*4.2. Peripheral Mechanism of Pain Regulation*

The enteric nervous system (ENS) is formed by about 200–600 million neurons and is often referred to as the 'second brain'. This network plays a part in maintaining GI tract function and reaches the lamina propria of the mucosa. ENS neurons form the subserous, myenteric, and submucosal plexuses and carry impulses to and from the brain. Intrinsic primary afferent neurons (IPANs) initiate secretory, motor, and vasomotor reactions from stimuli within the mucosa and from the central nervous system (CNS) [104]. Enteric sensory neurons receive the information through neurotransmitters and hormones released by enteroendocrine (EEC) and enterochromaffin (EC) enteric cells.

Enteric hormones such as serotonin (5-HT), glucagon-like peptide 1 (GLP-1), or peptide YY (PYY) are thought to have an impact on visceral pain and its management [104]. 5-HT excreted by EC cells activates receptors on EC cells and extrinsic primary afferent nerve (EPAN) terminals. This triggers enteric reflexes such as secretion, peristalsis, and perception of pain and inflammation [105–107].

Microbes in the GI tract microbiome can synthesize various neurotransmitters and metabolites involved in gut–brain communication, as shown in recent studies [108–111]. This includes SCFAs, tryptophan metabolites, GABA, dopamine, and noradrenaline [104]. One of the SCFAs, butyrate, was proposed as an agent with an indirect effect on regulating inflammatory visceral pain. Its injection in rat and mouse brains stimulated the production of brain-derived neurotrophic factor (BDNF), which favors neurogenesis, memory formation, and mood stabilization [112–114].

Bacteria such as *Escherichia*, *Fusobacterium*, *Prevotella*, *Enterococcus casseliflavus*, or *Bacteroides* can produce tryptophan, which later passes the blood–brain barrier (BBB), influencing serotoninergic neurotransmission in the CNS. In a study by Agus et al., it was shown that during gut inflammation, an increase in tryptophan conversion to kynurenine may be responsible for the development of anxiety and mood shifts [115]. During inflammation, there is an enhanced plasma level of kynurenine, which may favor its passage through the BBB and later metabolism into kynurenic acid (KynA) and quinolinic acid (QuiA), the latter of which is described as a neurotoxic agent [111].

Another microbial product, glutamate, is produced by certain microbial strains in the healthy GI tract [116–119]. It is a major neurotransmitter in the CNS and acts as a neuroactive molecule. A recent study suggested that glutamate may also regulate gut sensory and motor functions via receptors in the ENS [120,121]. During stress-induced dysbiosis, glutamate receptor expression is altered. In antibiotic-treated mice with dysbiosis, there were decreased levels of hippocampal NMDA and BDNF, which were later restored by prebiotic treatment [31,122–124].

GABA is an important neurotransmitter in the brain. Bravo et al. studied its role in pain management and suggested that GABA can inhibit visceral hypersensitivity, altering abdominal pain [125]. Oral administration of *Lactobacillus rhamnosus* in mice increased GABA levels in the CNS. Additionally, in a study by Perez-Berezo et al., administration of the *E. coli* Nissle 1917 (EcN) strain showed an increase in analgesic lipopeptide production, activation of GABA receptors on IPANs, and inhibition of visceral hypersensitivity [126].

#### **5. Headache and Its Association with Drugs**

Headache is one of the most frequently reported symptoms [127], and various types have been described. Primary headaches can be divided into four groups: migraine, tension headache, trigeminal autonomic cephalgia, and other primary headache disorders [127]. Migraine, a neurological disorder characterized by headache, nausea, vomiting, and photophobia or phonophobia [128,129], is one of the most common types of headaches [17]. The hemicrania occurs due to hypothalamus activation and further pituitary adenylate cyclase-activating polypeptide (PACAP) secretion, which is responsible for vasodilatation [17]. Moreover, migraine is related to GI illnesses, which include celiac syndrome, irritable bowel syndrome, or infection by *Helicobacter pylori* [12,30]. There is also an association between the gut microbiome and the pathogenesis of migraine. The gut–brain axis triggers the migraine attack through pro-inflammatory factors, gut microbiome composition, neuropeptides, serotonin pathways, stress hormones, and nutritional substances. The physical or psychological stress factors may lead to gut microbiome changes such as dysbiosis [30]. This, in turn, causes an increase in calcitonin gene-related peptide (CGRP) secretion [17], which is correlated with migraine symptoms and has antibacterial effect on strains such as *E. coli*, *E. faecalis*, and *L. acidophilus* [17,30]. This particular type of headache is associated with pro-inflammatory factors. During migraine attacks, increased secretion of serum cytokines such as IL-1b, IL-6, IL-8, and TNF-a was observed. Moreover, Arzani et al. reported that in germ-free mice, the hypernociception induced by inflammatory mediators is reduced [30]. These could result from increased expression of IL-10 in germ-free mouse models [130]. This cytokine is an important regulator of inflammatory responsiveness [130]. These lines of evidence emphasize the importance of the gut microbiome in migraine and have prompted research on whether probiotic supplementation is a beneficial therapy for the condition [12]. The data on the efficacy of probiotic supplementation in migraine are

incoherent. Sensenig et al. showed that most patients who were given probiotics, such as *L. bulgaricus*, *L. acidophilus*, *E. faecium*, and *B. bifidum*, reported an improvement in quality of life [131]. By contrast, another study showed no significant differences between a group of patients who suffered from migraine and were supplemented with probiotics and the one that was not supplemented with probiotics [12,132].

To summarize, the association between the gut microbiome and migraine is clear. Studies show not only a correlation in pathogenesis but also a possible way of treating migraine with probiotics. However, there is still a lot to be discovered [12].
