Through immunohistochemistry and intestinal motility analyses, this study demonstrated the effects of PB treatment on EGCs subjected to different periods of ischemia and reperfusion. PB treatment induced the recovery of GFAP-positive EGCs. However, treatment with PB did not result in the recovery of S100β-positive EGCs positive. Neurons positive for HuC/D showed recovery after treatment with PB. The analysis of intestinal motility showed that PB was effective. We visually confirmed that our 45-min ileal vessel clamping technique efficiently induced ischemia, in that the intestine became purple in color during vessel clamping and regained its normal color after the removal of the clamp, as has been previously described [29–32]. The use of immunohistochemistry to identify EGCs using GFAP or S100β as a marker was efficient and demonstrated the presence of two different glial cell phenotypes . The antibodies used in this work were effective for the identification of enteric neurons and pannexin-1 channels. The organ bath method was very useful for studying intestinal motility after treatment with PB and the application of carbachol and electrical stimuli. Additionally, the use of the antagonist PB did not cause harmful functional changes in the animals studied.
Regarding the pannexin-1 channel, this study found that this channel was present in 100% of EGCs. In the presence of injury or pathological conditions, the pannexin-1 channel can open and thus allow the release of intracellular ATP into the extracellular environment, and this action can further aggravate the inflammatory process [33–35].
In the present study, double labeling of GFAP and S100β was performed to study the phenotype of EGCs. The results demonstrated that in all the groups studied, 70% of EGCs showed GFAP/S100β colocalization, and 29% of EGCs were immunoreactive for only S100β or GFAP. Previous studies have demonstrated the phenotypic heterogeneity of EGCs, and the differential expression of S100 and GFAP in these cells indicates their high plasticity [36–38]. Boesman et al. (2014)  morphologically classified EGCs into types I, II and III and then performed experiments to assess the colocalization of GFAP and S100β. Among type I EGCs, 55.8 ± 5.2% were GFAP+/S100β+, whereas GFAP-/S100β + and GFAP+/S100β- cells accounted for 29.9 ± 4.4% and 14.3 ± 3.9%, respectively. Among type II EGCs, the percentage of GFAP-/S100β + cells was 59.6 ± 1.6%; among type III EGCs, the percentage of GFAP-/S100β + cells was 78.9 ± 0.7%. Grundmann et al. (2016)  also showed colabeling of S100β and GFAP.
Ischemia–reperfusion injury reduces the numbers of enteric neurons and EGCs expressing HuC/D and S100β, respectively, per unit area [29, 30]. In the present study, treatment with the antagonist PB resulted in the recovery of HuC/D staining (pan-neuronal). Additionally, the use of PB has been shown to exert protective effects against oxygen and glucose deprivation injuries by regulating inflammatory activity . Wei et al. (2015)  showed that PB has a neuroprotective effect against brain ischemia and reperfusion injuries, and another study also found that PB suppresses inflammatory processes and reduces the release of high-mobility group box 1 protein (a proinflammatory cytokine) in neurons after cerebral ischemia .
The analysis of EGCs per unit area revealed that this variable increased in the various IR groups. The increase in EGCs positive for GFAP and S100β in the present study could be explained by the neuroprotective action of EGCs after injury to the ENS [29–30]. Previous studies have reported the importance of EGCs in the biology and function of enteric neurons as well as in neurochemical coding, the release and degradation of neuroactive substances, and neuroprotection; all of these mechanisms have been shown to be modulated by EGCs [1–3].
In our study, we observed that treatment with PB exerted different effects on GFAP-immunoreactive and S100β-immunoreactive cells. The treatment of GFAP-positive cells with PB has been shown to be effective, and the effects are similar to those obtained after treatment with Brilliant Blue G (BBG) . However, a marked decrease in S100β-positive cells was found in the PB-treated groups compared with the other groups. S100 is implicated in the regulation of Ca2+ and has been reported to act as a Ca2+ sensor [41, 42]. Other previous studies have shown that intracellular Ca2+ is translocated to the extracellular fluid via pannexin channels . Additionally, Ca2+ enters the cell from the extracellular fluid, and the increase in intracellular Ca2+ stimulates the opening of pannexin channels to release Ca2+ . PB is known to directly reduce Ca2+ overload .
In addition, several studies have demonstrated that P2X7 and pannexin-1 receptors can act synergistically [9, 18, 28, 33] and that they are associated with the same harmful signaling cascade that leads to neuronal and tissue death . Another study showed that PB also exerts a direct effect as an inhibitor of the P2X7 receptor . Additionally, Gulbransen et al. (2012)  administered PB to mice with intestinal inflammation and observed a recovery of enteric neurons. Extracellular ATP can activate the P2X7 receptor, which stimulates an inflammatory response in immune cells, such as the release of IL-1b and other cytokines .
In the present study, the profile area of neurons positive for HuC/D was assessed. The results revealed a wide variety of neuron sizes, ranging from small to medium and large, among the groups analyzed, and no difference was detected between the groups. However, Palombit et al. (2019)  observed different results because different classes of neurons are analyzed using different ischemia protocols, and an increase or decrease in the positive area can be detected depending on whether nNOS, ChAT or NF-200 positivity as assessed. For EGCs specifically, the profile area was reduced in the IR groups and restored in the PB groups.
In the present study, decreases in spontaneous activity, activity after the application of carbachol, and contraction amplitude after electrical field stimulation were observed in the IR groups. Regarding the contractile activity of the intestine, some studies suggest that intrinsic neural circuits might be impaired as a result of ischemia–reperfusion injury; some neurons might die, and others might change after injury [25, 27, 32, 46]. Additionally, ATP causes the death of enteric neurons and changes in intestinal motility during P2X7 receptor–dependent intestinal inflammation, which affects glia, neurons and the expression of connexin-42 .
The PB groups, particularly the 24h-PB group, did not respond to low carbachol concentrations or electrical field stimulation at 5 Hz, 10 Hz or 20 Hz. Contraction amplitudes were observed to recover after the application of low concentrations of carbachol and electrical field stimulation in the 14-day and 28-day PB groups. ATP exits through the pannexin channel into the extracellular fluid [28, 48] and exerts autocrine and paracrine effects on the P2X7 receptor . Additionally, fast excitatory synaptic potentials (EPSPs) have been shown to participate in P2X receptor activity [49, 50]. It has been reported that a decrease in the number of EGCs can result in disturbances in intestinal motility [51, 52].