The Analgesic Effects Of Different Doses Of Morphine Biology Essay

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Morphine is a potent opiate analgesic and it causes changes in mood, mental clouding, drowsiness, respiratory depression, euphoria and physical dependence on chronic application. Endogenous opioids such as endorphins are responsible for analgesia and morphine appears to be mimicking the effects of endorphins. It is a strong exogenous opioid agonist, which preferentially binds to μ opioid receptors in nociceptive circuits. μ receptors are found in areas, which are involved in descending analgesia. These include, the periaqueductal gray (PAG), rostral ventral medulla (RVM), and dorsal horn of the spinal cord. They are also found in areas where they are responsible for the reinforcing effects of opiates, areas such as ventral tegmental area (VTA) of the mid-brain and the nucleus accumbens (ventral striatum). Opiate receptors are also located in the locus ceruleus (LC) and other areas, which mediate physical dependence (Nestler, Hyman & Malenka , 2009). Morphine activates off cells in PAG to inhibit pain, they cause disinhibition so that GABA inhibition of off cells is reduced so that the on cell would not be switched on (Fields H, 2004). Solution X, which was used in this investigation, appears to antagonise the effects of morphine. Solution X causes on cells to be activated and produce hyperalgesic state.

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The purpose of this investigation is to measure the analgesic effect of different doses of morphine 1.5 mg/kg, 2.5 mg/kg, 5 mg/kg and 7.5 mg/kg on the response to mechanical pain in QS mice. It was concluded that morphine analgesia in female mice affects the response time to the mechanical pressure test as the dosage of morphine increases. However, in male mice it was inconclusive whether the dosage of morphine affected the response time.

Methods

Each group in the class selected five mice of the same gender. Total number of males (n= 25) and females (n=24) in the study. The mice were labelled A-E as the dose of morphine given was blinded. The mice were weighed so that the volume of the morphine to be injected could be calculated for each one. To determine if the mice could be included in the study, the mechanical pressure test was performed. A tail clip was placed on the base of the tail of each mouse and the response time, which is the time taken in the attempt by the animal to dislodge the clip, had a cut-off of 15 seconds. Once a response was observed the clip was removed. Each mouse was injected with 0.1ml/10g intraperitoneally one of the following solutions: saline, 1.5mg/kg, 2.5mg/kg, 5mg/kg and 7.5mg/kg of morphine. The time of injection was noted for each mouse and they were monitored according to the Animal Monitoring Sheet in (PHAR3202, 2010). At 30 minutes the mechanical pressure test was done, with a cut-off of 60 seconds. The mouse that took the longest time to respond was injected with solution X 0.1ml/10g. After 15 minutes has lapsed the mechanical pressure test was performed and the time of response was recorded, with a cut-off of 60 seconds.

Results

Male mice

The one-way ANOVA analysis and Dunnett's multiple comparison test of the saline versus the individual treatments of morphine did not show any significant P-values. In figure 1 the variance at 5mg/kg is large this was due to the raw data being widely spread from 0±6.76 seconds to 33±6.76 seconds as shown in table 1. In addition, group 4 results in table 1 seem to be outliers as the response time was zero for all of the treatments except at 1.5mg/kg of morphine. Overall, the sample size was n=25 for male mice. A rising trend was not observed in the mean values of response time in figure 1 when the dosage of the drug increases. This is further confirmed by figure 2 the morphine concentration log[dose] response curve.

The mice given solution X were the ones, which took the longest time to respond to the tail clip test after morphine analgesia and the mean value is 18±5.81 seconds with a SD value of 12.98 (Table 2) indicating that the data is largely variable. The effect of solution X was to reduce the response time of the mechanical pressure test after pretreatment with morphine. In table 2 the mean response time lowered to 2.6±1.47 seconds.

Female mice

Figure 3 displays a rise in the mean values of response time as the dosage of the morphine increases. At 5mg/kg and 7.5mg/kg the variance is large. The SD values are 25.56 and 32.49 respectively (Table 3). The raw data in table 3 shows a large range of response time at 5mg/kg from 0±11.43 to 60±11.43 and at 7.5mg/kg the response time varies largely from 0±14.53 to 60±14.53. The total number of female mice were n=24 as the X in table 3 suggests that the mouse died. The morphine concentration log[dose] response curve for female mice (figure 4) shows a rising trend with an almost linear relationship. Solution X was administered to mice that took the longest time to respond to the mechanical pressure test after certain dose of morphine analgesia. The mean response time reduced from 31.6±11.74 seconds to 0.8±0.36 seconds (Table 4). The one-way ANOVA and Dunnett's multiple comparison tests were insignificant.

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Figure 1 Response time in seconds after injecting male mice with 0.1ml/10g of saline, and morphine with the following concentrations 1.5mg/kg, 2.5mg/kg, 5mg/kg and 7.5mg/kg. For each treatment n=5. A clip was applied to the tail of the mouse at 30 minutes after drug administration; the time taken in an attempt to dislodge the clip was measured. The time cut-off for dislodging the clip was 60 seconds. This was a single blinded study.

Figure 2 Morphine concentration log[dose] response curve of male mice. Mean response times in seconds by the male mice caused by different doses of morphine after 30 minutes of exposure time. The log of the following doses was taken: 1.5mg/kg, 2.5mg/kg, 5mg/kg and 7.5mg/kg. For each treatment n=5. The response time was measured after an attempt by the mouse to dislodge the tail clip within 60 seconds as of application of the clip. The mean response time is the average of the individual response times at a given dose. This was a single blinded study.

Table 1 Response time (sec) of male mice injected with saline and the following morphine concentrations 1.5mg/kg, 2.5mg/kg, 5mg/kg and 7.5mg/kg

Soln

A

B

C

D

E

Concentration of morphine

1.5 mg/kg

Saline

2.5 mg/kg

7.5 mg/kg

5 mg/kg

mouse 1

mouse 2

mouse 3

mouse 4

mouse 5

Group 1

4

9

0

6

15

Group 2

7.8

6

7

14

1

Group 3

9

2

2

7

28

Group 4

4

0

0

0

0

Group 5

6

1

11

8

33

Mean

6.16

3.6

4

7

15.4

SD

2.24

3.78

4.85

5

15.11

SEM

1.00

1.69

2.17

2.24

6.76

P value

ns

ns

ns

ns

ns=not significant

Table 2 Response time after the injection of solution X in male mice that took the longest time to dislodge the tail clip after morphine administration

mouse no.

morphine solution injected

response time after injection of solution X

Group 1

5

E

0

Group 2

4

D

0

Group 3

5

E

2

Group 4

5

E

8

Group 5

5

E

3

Mean

(18)

2.6

SD

(12.98)

3.29

SEM

(5.81)

1.47

Values in parenthesis are the mean, SD and SEM values derived from the male mice that took the longest to respond in the mechanical pressure test after morphine analgesia across groups 1,2,3,4 and 5.

Figure 3 Response time in seconds after injecting female mice with 0.1ml/10g of saline, and morphine with the following concentrations 1.5mg/kg, 2.5mg/kg, 5mg/kg and 7.5mg/kg. For each treatment n=5 except for saline, n=4. The response time was measured after an attempt by the mouse to dislodge the tail clip with 60 seconds as cut-off time from the application of the clip. The tail clip was applied 30 minutes after the drug administration. This was a single blinded study.

Figure 4 Morphine concentration log[dose] response curve of female mice. Mean response time in seconds by the female mice caused by different doses of morphine after 30 minutes of exposure time. The log of the following doses was taken: 1.5mg/kg, 2.5mg/kg, 5mg/kg and 7.5mg/kg. For each treatment n=5, except for saline n=4. The response time was measured after an attempt by the mouse to dislodge the tail clip within 60 seconds as of application of the clip. The mean response time is the average of the individual response time at a given dose. This was a single blinded study.

Table 3 Response time (sec) of female mice injected with saline and the following morphine concentrations 1.5mg/kg, 2.5mg/kg, 5mg/kg and 7.5mg/kg

Soln

A

B

C

D

E

Concentration of morphine

1.5 mg/kg

Saline

2.5 mg/kg

7.5 mg/kg

5 mg/kg

mouse 1

mouse 2

mouse 3

mouse 4

mouse 5

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Group 6

9

2

6

>60

3

Group 7

0.1

0.1

17

0.1

0.1

Group 8

2

5

6

0

0

Group 9

1

4

13

>60

>60

Group 10

1

X

1

2

>15?

Mean

2.62

2.77

8.6

24.42

15.62

SD

3.63

2.18

6.35

32.49

25.56

SEM

1.62

1.09

2.84

14.53

11.43

P value

ns

ns

ns

ns

ns=not significant

Table 4 Response time after the injection of solution X in female mice that took the longest time to dislodge the tail clip after morphine administration

mouse no.

morphine solution injected

response time after injection of solution X

Group 6

4

D

1

Group 7

3

C

0

Group 8

3

C

0

Group 9

5

E

1

Group 10

5

E

2

Mean

(31.6)

0.8

SD

(26.25)

0.84

SEM

(11.74)

0.36

Values in parenthesis are the mean, SD and SEM values derived from the female mice that took the longest to respond in the mechanical pressure test after morphine analgesia across groups 6,7,8,9 and 10.

Discussion and conclusions

The present findings indicate that sex differences influence the response to morphine analgesia and this can be explained by the neuroanatomical substrates of the particular nociceptive stimuli or procedural factors unique to the nociceptive stimulus applied (Barrett, Smith & Picker, 2002). Thermal stimulation procedures involve the hindpaws of the mice and male hindpaws are larger than female ones introducing confounding factors. Also, the intensity of thermal stimulus whether low or moderate may influence to observe sex difference especially in μ opioid analgesia (Craft, 2003). Electrical and thermal nociceptive procedures mainly produce cutaneous stimulation compared to mechanical and chemical, which stimulate deeper structures.

Male mice are more sensitive to the analgesic effects of morphine than female mice; hence males will be less sensitive to pain (Barrett AC, Smith ES & Picker MJ 2002). This suggests that the response time for males should be longer than that of females. Female mice are less sensitive to morphine analgesia, however their response to mechanical pain was slower compared to males at 2.5 mg/kg and 7.5mg/kg of morphine. Those results are not consistent with the literature as females are more sensitive to painful stimuli than males (Lee-Parritz, 2007) hence they should have a quicker response time. Female mice may have fewer μ receptors or less efficient μ opioid receptor-mediated signal transduction than males (Craft, 2003). Another factor that differs between the two sexes is gonadal steroid hormone state of the mice. Across the estrus cycle in female mice, the potency of morphine changes so that during estrus, females where mostly different from males as the morphine analgesia potency is lowest (Craft, 2003) hence less time taken to respond compared to males.

Age is another factor that can affect morphine's analgesic effects. Older mice had greater sensitivity to pain that may lead to reduced analgesic response to morphine in older animals (Webster & Shuster 1976). Old mice have slower distribution and absorption of the morphine, which may account for this difference. Perhaps the male mice were older than females.

Solution X in this experiment is an antagonist because in both male and female mice the response time to dislodge the clip reduced as seen in tables Table 2 and Table 4. Therefore, solution X can be naloxone, a competitive antagonist that reverses the effects of μ-receptor agonists hence it increases pain perception. It has a very high affinity for μ-opioid receptors in the central nervous system (CNS), (Goodman & Gilman, 2008). Naloxone is used to precipitate acute abstinence so that on cells are activated in the PGA producing hyperalgesic state (Fields H, 2004). Naloxone might increase activity in interneurones, which gate pain impulses (Melzack & Wall, 1965). The potency of naloxone is increased following pretreatment with morphine (Wong & Bentley, 1977).

In this study the log[dose] response curve (figure 4) in female mice showed that with increased dose of morphine the response time to the mechanical pressure test was longer which is what is expected. However, this was not the case in male mice. Due to some of the experimental flaws, there does not seem to be a significant difference between the control and the different doses of morphine.

The discrepancy of the results with the literature can be explained by several experimental errors. The response time in females was much longer than males, which is unexpected as males are more sensitive to morphine analgesia. This anomaly can be explained by experimental errors, which can be improved in future studies. Having a larger sample size to eliminate the large standard errors for the different parameters. In addition, having the same experimenter to inject the mice to avoid technical issues. In addition, recording the data by multiple experimenters so that mistakes can be avoided. Less people in the room so that stress-induced analgesia where the animals exhibit elevated pain thresholds does not become a problem. Also the male and female mice experiments should not be performed in the same laboratory, perhaps putting them in separate rooms may have reduced the stress on the animals. A negative control should be added to this experiment i.e. mice that have not been injected to make sure that our protocol is not observing an effect, which may be unrelated so that false positives can be minimised. Lastly the effect of morphine analgesia can be influenced by the type of pain induced and pain is dependant of many factors such as the intensity, quality, duration and referral.