Morphine is one of the primary drugs used to alleviate persistent or severe pain both in clinical and preclinical studies. Although it is identified as a primary drug, females require approximately 2 to 3 times more dosage than males to attain a similar level of analgesia. The study identifies that microglia cells present in the periaqueductal gray (PAG) of all mammals undergo activation sexual dimorphic. Morphine also binds innate immune receptor toll-like receptor 4 (TLR4) situated in the microglia. Both sexes had similar microglial cells where dimorphic cells stem from varying activation level. Both sexes were injected with morphine to identify the microglial activity. Nickel sulfate was used to visualize microglia immune reactivity which either formed purple or black. The morphological activity of microglial produces a highly indicative nonramfied functional state of the cells forming a large oval and circular shaped.
Sexual Dimorphism in Toll-Like Receptor Four (TLR4)
Sex Differences in Microglia Activity within Rat Periaqueductal Gray: Potential Mechanism Driving the Dimorphic Effects of Morphine
Chronic and severe pain in adults in the United States affects approximately 3-4% of adults and can easily be managed using opioid therapy (Doyle et al., 2017). Czerniawski and Guzowski (2014) have identified that morphine can be easily used to manage pain but its effectiveness is more in males than in females. The reduction of analgesic efficacy is contradicted by escalating dose resulting in reduced risk to negative effects like an overdose, tolerance, and respiratory depression (Doyle et al., 2017). Morphine, as well as, other opioids bind myeloid differentiation factor 2 (MD-2) coreceptor of the innate TLR4. The TLR4 is found on the microglia where it activates the MD2-TLR4, promoting the anti-inflammatory and pro-inflammatory that have cytokines like prostaglandin E2 (PGE2), chemokines (CXCL3), interleukins (IL-1β, IL-6, IL-10), and tumor necrosis factor-alpha (TNFα) (Dowell, Haegerich, & Chou, 2016).
Marinelli et al. (2015) explain that the process of TLR4 activation and the release of proinflammatory signaling molecules increase neuronal edginess through reduced γ-aminobutyric acid (GABAA) receptor expression, down-regulation of the glutamate transporter, and up-regulation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor. Sorge et al. (2015) elaborate that TLR4-mediated inflammation is used to reduce the analgesic efficacy of morphine. The antinociceptive effect of morphine mediates the neural locus of ventrolateral midbrain periaqueductal gray (vlPAG). However, long-lasting analgesia effect occurs through direct PAG morphine administration. Nonetheless, antinociceptive effects of systemic morphine are abolished by lesions of PAG μ-opioid receptor (MOR) or opioid antagonist (−)-naloxone. The antinociceptive effect of intra-PAG are highly influenced by gender wherein females, the half-maximal antinociceptive dose (ED50) of morphine range from 16 to >50 μg/μl while a similar dose in male ranges from 1.2 to 1.6 μg/μl (Morrison & Filosa, 2016; Zhang et al., 2013).
PAG administration in morphine is expressed in MD-2 where it activates the PAG microglia where it opposes the analgesic effects of morphine. It also induces tolerance in a TLR4-dependent manner. The tolerance and inhibition of analgesic effect following the blockade of PAG microglial activation are demonstrated in rats. The hypothesis of the research is to examine if the reduced effect of morphine in females is due to heightened microglia activation in a region of the brain called the periaqueductal gray (PAG).
Materials and Methods
The population for the study was intact male and cycling female Sprague Dawley rats aged 60-90 days old. The male and the female rats were put in rooms on a 12:12h dark and light cycle with lights on from 8.00. The rats were provided with food and water during the behavioral test. To ensure that all females were cycling, the researcher conducted vaginal lavages every day and cycle stage was recorded each time. The predominance of cornified epithelial cells was estrus while the predominance of nucleated epithelial cells was proestrus. Presence or absence of leukocytes differentiated Diestrus 1 from Diestrus 2. A specimen between phases was recorded as being in an advanced stage.
In experiment 1, the researcher observed whether morphine activation of microglia is sexually dimorphic by exposing 48 female and 54 male rats with ED50 subcutaneous injection with saline (135/kg) or 5mg/kg of morphine sulfate. The microglia activation was measured using ionized calcium binding adaptor molecule. This was followed by counting the Iba-1 positive and classified as 1, 2, or 3 meaning non-ramified, intermediate, and ramified. The author identified the effect of treatment and sex on morphology and microglia cell count.
The sexual relationship was determined by conducting a study among 8 female and 15 male rats between microglia’s morphology and the amount of morphine administered. The rats were administered with 1.8mg/kg to 18mg/kg of increasing morphine while 1Ml/kg was administered to the control group. After every 15 minutes, a paw thermal stimulator was used to assess thermal nociception. The duration in seconds taken by the rat to remove its paw from the stimulator was recorded as paw withdrawal frequency (PWL). The correspondence of the percentage maximal PWL was the ED50 calculation.
The purpose was to determine if morphine was the cause for the difference between the males and the females or sex activation. This was attained by exposing 6 female and 7 males to saline injection or TLR4 agonist Lipopolysaccharide (LPS). The microglia was observed by the use of qPCR to identify the amount of cytokine and ANOVA was used to analyze the impact of cytokine mRNA levels and treatment and sex on microglia morphology.
The purpose of the fourth experiment was to determine the impact of inhibition and activation of PAG TLR4 on morphine analgesia. This was attained by implanting cannulae on the viPAG to anesthetized rats. The inhibition and activation properties were observed. Additionally, naloxone 5ug was injected to calculate the antinociceptive effect.
In the first experiment that was determining if sex influences microglia activation without administering morphine, the study identifies that there was no difference in the total number of viPAG microglia. However, there was more intermediate microglia and non-ramification among females than the males irrespective of the treatment. In the second experiment, the aim was to determine whether there is a relationship between the administration of morphine and microglia morphological type. The results identified a significant variation between males 6.01 and females 8.69. The study also identified a significant variation between ED50 and ramified microglia, as well as sex-specific correlation with microglia activation.
Figure 1: Microglia immunoreactivity of males and female and classified non-ramified, intermediate and ramified.
In the third experiment, the study identifies that LPS in females have an increased intermediate and nonramfied microglia and it reduces the anti-inflammatory effect in females through dimorphic activation. On the fourth experiment, the study identifies that administering LPS reduces the antinociception of morphine in both males and females.
In comparison to males, the females recorded a higher ratio of nonramfied and intermediate microglia. This is an indication that females in viPAG have a more activated microglial state. vlPAG microglia produces sexual dimorphic after activating with LPS. Lastly, the attenuated difference between females and males is based on TLR4 signaling.
This study did not include the aspect of long-term pain but in mice chronic spinal pain is persistent in non-TLR dependent. The TLR is involved in developing the tolerance effect observed in morphine.
Female rats experience reduced pain sensation following the activation of PAG microglia. The study also identifies sex-specific differences in pain modulation through TLR4. The LPS was used as a form of chronic pain for the study, which is an indication that females have a lower activation threshold providing a more vigorous pro-inflammatory response contrary to males. The rate of pain response can be increased through the inhibition of TLR4, which is higher among females than males.
Czerniawski, J., & Guzowski, J. (2014). Acute Neuroinflammation Impairs Context Discrimination Memory and Disrupts Pattern Separation Processes in Hippocampus. Journal Of Neuroscience, 34(37), 12470-12480. doi: 10.1523/jneurosci.0542-14.2014
Dowell, D., Haegerich, T., & Chou, R. (2016). CDC Guideline for Prescribing Opioids for Chronic Pain—United States, 2016. JAMA, 315(15), 1624. doi: 10.1001/jama.2016.1464
Doyle, H., Eidson, L., Sinkiewicz, D., & Murphy, A. (2017). Sex Differences in Microglia Activity within the Periaqueductal Gray of the Rat: A Potential Mechanism Driving the Dimorphic Effects of Morphine. The Journal Of Neuroscience, 37(12), 3202-3214. doi: 10.1523/jneurosci.2906-16.2017
Marinelli, C., Di Liddo, R., Facci, L., Bertalot, T., Conconi, M., & Zusso, M. et al. (2015). Ligand engagement of Toll-like receptors regulates their expression in cortical microglia and astrocytes. Journal Of Neuroinflammation, 12(1). doi: 10.1186/s12974-015-0458-6
Morrison, H., & Filosa, J. (2016). Sex differences in astrocyte and microglia responses immediately following middle cerebral artery occlusion in adult mice. Neuroscience, 339, 85-99. doi: 10.1016/j.neuroscience.2016.09.047
Sorge, R., Mapplebeck, J., Rosen, S., Beggs, S., Taves, S., & Alexander, J. et al. (2015). Different immune cells mediate mechanical pain hypersensitivity in male and female mice. Nature Neuroscience, 18(8), 1081-1083. doi: 10.1038/nn.4053Zhang, X., Zhang, Y., Asgar, J., Niu, K., Lee, J., & Lee, K. et al. (2013). Sex differences in μ-opioid receptor expression in trigeminal ganglia under a myositis condition in rats. European Journal Of Pain, 18(2), 151-161. doi: 10.1002/j.1532-2149.2013.00352.x
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