Fig 1.
Gross macroscopic changes in stomachs and loss of body weight following vertical sleeve gastrectomy (VSG).
Panels A&B, Gross macroscopic images reveal the differences between stomachs that have undergone VSG compared to non-surgical controls. Panel A, An intact sham-operated control stomach reveals normal gross anatomy including fundus (F) corpus (C) and antrum (A). The greater curvature (GC), lesser curvature (LC), pyloric sphincter (PS), duodenum (D), and esophagus (E) are also identified. A prominent fundus is readily discriminated from the corpus by the change in tissue color (arrow). Panel B, Following VSG, up to 80% of the greater curvature including fundus and corpus was surgically removed. Dashed line represents where the greater curvature was surgically removed. The image presented was from a 3-week post-VSG stomach. Panels C&D, Diagrammatic representation of where the surgical procedure line and removal of fundus and corpus along the greater curvature was performed. Panel C, Ventral view of the stomach with identified regions (above). The surgical procedure line is identified by crisscross lines. Panel D, After opening of the stomach along the greater curvature, experimental sites (#1) are identified. Studies focused on an area distal and relatively distant from the sutured area in the antrum (site #1) and more proximal and closer to the sutured area in the corpus (site #2). Panel E, Body weight of mice that underwent VSG decreased dramatically over 7 days and then slowly returned within 28 days (solid circles), compared to sham controls (open circles). * P<0.05; *** P<0.001 compared to sham controls.
Table 1.
Details of primers used.
Fig 2.
Disruption of gastric pacemaker activity and its recovery post-VSG in the gastric antrum at site #1.
Panel A, Typical spontaneous electrical activity of gastric tissues following VSG. Intracellular microelectrode recordings of antral pacemaker activity performed at site #1 in sham controls (Ai, 0 week), compared to 1 week—4 months post-VSG (Aii-Av). Pacemaker activity was disrupted 1–3 wks post-VSG and recovered at 2 & 4 months. Panel B, There was a significant depolarization in RMP post-VSG compared to controls that slowly recovered by 4 months at site #1. ** P<0.01; *** P<0.001; **** P<0.0001 compared to sham controls.
Fig 3.
Analysis of changes in pacemaker waveform parameters post-VSG at site #1.
Panel A, Diagrammatic representation and electrical parameters of slow wave activity of electrical parameters analyzed. Upstroke amplitude of slow waves (Aa), slow wave plateau amplitude (Ab), half maximal duration of slow waves (Ac) and inter-slow wave period (Ad). Panels Ba-Bd, Summary of the changes in slow wave parameters at 3 weeks, 2 months and 4 months compared to sham-operated controls. * P<0.05; ** P<0.01; **** P<0.0001 compared to controls.
Fig 4.
Different types of aberrant gastric electrical activity recorded at site #1, 1-week post-VSG.
Normal antral slow waves were replaced with either (i) Panel A, no activity, (ii) Panels B&C, rapid oscillations in membrane potential, Panels D-F, slow waves with little or no plateau phase and Panels G&H, electrical activity that displayed single or Panel I, spike complexes.
Fig 5.
Loss or disruption of gastric pacemaker activity and its recovery following VSG in the gastric corpus at site #2.
Panel A, Intracellular electrical recordings of gastric pacemaker activity at site #2 under control conditions (Ai, 0 week). Panels Aii-Aiii, Slow waves were absent in gastric tissues 1-3weeks post-VSG, were disrupted at 2 months (Panel Aiv) and had recovered to sham control activity by 4 months (Panel Av). Panel B, Changes in resting membrane potential (RMP) at different times following VSG. A marked depolarization in RMP was observed at site #2 but this recovered to control levels by 2 months. * P<0.05; ** P<0.01 compared to controls (n = 6).
Fig 6.
Loss or disruption of pacemaker activity was limited to the stomach.
Panel A, Pacemaker activity recorded from the jejunum of a sham-operated animal and Panel B, 1-week post-VSG. Panel C, Jejunal slow waves 4-months following VSG. There was no difference in pacemaker activity recorded from jejunums post-VSG at any time period compared to controls (n = 4).
Fig 7.
Changes in post-junctional neural responses in the gastric antrum post-VSG.
Panel A, In control sham operated animals under control conditions (i.e. no drugs), stimulation of motor nerves (0.3 ms duration @1 Hz, for 1 second; arrows) by electrical field stimulation (EFS) evoked a fast inhibitory junction potential (fIJP; red arrow, Panels Ai&F). In the presence of the nitric oxide synthase inhibitor L-NNA (100 μM, Panels Aii&F) the amplitude of the fIJP was not affected. The P2Y1 purinergic receptor inhibitor MRS2500 (1μM) in the presence of L-NNA, inhibited the fIJP (Panels Aiii&F). Application of the muscarinic antagonist atropine had no further effect on post-junctional responses (Panels Aiv&F). Panels Bi&G, 1-week post-VSG the amplitude of the fIJP was reduced. Panels Bii&G, L-NNA (100 μM), had little or no effect on the amplitude of the fIJP. Panels Biii&G, MRS2500 in the presence of L-NNA, inhibited the fIJP. Panels Biv&G, Application of atropine in the presence of L-NNA and MRS2500 had no further effect on the post-junctional response. Panels Ci&H, Recovery of post-junctional inhibitory responses at 3 weeks, 2 months (Panels Di&I) and 4 months (Panels Ei&J). A similar pharmacological dissection was performed as in Panel A. Panels F-J, Summary of the changes in post-junctional inhibitory responses at 1 week (G), 3 weeks (H), 2 months (I) and 4 months (J), compared to controls (F). * P<0.05, compared to sham controls; ### P< 0.001, #### P<0.0001 for drug treatments.
Fig 8.
Changes in post-junctional responses in the gastric antrum in response to intense neural stimulation.
Increasing the frequency of EFS (0.3 ms duration @ 5Hz and 10sec train durations) generated several distinct post-junctional neural responses in sham operated animals. Panels Ai,F&K, Under control conditions EFS activation of enteric motor nerves evoked an initial fIJP (red arrow) followed by a slow IJP (sIJP; blue arrow). In the presence of L-NNA (100 μM, Panels Aii,F&K) the amplitude of the fIJP was not affected but the sIJP was replaced by an excitatory junction potential (EJP). At the termination of EFS, the EJP was immediately followed by a slow wave. Panels Aiii,F&K, In the presence of L-NNA, MRS2500 (1μM), inhibited the EFS evoked fIJP but evoked an EJP and slow wave during EFS. Panels Avi,F&K, Atropine (1 μM) in the presence of L-NNA and MRS2500 inhibited the EFS evoked EJP and slow wave. Panels Bi,G&L, 1-week post-VSG post-junctional neural responses were greatly attenuated. EFS evoked a small fIJP and after its termination an EJP were recorded. Panels Bii&G&L, Addition of L-NNA did not affect post-junctional neural responses. Panels Biii,G&L, MRS2500 in the presence of L-NNA inhibited the fIJP but did not affect the late developing EJP. Panels BivG&L, Atropine in the presence of L-NNA and MRS2500 inhibited the late developing EJP. Panels C-E, reveal a recovery of neural responses such they were similar to control responses by 2 and 4 months. A similar pharmacological dissection was performed as in Panels A&B. Panels F-O, summarized data of the effects of VSG on the fIJP (Panels F-J) and the sIJP (Panels K-O).). # P<0.05, ### P< 0.001, #### P<0.0001 for drug treatments.
Fig 9.
Disruption of Ca2+ transients in gastric ICC following sleeve surgery.
Panel A, Diagrammatic representation of Ca2+ imaging sites in gastric tissues of sham and VSG KitCreGCaMP6f animals. Panel B, Images of ICC expressing the Ca2+-sensor, GCaMP6 in gastric tissues. Three sites were chosen for Ca2+ imaging (sites #1, as described in Fig 2). A 3rd. site proximal and closer to the surgical line than site #1 was also chosen for imaging and identified in Panel A. Panel C, Spatio-temporal Ca2+ map (STMap) obtained from sham operated mice showing Ca2+ transient firing patterns in ICC-MY at site #1. Panels D&E, STMaps from sites #2 and site #3, respectively. A color-coded hue was added as an overlay to enhance visualization; color scale indicates intensity of Ca2+ transients (i.e., dark blue represents low Ca2+ transient intensity; light yellow to red indicates high Ca2+ transient intensity). Panels F-H, STMaps of ICC-MY-dependent Ca2+ transients obtained 3 weeks post-VSG. Panel F, ICC-MY-dependent Ca2+ transients were clustered and relatively normal at site #1, Panel G, Ca2+-transients were absent at site #2, Panel H, Ca2+-transients were greatly disrupted at site #3 compared to organized Ca2+ transient clusters (CTCs) in sham control mice (Panel E). Panels I&J, Summary of Ca2+ transient parameters recorded from the 3 identified sites. Panel I shows CTC frequency and Panel J CTC duration. *denotes significant difference between sham and sleeve operated animals. ** P<0.01 and **** P<0.0001. Scale bar in B is 50 μm and pertains to all images.
Fig 10.
Changes in gene transcripts of gastric tissue following VSG.
Panels A-E, Changes in Kit (ICC), Ano1 (ICC), Pdgfra (PDGFRα), Nos1 (nNOS; nitric oxide synthase inhibitory nerves), Myh11 (smooth muscle myosin), in control (0 week), 1 week, 3 weeks, 2 months, and 4 months, respectively from site #1 post-VSG. Kit, Ano1, Pdgfra Nos1 and Myh11 transcripts were markedly decreased in antral muscles 1-week post-VSG. Cell-specific gene transcripts showed partial recovery over the 4-month examination period. * P <0.05 and ** P <0.01.
Fig 11.
Comparison of changes in expression of gene transcripts in antum versus corpus in gastric tissues post-VSG.
Panels A-D, Transcript expression of Kit, Ano1, Pdgfra, and Nos1 genes at sites #1 and #2, 1- and 3- weeks post-VSG, respectively. At site #1, all cell-specific genes were significantly downregulated by 1 week and had shown only a partial recovery by 3 weeks. All of the gene transcripts that were examined at site #2 were downregulated, to a level that the expression was negligible, and these did not recover by 3 weeks (** P <0.01 and *** P <0.001 for all genes).
Fig 12.
Immunohistochemical labeling of ICC and enteric nerves following VSG.
Panels A-C, double labeling of Kit+ ICC at the level of the myenteric plexus (A; *) and intramuscular ICC (arrowheads, green). B, PGP9.5+ enteric nerve ganglia and fibers (arrows, red), in control gastric antrum tissues. Merged image is shown in Panel C. Panels D-F, double labeling of Kit+ ICC-IM (D; arrowheads, green) and PGP9.5+ enteric nerves (E; arrows, red), including the myenteric plexus, 1-week post-surgery. Note the absence of ICC-MY at the level of the myenteric plexus. Merged image is shown in Panel F. Panels G-I, 3-weeks after surgery ICC-MY were still disrupted or absent, whereas ICC-IM (Panel G; arrowheads) and enteric nerves (Panel H; arrows) were evident. Panel I, merged image of panels G&H. Panels J-L, 4 months post-surgery ICC-MY networks had recovered (*, green Panel J). ICC-IM (arrowheads) were also evident. Panel K, PGP9.5+ enteric nerves (red; arrows) were widely distributed within the circular muscle layer. Merged image is shown in panel L. Scale bars in Panels C,F,I&L = 50 μm and applies to their respective panels.