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Pharmacology Of The Spinal Cord Practical Biology Essay

Pain is a subjective experience which frequently involves sensory and psychological components. The threshold, intensity, and location of a stimulus is relayed by the primary afferent nociceptive fibres that form the sensory aspect of pain. The psychological aspect acts upon the sensory input to classify the stimulus as unpleasant and threatening. This psychological component of pain determines the decision of the action which is to be taken in response to avoid or recover from pain. Pain can be facilitated and suppressed by the descending serotonergic and noradrenergic pathways which act on the neurones in the dorsal horn of the spinal cord to enhance/reduce and reduce neuronal activity respectively in the spinal cord. In this experiment we observed the actions of a serotonin and noradrenaline reuptake inhibitor (SNRI), Milnacipram and its effect in modulating the transmission of pain. The result showed that Milnacipram was able to reduce the neuronal activity in the spinal cord therefore was able to suppress the transmission of pain.

Introduction

Pain is defined as “an unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage” by the International Association for the Study of Pain (IASP). Pain is classified into two broad terms acute and chronic pain based upon its aetiology, mechanism and pathophysiology. Acute pain has a fast onset and short duration period, which can be treated and cured within days or weeks. On the other hand chronic pain is persistent long term sensation of pain, which may go on for months; in general, pain lasting for more than three months is considered as a transition from acute to chronic 1. Chronic non malignant pain can be classified as inflammatory/nociceptive pain (following tissue damage), neuropathic pain (following nerve injury), and generalised pain (no tissue or nerve damage).

Peripheral Mechanisms in initiation of pain

Pain is initiated by the activation of specialised sensory receptors, nociceptors, located on the peripheral nerve endings of the primary afferent (sensory) fibres, which respond to noxious stimuli (mechanical, thermal and/or chemical) which may damage normal tissue or non-noxious stimuli which may become noxious if prolonged 1. The cell bodies of these sensory neurones are located in the dorsal root ganglion and the afferent fibres terminate in the dorsal horn of spinal cord located in the central nervous system (CNS) 2. The perception of acute pain is proportional to the magnitude of noxious stimulation and diminishes when the stimuli are removed.

There are three different types of primary afferent nerve fibres, A-beta (Aβ), A-delta (Aδ) and C-fibre, which are classified based upon the type of stimuli they respond to and their speeds of transduction. Aβ fibres are large diameter, myelinated, fast conducting primary afferent fibres, which respond to low threshold innocuous stimuli (e.g. light touch) and terminate deep in the spinal cord. Aδ fibres are smaller in diameter when compared to Aβ fibres, and thin myelinated thereby contribute to intermediate speed of conduction. These fibres respond to both noxious and innocuous sensory information and therefore convey sensory information in reference to pain. Aδ fibres generally respond to mechanical and thermal stimuli as these fibres have high threshold mechanoreceptors, thermal, and mechano-thermal nociceptors on their peripheral nerve free endings. C-fibres are small diameter, slow conducting unmyelinated primary afferent fibres which have polymodal nociceptors located on their periphery nerve endings that convey high threshold noxious sensory information to the superficial laminae (I and II collectively known as Substantia Gelatinosa (SG)) of spinal cord 1, 2. Therefore it is evident that the primary afferent fibres which convey nociceptive information to the spinal cord are the A-delta (Aδ) and C-fibres.

Following tissue damage or nerve injury there are various chemical mediators released by neuronal and non-neuronal cell which interact with C-fibres to sensitize nociceptors to other chemical mediators or cause direct activation. There are various membrane bound receptors (e.g. B2-receptor, EP3, P2X3 and etc.) on the primary afferent fibres in the periphery, which upon activation by their respective mediators (e.g. bradykinin, prostaglandins, ATP and etc.) increase central sensitization 1. The result of increase in central sensitization can be a reduced threshold for nociceptor activation, an increase in response to a given stimulus, or increase in spontaneous activity. These events would lead to generation of hyperalgesia (an increased response to a stimulus which is normally painful), allodynia (pain due to a stimulus which does not normally provoke pain), and spontaneous pain 3.

Central Mechanisms in pain transmission and modulation

Upon activation of nociceptors, the primary afferent fibres convey the sensory information from the periphery to the dorsal horn of the spinal cord where sensory information is integrated, modified, and amplified before it is propagated to the brain via various ascending pathways 3. The major ascending pathways include the spinothalamic tract, which conveys nociceptive information to the somatosensory cortex of the brain via the thalamus 2.

Ascending pathway: Spinothalamic tract

The nociception transmitting primary afferent fibres acts as the first order neurone which terminate in the SG dorsal horn of the spinal cord where they transmit sensory information to second order neurones located in laminae IV to VI, collectively known as nucleus proprius (principle sensory nucleus) 2. Nociceptive information transmitted by first order neurone via release of the excitatory neurotransmitter, glutamate and substance P from its nerve terminals into the synapse. The glutamate activates post-synaptic α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) and N-methyl-D-aspartic acid (NMDA) glutamate receptors in the spinal cord. The activation of AMPA receptor is fast onset and short duration, which causes post-synaptic neurone to depolarize. However NMDA receptor activation is slow and persistent, and due to the magnesium (Mg2+) channel block at rest, this receptor requires depolarization in order to remove the block. NMDA receptor therefore, is only activated following repetitive pain sensory inputs as this would increase activity of AMPA receptors, which depolarize post-synaptic cell thereby, remove Mg2+ block and activate the NMDA receptor. Furthermore, substance P also acts on the post-synaptic neurone via its receptor NK-1 also aid the removal of the Mg2+ block from the NMDA receptor channel hence allowing activation of NMDA 1. The second order neurones ascend to the thalamus from contralateral spinal cord, where the synapse onto third order thalamo-cortical neurones to convey nociceptive information to the primary somatosensory cortex of the brain 2.

Descending pain pathways

Pain sensations can be inhibited and facilitated by the descending pathways which act on the spinal cord in order to regulate the transmission and transduction of nociceptive information. Studies have revealed that these pathways arise from the brainstem from regions and nuclei such as the Periaquaductal Gray (PAG) in the midbrain, Nucleus Raphe Magnus (NRM) in the rostra-ventral medulla (RVM), and the locus ceruleus (LC) which is located in the pons. Electrical stimulation of the PAG leads to excitatory neuronal projections to the RVM 2 which is regulate the activity of nociceptive mechanisms as the nucleus contains 5-HT containing neurones which project directly to spinal cord. 5-HT-containing neurones can mediate excitatory and inhibitory influence in order to enhance or suppress the dorsal horn activity. 5-HT can cause facilitation of nociception via interaction with the 5-HT2A AND 5-HT3 receptors on the primary afferent central terminal thereby increasing the release of glutamate and substance P from presynaptic neurone. 4

However, 5-HT can also cause inhibition of nociception via interaction with 5-HT3 receptors located on the inhibitory interneurones that have axo-axonal synapse on the primary afferent fibres, and axo-dendritic synapse on the second-order dorsal horn neurones mediating the pain sensation. Hence activation of inhibitory interneurones by 5-HT causes release of neurotransmitters such as GABA and enkephalin, which act on their respective receptors to increase conductance of potassium ions (K+). This leads to decrease in calcium (Ca2+) influx in primary afferent central terminal as a result inhibits release of glutamate and substance P, and also causes hyperpolarization of the postsynaptic second order neurone involved in the ascending pathway as a result inhibition the transmission of pain 2. Furthermore, 5-HT can inhibit the release of glutamate from central terminals of primary afferent fibres via its interaction with the 5-HT1A receptor located on the central terminals of nociceptive fibres 4.

It has been established that this facilitation and inhibition of nociception is caused by two cell types “On” and “Off” respectively. The “On” cells fire in response to peripheral noxious stimuli as a result causing enhanced nociception whereas the “Off” cells pause in firing in response to peripheral noxious stimuli and thereby suppress nociceptive mechanisms. 4

Furthermore, it has been established that the noradrenergic descending pathway arising from LC is also involved in modulating nociceptive input to the spinal cord via similar mechanisms as described earlier. The difference being that in the spinal cord noradrenaline (NA) interacts with α1-adrenoreceptor to excite the inhibitory interneurones, which in turn mediate actions as described above, and can directly inhibit release from central terminal of primary afferent fibres via action on the α2-adrenoreceptor (in particular α2A-adrenoreceptor). Furthermore NA can interact with α2-adrenoreceptor (in particular α2C-adrenoreceptor) located on the membrane of post-synaptic second order neurone 4, which upon activation hyperpolarizes the postsynaptic neurone thereby inhibiting postsynaptic transmission.

Aim of study

In this experiment I will be analysing the effect of Milnacipram, which is a Serotonin and Noradrenaline Reuptake Inhibitor (SNRI) and how it affects the excitability of wide dynamic range (WDR) neurones in laminae V of the spinal cord. These WDR neurones are sensory neurones which respond to touch, heat and noxious stimuli; and receive direct and indirect inputs (via interneurones) from all primary afferent fibres. Also we will observe the effect of a non-selective α-adrenoreceptor antagonist, Phentolamine, and the selective α2-adrenoreceptor antagonist, Atipamezole; and analyse their effects on the excitability of WDR neurones in the dorsal horn of the spinal cord.

Method

Spinal cord neuronal recordings:

Sprague-Dawley rats (200-250 g) were anaesthetised with isoflurane in 66% N2O / 33% O2. Extracellular recordings of dorsal horn neurones were made with parylene coated tungsten electrodes. To isolate a neurone, electrode was moved incrementally through the cord whilst the ipsilateral hindpaw (receptive field) is stimulated with a light tap.

Neurones respond to A-beta (Aβ), A-delta (Aδ) and C-fibre cutaneous afferent inputs from the hindpaw following transcutaneous electrical stimulation. Responses were elicited by a train of 16 electrical stimuli at 0.5 Hz (3 times the threshold current required for C-fibre evoked activity and for Aβ/δ-fibre evoked activity) at 10 minutes intervals and post-stimulus histograms constructed.

Repetitive stimulation at C-fibre strength resulted in the NMDA receptor mediated wind-up, an enhanced neuronal response, and a resultant post-discharge (PD). The evoked responses were separated and quantified on the basis of threshold and latency: Aβ-fibre evoked activity at 0-20ms post stimulus; Aδ at 20-50 ms; afferent C-fibre evoked activity at 90-300ms and post-discharges at 300-800ms.

The non-potentiated response of the dorsal horn neurones evoked by C-fibre stimulation was calculated as the number of action potentials produced by the first stimulus multiplied by the total number of stimuli (sixteen). Wind-up can be calculated as the overall response to the 16 stimuli minus this non-potentiated response.

Responses to the following natural stimuli were also measured:

Mechanical stimuli: von Frey filaments with forces of 2 (non-noxious) 6 (about threshold), 26 and 60g, plus brush.

Thermal stimuli: 40 (non-noxious), 45 and 48ºC.

Responses to the stimuli following these drug administrations were also measured:

Milnacipram was administrated at 1μg, 10μg and 100μg directly onto the spinal cord where it could interact with the terminals of the descending noradrenaline pathways.

Phentolamine, which is a non selective α-adrenoreceptor antagonist or Atipamezole, which is a selective α2-adrenoreceptor antagonist was administrated.

Results

Electrical Response Results

Table 1: effect of different doses of Milnacipram on the average number of spikes generated by primary afferent nerve fibres, input, and PD

Number of Spikes

Control

1μg Milnacipram

10μg Milnacipram

100μg Milnacipram

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

Aβ fibres

116

23

65

17

72

16

55

22

Aδ fibres

163

26

103

33

65

25

32

16

C-fibres

421

42

293

119

119

42

88

30

PD

334

66

221

124

120

48

67

51

Input

440

104

323

196

145

59

51

27

Figure 1: a graph to show the effect of different doses of Milnacipram on primary afferent nerve fibres, input, and PD

The results from Table 1 and Figure 1 outline that in general, as the dose of Milnacipram increases, the average number of spikes generated by the afferent nerve fibres, input, and PD decrease. However this is not the case when we observed Aβ fibres as following 10μg administration of Milnacipram we observe a greater number of spikes when compared to the mean number of spikes generated by administration of 1μg of Milnacipram.

Table 2:

Response Change (%)

1μg Milnacipram

10μg Milnacipram

100μg Milnacipram

Mean

SEM

Mean

SEM

Mean

SEM

Aβ fibres

67

19

65

16

62

20

Aδ fibres

62

16

31

8

23

11

C-fibres

65

29

35

13

30

10

PD

89

63

65

28

17

9

Input

42

11

39

18

18

7

Figure 2

The results from Table 2 and Figure 2 shows that as the dose of Milnacipram increases, the mean percentage change in response decreases is observed for PD, Input, C-fibres, and Aδ fibres. This suggests that Milnacipram causes inhibition of spinal neuronal activity thereby suppressing nociceptive mechanisms. However we observed that there the response of Aβ fibres decreases after administration of 1μg Milnacipram when compared to control, however there is no significant decrease in mean percentage response following further administration of higher doses Milnacipram, 10μg and 100μg. This therefore suggests that decrease in the mean percentage change in response of Aβ fibres is dose-independent.

Table 3

Response Change (%)

100μg Milnacipram

Phentolamine

Atipamezole

Mean

SEM

Mean

SEM

Mean

SEM

Aβ fibres

62

20

54

17

64

9

Aδ fibres

23

11

12

7

58

NA

C-fibres

30

10

20

18

NA

NA

PD

17

9

8

7

56

NA

Input

18

7

15

11

NA

NA

Figure 3

The result from Table 3 and Figure 3 outlines administration of Phentolamine following 100μg Milnacipram does not reverse the effects of the SNRI. This is because Phentolamine administration caused further reduction in percentage mean change in response, thereby indicating further inhibition of spinal neuronal activity. Whereas, with the data obtained for Atipamezole, we can analyse that the drug causes partial reversal of the effect caused by Milnacipram. As a result it shows that Atipamezole acts to increase spinal neurone activity but does not restore activity to control level (100%).

Thermal Stimulation Results

Table 4: effect of different doses of Milnacipram on the average number of spikes generated in response to a thermal stimulus

Number of Spikes

Temperature (ºC)

Control

1μg Milnacipram

10μg Milnacipram

100μg Milnacipram

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

40

182

60

290

133

126

46

67

60

45

492

128

311

124

212

89

229

114

48

768

71

618

158

1181

906

206

177

Figure 4: a graph to show the effect of different doses of Milnacipram on thermal response

The results from Table 4 and Figure 4 show that as the temperature increases the average number of spikes in general increases. Also, administration of 1μg of Milnacipram when response to non-noxious thermal stimuli, 40ºC, caused an increase in average number spikes thereby indicating greater response when compared to control. This suggests that 1μg of Milnacipram at 40ºC causes increase in spinal neurone activity. Administration of 10μg Milnacipram and the response observed at noxious thermal stimuli, 48ºC, we also observed that the average number of spikes were greater than control, suggesting greater response when compared to control, thereby indicating hyperexcitability of spinal neuronal activity. Apart from these two observations, we generally see a decrease in average number of spikes as the dose of Milnacipram increases.

Table 5

% Response Change

Temperature (ºC)

1μg Milnacipram

10μg Milnacipram

100μg Milnacipram

Mean

SEM

Mean

SEM

Mean

SEM

40

128

66

70

20

34

18

45

65

30

31

8

41

19

48

75

16

144

111

19

16

Figure 5

The result from Table 5 and Figure 5 outlines that in general as dose of Milnacipram increases, the mean response change percentage decreases. This suggests that increasing Milnacipram dose, inhibits spinal neuronal activity. However, we do observe a large increase in % mean response change following administration of 10μg Milnacipram from 45ºC to 48ºC, indicating that at higher temperature 10μg Milnacipram is unable to suppress neuronal activity as effectively.

Table 6

% Response Change

Temperature (ºC)

100μg Milnacipram

Phentolamine

Atipamezole

Mean

SEM

Mean

SEM

Mean

SEM

40

34

18

23

15

120

NA

45

41

19

15

8

8

NA

48

19

16

48

34

19

NA

Figure 6

The results from Table 6 and Figure 6 shows that Atipamezole is able to reverse the effect of Milnacipram at non-noxious thermal stimuli, 40ºC, but unable to reverse the actions of the SNRI, at higher temperatures. In contrast, Phentolamine is observed to have reversed the action of Milnacipram at the noxious thermal stimuli, 48ºC, but unable to reverse the actions of SNRI at lower temperatures.

Mechanical Stimulation Results

Table 7: effect of different doses of Milnacipram on the average number of spikes generated in response to a mechanical stimulus

Number of Spikes

Mechanical Force

Control

1μg Milnacipram

10μg Milnacipram

100μg Milnacipram

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

Brush

420

93

294

94

213

61

147

56

von Frey 2g

50

27

31

16

18

9

5

3

von Frey 6g

240

85

280

176

52

22

38

19

von Frey 26g

647

137

324

132

285

83

141

70

von Frey 60g

851

97

467

96

444

101

409

158

Figure 7: a graph to show the effect of different doses of Milnacipram on mechanical response

The result from Table 7 and Figure 7 show that the mean number of spikes decreases as the dose of Milnacipram increase indicating that the drug reduces neuronal activity in the spinal cord. However, there was only one value observed above control following administration of Milnacipram, which was at mechanical force von Frey 6g following administration of 1μg of Milnacipram indicating increase in neuronal activity in the spinal cord. Also we observed that as the von Frey filament mass increases, the mean number of spikes increase.

Table 8

% Response Change

Mechanical Force

1μg Milnacipram

10μg Milnacipram

100μg Milnacipram

Mean

SEM

Mean

SEM

Mean

SEM

Brush

57

25

58

16

105

51

von Frey 2g

161

79

155

84

31

11

von Frey 6g

73

27

38

12

40

19

von Frey 26g

56

16

62

18

28

12

von Frey 60g

47

4

51

13

56

25

Figure 8

The results from Table 8 and Figure 8 outline that there is a general decrease in the mean % response as dose of Milnacipram increases, apart from one set of results which were obtained for brush, which show increase in % response as Milnacipram dose increases. This means that Milnacipram is ineffective in suppressing neuronal activity following the mechanical stimuli, brush. We also observed that von Frey 2g filament produced response greater than control (above 100%), following administration of 1μg and 10μg of Milnacipram.

Table 9

% Response Change

Mechanical Force

100μg Milnacipram

Phentolamine

Atipamezole

Mean

SEM

Mean

SEM

Mean

SEM

Brush

105

51

75

37

107

NA

von Frey 2g

31

11

33

31

471

NA

von Frey 6g

40

19

22

20

9

NA

von Frey 26g

28

12

47

19

41

NA

von Frey 60g

56

25

44

22

109

NA

Figure 9

The results from Table 9 and Figure 9 outline Phentolamine and Atipamezole do not significantly reverse Milnacipram effect on mechanical response when the mechanical stimuli was von Frey 6g. However, we did observe reversal of the effect mediated by Milnacipram by Atipamezole when stimulated with brush (minimal increase); von Frey 2g (marginal increase), and von Frey 60g (significant increase). We also observed reversal of action of Milnacipram following administration of Phentolamine when stimulated by von Frey 2g (minimal increase) and von Frey 26g (significant increase).

Discussion

The aim of this study was to outline the effect of the SNRI, Milnacipram, on the neuronal activity in the dorsal horn of the spinal cord, and whether this drugs acts to facilitate or suppress the response to a given stimulus. In order to observe this, the drug was assessed by its effect on primary afferent fibres firing associated with noxious electrical stimuli, in addition to non-noxious and noxious thermal and mechanical stimulations. We used three doses of Milnacipram, 1μg, 10μg, and 100μg respectively in order to compare the dose-dependent nature of the drug.

From our electrical stimulation results (Tables 1-2 and Figures 1-2) we were able to observe that the administration of Milnacipram was able to reduce the mean number of spikes Aδ fibres, C-fibres, input, and PD as the dose of drug increased. This indicates a dose-dependent decrease in neuronal activity in the spinal cord, which means that the drug is able to suppress the nociceptive neuronal mechanisms in the spinal cord as a result reduce pain transmission. However this is not clearly evident for Aβ fibres, as we did observe a decrease in the mean number of spikes observed for Aβ fibres following administration of 1μg Milnacipram when compared to control, but following the administration of 10μg of Milnacipram we observe an increase in the mean number of spikes as shown in Figure1. In addition, Figure 2 outlines that following the initial decline in the mean response change of Aβ fibres after administration of 1μg Milnacipram, there is no significant change in Aβ fibres firing at higher doses of Milnacipram. This may be due to the fact that the Aβ fibres respond to innocuous stimuli therefore their activity is not as significantly affected as the nociceptive Aδ fibres and C-fibres; this can be supported by the very low mean number of spikes as well caused by Aβ fibres indicating very little response to noxious stimulus.

The results from the thermal stimulation experiment (Tables 4-5 and Figures 4-5) we observed that as the temperature increased from non-noxious 40ºC to noxious 48ºC, the mean number of spikes increased for control. This suggests that at noxious stimuli, there is hyperexcitability in the dorsal horn of the spinal cord, therefore increase in sensation and transmission of pain. Administration of Milnacipram decreased the mean number of spikes to some extent. However we did observe that following administration of 1μg Milnacipram and 10μg Milnacipram at non-noxious 40ºC and noxious 48ºC respectively, there was an increase in number of spikes outlining a greater excitability of neurones in the spinal cord. This could be due to the large standard error in mean that led to such variability in results hence it is important to repeat the experiment for validation.

Finally, the effect of Milnacipram was observed via the mechanical stimulation experiment (Tables 6-7 and Figures 6-7) from which we were able to observed that after application of von Frey 6g (about threshold) the mean number of spikes increased significantly for control suggesting increase in neuronal activity thereby increase in pain sensation. By administrating Milnacipram we were able to see a decrease in the mean number of spikes, which revealed that the drug was suppressing the firing of neurones in the dorsal horn, thereby inhibiting transmission of pain. However, one observation, application of von Frey 6g with 10μg Milnacipram administrated, we observed that man number of spikes were greater than the control, indicating facilitation of neuronal activity when compared to control. This may be due to the large standard error in mean that led to such variability in results hence it is important to repeat the experiment for validation.

Overall, the results we obtained we did observe that Milnacipram does reduce the neuronal activity in the spinal cord following electrical, thermal, and mechanical stimuli, when compared to control. This can be explained by the pharmacological understanding of the mechanism of action of Milnacipram, as we already know that it is a SNRI, thereby it interacts and blocks the membrane transporters involved in reuptake of NA and 5-HT. As a result of this, there is an increased level of 5-HT and NA at the synapse, which in turn leads to a prolonged action of these monoamines. It has been revealed by studies that Milnacipram inhibits reuptake of NA to a greater extent when compared to 5-HT reuptake inhibition 5, as a result enhancing the descending noradrenergic pathway which mediates its actions in order to suppress neuronal activity in the spinal cord by mechanism explained earlier. In addition, Milnacipram has been identified to mediate weak inhibitor of the NMDA receptor so therefore this drug can inhibits the transmission of pain. 5

So therefore, the action of Milnacipram in inhibiting NA reuptake and NMDA receptor play a vital role in the inhibition of pain transmission. For this particular reason Milnacipram is being used to treat chronic widespread pain conditions such as fibromyalgia, in which the patient experiences hyperalgesia and allodynia due to the sensitization of the CNS. The symptoms of this condition include tenderness, sleep disturbances, fatigue, cognitive impairment, and mood disorders. The causes for this condition are not clearly understood however there may be some involvement of abnormal “wind-up” in the spinal cord, peripheral sensitization, central sensitisation, and abnormal descending pathways modulating pain transmission. 6

The increase in peripheral sensitization may be caused by chemical mediators as described earlier which may induce sensitization of polymodal C-fibre nociceptors, lowered thermal stimuli threshold, and increased response to mechanical stimuli. As a result, central sensitization is caused due to the events that take place in the periphery which in turn lead to a lowered nociceptive threshold at the dorsal horn of the spinal cord. This sensitization of the periphery and CNS lead repetitive high-frequency stimulation of C-fibres which release glutamate and substance P from the central terminals in the dorsal horn of the spinal cord 6. This repetitive stimulation amplifies and prolongs response of neurones in spinal cord via activation of NMDA receptors as described earlier which induce “wind-up”. Therefore activation of NMDA receptors and the induction of “wind-up” are seen to play a vital role in hyperalgesia and enhancement of pain transmission 1 seen in patients with persistent pain condition as fibromyalgia.

In addition, it is also thought that the dysfunction of the descending modulating pathway may also contribute to pathogenesis of fibromyalgia. There in no clear understanding of this, however, it may be due to the enhanced 5-HT transmission from the brainstem which acts on 5-HT3 receptors to facilitate pain transmission and reduced noradrenergic transmission thereby reduced inhibitory control 6. As a result the use of Milnacipram would be effective to treat patients that experience fibromyalgia as the drug is able to partially inhibit NMDA receptor and also block NA reuptake effectively. As a result Milnacipram will inhibit the “wind-up” caused by NMDA receptor activation and restore the inhibitory influence of the descending modulating pathway, which will consequently improve pain via reducing its transmission.

In this experiment we also observed the effect of a non-selective α-adrenoreceptor antagonist, Phentolamine, or a selective α2-adrenoreceptor antagonist, Atipamezole, which was administrated after 100μg of Milnacipram; this was to observe if the inhibiting the noradrenergic action on its respective receptors can reverse the effect of Milnacipram and to identify the receptors involved. It is already established that the noradrenergic descending pathway modulates nociceptive input to the spinal cord thereby inhibiting its action would consequently cause facilitation of pain transmission.

Phentolamine results (Tables 3, 6, and 9; and Figure 3,6 and 9) outline that administration of Phentolamine did not reverse the effect of 100μg Milnacipram following electrical stimulation as the mean percentage response change mediated by Phentolamine was less than that of Milnacipram thereby suggesting Phentolamine has an analgesic effect. This was also observed following thermal stimuli of 40ºC and 45ºC; and also mechanical stimuli brush von Frey 6g, and von Fey 60g filaments. However, previous studies have revealed that Phentolamine enhances pain transmission 10 and this can be explained by its mechanism of action, which is to inhibit α1-adrenoreceptor and α2-adrenoreceptor 8. This in turn inhibits noradrenergic descending modulating pathway as it inhibits the α1-adrenoreceptor (located on interneurones which go onto inhibit neuronal activity in pain processing in the spinal cord), in addition inhibition of α2-adrenoreceptor (located on primary afferent presynaptic terminal and second-order post-synaptic neurones in the spinal cord) the net effect being reversal of the analgesic effect mediated by Milnacipram. In addition, a possible explanation of the results obtained could be due to the large SEM therefore the experiment should be repeated for validation.

Atipamezole results (Tables 3, 6, and 9; and Figure 3,6 and 9) outline that administration of this drug was able to reverse the effect of Milnacipram following electrical stimulation as we observed an increase the mean response change percentage when compared to 100μg Milnacipram. This was also observed following thermal stimuli 40ºC; and mechanical stimuli brush, von Frey 2g, and von Frey 60g. However the remaining results in the thermal and mechanical stimulations outline no reversal mediated by Atipamezole. This is due to the limited number of results obtained for Atipamezole effect; therefore in most cases the SEM could not have been calculated. So therefore we did not have sufficient evidence to conclude on the study of Atipamezole. However, Atipamezole being a selective α2-adrenoreceptor antagonist has been identified by previous studies to enhance hyperalgesia as they inhibit the inhibitory action mediated by the receptor. As a result enhances the activity of dorsal horn neurones which facilitates the transmission of pain 7. Furthermore studies have shown that Atipamezole enhances response of spinal cord neurones to low frequency mechanical stimuli thereby suggesting that the drug may control the descending inhibition via the α2-adrenoreceptor in the spinal cord. It is also observed in previous studies that Atipamezole shows no effect on the neuronal activity in the spinal cord following noxious mechanical, and non-noxious and noxious thermal stimulation 9. So therefore, it is essential to repeat the experiment for result validation.

On whole, previous studies and to some extent our results have outlined that inhibition of the α-adrenoreceptor and in particular α2-adrenoreceptor causes facilitation of pain transmission. Therefore we are able to say that α2-adrenoreceptor plays a vital role in mediating the inhibitory control on spinal cord neurones via actions of NA that is transmitted to the spinal cord via descending noradrenergic pathways from the brainstem. As result previous studies have allowed us identify the importance of α2-adrenoreceptor in suppression of pain transmission.

Conclusion

From the results obtained, we can conclude that the administration of Milnacipram causes an analgesic effect by suppressing neuronal activity following electrical, thermal, and mechanical stimuli and therefore is potentially an effective drug in treatment for fibromyalgia. In addition, the noradrenergic descending pathway plays an important role in modulating nociceptive inputs therefore remain an attractive target for analgesic drug actions. Furthermore, there was not sufficient evidence from the results obtained to conclude on the effect of the α-adrenoreceptor antagonists on neuronal activity in the spinal cord, but we do know from previous studies that α-adrenoreceptor antagonists induce hyperalgesia via inhibition of NA action on its respective receptors in the dorsal horn of the spinal cord. So therefore further experiments should be conducted in order to validate the results for α-adrenoreceptor antagonists and further research is required in order to produce drugs which can suppress and therefore improve pain via modulation mediated by the descending pathway.


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