia Intensiva da Universidade Federal de São Paulo (UNIFESP), São Paulo, SP

Publication Dates

• Publication in this collection
  06 May 2004
• Date of issue
  Feb 2004

History

• Accepted
  06 May 2003
• Received
  04 Feb 2003