Gastroparesis is a chronic, symptomatic disorder of the stomach that is characterized by delayed gastric emptying in the absence of mechanical obstruction [
Diabetic gastroparesis is common but without satisfactory treatment in clinical practice. Gastric electrical stimulation (GES) is a potentially alternative therapy for the medical or surgical treatment of difficult gastroparesis [
Gastric emptying is mediated via vagovagal reflex pathway. Earlier studies had found that delayed gastric emptying was presented after truncal vagotomy, but there were about one-half of the patients returning to normal one month later; this finding implied that neural pathways other than the truncal vagus compensated impaired motility functions [
Therefore, the aims of this study were to better describe the distribution and morphology of the EGC in gastric longitudinal muscle myenteric plexus (LMMP) of rats with different periods of diabetic rats by using antibodies against S100B to explore the possible mechanisms of the effects by observing the changes of EGC in the stomach.
Forty-six adult male Sprague Dawley rats weighing 250–350 g were used in this study. These rats were purchased from the Experimental Animal Center of Tongji Medical College of Huazhong University of Science and Technology (Wuhan, Hubei Province, China). The rats were divided into two large groups randomly including the age-matched control group (CN,
After an overnight fast, all rats were deeply anesthetized with 1% Nembuta (40 mg/kg, ip) and the maintenance anesthetic was given whenever necessary. The surgical procedure was performed through a midline incision with small scissors. Two pairs of temporary cardiac pacing wires (United States Surgical, a division of Tyco Healthcare Group LP) were placed on the serosal surface of the greater curvature of the stomach by unabsorbable sutures. One pair in the middle of the stomach was used for stimulation, with the other in the middle between the proximal pair and the pylorus for recording. The electrodes in each pair were 0.3 cm apart. The insulated connecting wires were subcutaneously brought out to the back of the neck for connection to the recorder or the stimulation equipment. The abdominal wall and skin were then closed in a simple interrupt pattern, and the study was initiated after the rats had fully recovered from the operation, about 10–14 days afterward. This procedure was applied in all groups of rats.
After a complete recovery from the surgery, diabetes was induced in overnight fasted rats by a one-time intraperitoneal injection of streptozotocin (STZ, 55 mg/kg, Sigma, USA) dissolved in a 1.5 mL citric acid buffer (PH 4.5 Sigma, USA). The control group rats were injected with 1.5 mL citric acid buffer. The blood glucose level was examined one week after STZ injection by cutting off the tip of the tail. Animals exhibiting blood glucose levels more than 16.7 mmol/L were considered diabetic; otherwise they were excluded from our study. The blood glucose was measured again on the day before sacrifice.
Intrinsic gastric slow waves were recorded from the implanted distal serosal electrodes on the greater curvature using a multichannel recorder (Acknowledge 3.7.1, MP100A-CE, Biopac System, Santa Barbara, CA, USA) in all groups before the injection of STZ or vehicle. Before recording, all rats should be quiet for 30 mins; the signals were displayed on a computer monitor and saved on hard disk. The definition of normal gastric slow wave frequency range was 4–6 cycles/min [
As is known to all, gastric electrical stimulation (GES) with short pulses improves nausea and vomiting in patients with gastroparesis; however, the effects of short pulses GES on gastric emptying or gastric slow waves were limited [
After SGES-60 mins or SGES-7 days, gastric emptying was assessed using a validated method as previously described [
Antrum of stomach tissue was fixed for 2 h in 2.5% glutaraldehyde (PH: 7.4) and rinsed with 0.1 M phosphate buffer and then fixed with 1% osmic acid. After a series of dehydration, embedding, sectioning, and polymerization, ultrathin sections were cut parallel to either the circular or the longitudinal muscle layers with ultramicrotome (Leica, Ultracut, UCT, Germany) and stained with uranium acetate. These sections were examined with a transmission electron microscope (Tecnai G2 12, FEI Company, Eindhoven, The Netherlands) and photographed for recording.
This method was used for evaluating the expression of S100B in EGC. After measuring gastric emptying, the rats were sacrificed by cervical dislocation. A median abdominal incision was made to expose the abdominal cavity and the whole stomach was harvested. The specimens of stomach were washed with PBS (PH = 7.4), and they were cut into small segments with a size of approximately 5 mm; then stomach samples were fixed in 4% paraformaldehyde for 4–6 hours at room temperature. Thereafter, tissue blocks were embedded in paraffin; the paraffin-embedded tissue samples were sliced into 5
Real-time polymerase chain reaction (RT-PCR) was used to measure the expression levels of S100B using GAPDH as internal control. Total RNA was isolated using TRIzol reagent (Invitrogen, USA). The obtained RNA samples were reverse transcribed according to instructions. Primer sequences used were S100B forward: 5′-GAGCAGGAAGTGGTGGACAAA-3′, S100B reverse: 5′-CACTCCCCATCCCCATCTT-3′, GAPDH forward: 5′-GTATGACTCTACCCACGGCAAGT-3′, and GAPDH reverse: 5′-TTCCCGTTGATGACCAGCTT-3′. The amplification of S100B and GAPDH fragments was performed using the SYBR Green PCR master mix. A 10
Results are presented as means ± SD. Paired Student’s
One week after STZ injection, all rats of diabetes groups presented polydipsia, polyuria, polyphagia. The random blood glucose level of the experimental group always more than 16.7 mmol/L, but the control group was in the normal range. Eight weeks later, the terminal diabetic rats became languid and extreme emaciation. The hair was withered without gloss. Diabetic rats’ models were induced successfully in our study (Table
Blood glucose and body weight in controls and diabetes mellitus rats.
Group |
|
Body weight, g | Blood glucose, mmol/L | ||
---|---|---|---|---|---|
Before injection | After injection | Before injection | After injection | ||
ECN | 5 | 276.5 ± 25.16 | 312.4 ± 32.35 | 6.92 ± 0.24 | 9.92 ± 2.11 |
EDM | 6 | 243.0 ± 39.79 | 222.7 ± 22.11b | 8.15 ± 2.63 | 26.00 ± 4.46a,b |
EDM + SGES | 12 | 269.2 ± 26.66 | 228.5 ± 25.48a,b | 10.14 ± 2.59 | 26.65 ± 4.51a,b |
TCN | 5 | 254.4 ± 31.60 | 437.0 ± 35.09 | 7.82 ± 1.47 | 7.68 ± 1.59 |
TDM | 6 | 256.8 ± 20.88 | 197.0 ± 20.06a,b | 8.78 ± 1.51 | 26.87 ± 3.72a,b |
TDM + SGES | 12 | 280.6 ± 25.17 | 208.6 ± 16.61a,b | 9.07 ± 3.15 | 26.57 ± 4.22a,b |
Data are presented as means ± SD. ECN: early control (7–14 days), EDM: early diabetes (7–14 days), TCN: terminal control (56–63 days), and TDM: terminal diabetes (56–63 days). SGES: synchronized gastric electrical stimulation of dual pulses.
The normal slow waves at baseline were shown in Figure
Gastric slow waves in ECN, EDM, TCN, and TDM. The impaired slow waves of TDM can be observed from above recording. While the rhythm in EDM was normal, the frequency of gastric slow waves increased; SGES normalized the gastric slow waves in EDM and TDM. Note: ECN: early control (7–14 days), EDM: early diabetes (7–14 days), TCN: terminal control (56–63 days), and TDM: terminal diabetes (56–63 days). SGES: synchronized gastric electrical stimulation of dual pulses.
Gastric slow wave in 7–14-day control rats (ECN group) and 7–14-day diabetes rats (EDM group)
Gastric slow wave in 56–63-day control rats (TCN group) and 56–63-day diabetes rats (TDM group)
Gastric emptying rate was significantly delayed in TDM (
The acute and chronic synchronized gastric electrical stimulation of dual pulses improved the delay of gastric emptying in TDM group (SGES-60 mins:
Effect of SGES on gastric emptying. The gastric emptying rates were significantly different between control groups and diabetic groups. Gastric emptying rate was faster in EDM than the age-matched control group but slower in TDM. ASGES and CSGES accelerated gastric emptying in TDM significantly but only had a little effect on EDM. There were no significant differences between EDM and EDM with SGES. Data are presented as means ± SD.
EGC were mainly located in myenteric plexus of the stomach. In the ECN and EDM, the electron microscope revealed that the smooth endoplasmic reticulum, Golgi apparatus, mitochondria, and filaments are abundant in cytoplast of EGC. While the mild vacuolization of mitochondria can be observed in cytoplast in TCN, the number of EGC significantly reduced in TDM. Dilation of endoplasmic reticulum and swelling of mitochondria in cytoplast can be observed in TDM and the filaments decreased seriously. Comparing with the age-matched diabetic groups, the number of mitochondria and filaments increased after SGES-60 mins and SGES-7 days both in EDM and TDM groups (Figure
(a) Ultrastructure images of EGC in rat myenteric plexus of ECN, EDM, EDM + ASGES, and EDM+CSGES. Smooth endoplasmic reticulum (letter E), Golgi apparatus (letter G), mitochondria (letter M), and filaments are abundant in cytoplast of ECN and EDM. The number of mitochondria and filaments increased after ASGES and CSGES. And lysosome can be observed in EDM after ASGES. (b) Ultrastructure images of EGC in TCN, TDM, TDM + ASGES, and TDM + CSGES. Mild vacuolization of mitochondria can be observed in cytoplast of TCN. Dilation of endoplasmic reticulum and swelling of mitochondria in cytoplast can be observed in TDM and the filaments decreased seriously. The number of mitochondria and filaments increased after ASGES and CSGES. Note: ASGES: SGES-60 mins; CSGES: SGES-7 days, 60 mins/day.
Figures
EGC in the rat myenteric plexus of the stomach (protein gene product S100B immunohistochemistry,
Immunohistochemical staining of S100B in the enteric plexus of stomach. Tissues with brown deposits were positive reaction. (a) S100B expression in ECN, EDM, EDM + ASGES, and EDM + CSGES. (b) S100B expression in TCN, TDM, TDM + ASGES, and TDM + CSGES. (c) Semiquantification of S100B-positive cells in each group. There were no significant differences between ECN and EDM. The expression of S100B in TDM and TCN groups was lower than in EDM and ECN groups, respectively. ASGES and CSGES had little effect on the expression of S100B in EDM. Comparing with TDM group, the immunopositive stain was decreased significantly in TDM group. But the immunopositive stain significantly increased after ASGES and CSGES. Data are presented as means ± SD.
The mRNA expression of S100B was shown in Figure
mRNA values of S100B in the enteric plexus of stomach. The mRNA expression of S100B was decreased with the course of diabetes, especially in TDM. But there were no differences between ECN and EDM. Furthermore, both mRNA expressions of S100B in TDM and TCN were significantly decreased compared with the age-matched early group. The expression of S100B mRNA in TDM increased both after ASGES and CSGES compared with TDM; statistical analysis showed significant differences between CSGES group and TDM group, but no statistical differences were observed between ASGES and TDM. Data are presented as means ± SD.
In this current study, the model of diabetes mellitus in rats was induced successfully by injection of streptozotocin (STZ). We found that gastric emptying rate was delayed in 56–63-day diabetes rats, but that in 7–14-day diabetes rats increased. The number of EGC in 56–63-day diabetes rats decreased and the ultrastructure changed. Additionally, we demonstrated the age-associated loss of EGC between 7–14-day control rats and 56–63-day control rats. SGES was able to not only accelerate gastric emptying in 56–63-day diabetes rats and 7–14-day diabetes rats but also normalize gastric slow waves. We also demonstrated that the expression of S100B protein which was used frequently as EGC marker decreased in 56–63-day diabetes rats. However, this marker expression increased after SGES and the ultrastructure of EGC was partially restored whether acute stimulation or chronic stimulation. The effect of SGES on gastric emptying may be associated with EGC activation.
Gastroparesis is a disorder in which the gastric emptying is delayed without mechanical obstruction [
As is known to all, the damage of ENS always occurred both in diabetic patients and animal models [
In gastrointestinal tract, the EGC mainly distributed in submucosal and myenteric plexuses and surrounded enteric neurons (Figure
S100B belongs to the S100 protein family; it is a kind of Ca2+ binding protein. S100B protein produced the best results in identifying EGC. This protein regulates cytoskeletal structure and function and calcium homeostasis in EGC. It is thought to be exclusively localized in these cells. The S100B in brain can promote neuronal survival, increase free Ca2+ levels in vitro, and participate in Ca2+ waves’ start and propagation, but the date on EGC is limited [
However, gastric emptying in 7–14-day diabetes rats increased; the number of EGC has no obvious change in our study. The pathogenesis of accelerated gastric emptying in rats with early diabetes is heterogeneous and the mechanism remains poorly understood. But it has been reported that fasting ghrelin plasma concentrations were elevated in the early stage of diabetes compared with those rats without diabetes [
To our surprise, both SGES-60 mins and SGES-7 days have no obvious effect on the expression of S100B in 7–14-day diabetes rats. The ghrelin plasma concentrations enhanced at 7–14-day diabetes rats as we mentioned above. And Lopez et al. have reported that ghrelin is capable of decreasing serum concentration of the astrocytic protein S100B in brain [
In summary, SGES not only normalizes gastric slow waves in 7–14-day diabetes rats and 56–63-day diabetes rats but also accelerates gastric emptying in 56–63-day diabetes rats. The effects of SGES on S100B expression in 7–14-day diabetes rats are little which indicates the other factors such as the fact that ghrelin is involved in S100B. Meanwhile, the impaired EGC can be partly restored which makes us have a good understanding of the plasticity of ENS. This study has highlighted a potential role of EGC in the pathophysiology of diabetic gastroparesis. Further studies such as patch clamp technique and calcium imaging experiment in EGC are needed to gain a better understanding of the signaling mechanism of EGC activation in the action of SGES.
The authors declare that there is no conflict of interests regarding the publication of this paper.
This study was supported by a Grant from the National Natural Science Foundation of China (Project no. 81170342). All authors read and approved this paper.